CA1184101A - Determination of lecithin - Google Patents
Determination of lecithinInfo
- Publication number
- CA1184101A CA1184101A CA000407494A CA407494A CA1184101A CA 1184101 A CA1184101 A CA 1184101A CA 000407494 A CA000407494 A CA 000407494A CA 407494 A CA407494 A CA 407494A CA 1184101 A CA1184101 A CA 1184101A
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- Prior art keywords
- buffer
- litre
- detergent
- lecithin
- borate
- Prior art date
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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
-
- 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
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
-
- 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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Analysing Materials By The Use Of Radiation (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
The present invention provides a process and reagent for the determination of lecithin in a sample, and for the splitting of lecithin; in particular lecithin is split with, for example, phospholipase-C
and alkaline phosphatase to give choline, this is reacted in the presence of adenosine triphosphate with the help of choline kinase again to give phosphoryl-choline and the thereby formed adenosine diphosphate is measured in known manner; in accordance with the invention the splitting of the lecithin is carried out in the presence of a borate/borax buffer and of a detergent, particularly a non-ionic detergent, based on a condensation product of polyethyleneglycol with isooctylphenol.
The present invention provides a process and reagent for the determination of lecithin in a sample, and for the splitting of lecithin; in particular lecithin is split with, for example, phospholipase-C
and alkaline phosphatase to give choline, this is reacted in the presence of adenosine triphosphate with the help of choline kinase again to give phosphoryl-choline and the thereby formed adenosine diphosphate is measured in known manner; in accordance with the invention the splitting of the lecithin is carried out in the presence of a borate/borax buffer and of a detergent, particularly a non-ionic detergent, based on a condensation product of polyethyleneglycol with isooctylphenol.
Description
's~
The present invention is concerned with a process and a reagent for the determination of lecithin in a sample.
A determination of lecithin is carried out, for example, in the investigation of foodstuffs. Lecithin is, in particular, present in egg yolk. The lecithin content of egg yolk is only subject to a narrow limit of variation so that it can be regarded as being a measure for the egg content of a foodstuff sample.
The determination of lecithin in amniotic fluid is of particular importance~ In this manner, the foetal development of the lungs can be substantially ascertained prior to birth and the possible risk of a respiratory failure syndrome assessed for a neonate. Respiratory failure syndrome is one of the most important complic-ations which can occur in premature neonates after birth.
It is brought about by a deficiency of surfactant ~ich, in the case of full term neonates, covers the lung alveoli in the form of a thin film and serves the purpose of lowering the surface tension on the boundar~
between air and fluid in the alveoli and thereby ensure~
the stability of the alveoli. The surfactant consists of proteins, carbohydrates and lipoids, the decisive surface-active properties being due to the phospho-lipids~ for example dipalmitoyllecithin, phosphatidyl-glycerol and sphingomyeline.
1 ~ 4~.~`d~
The present invention is concerned with a process and a reagent for the determination of lecithin in a sample.
A determination of lecithin is carried out, for example, in the investigation of foodstuffs. Lecithin is, in particular, present in egg yolk. The lecithin content of egg yolk is only subject to a narrow limit of variation so that it can be regarded as being a measure for the egg content of a foodstuff sample.
The determination of lecithin in amniotic fluid is of particular importance~ In this manner, the foetal development of the lungs can be substantially ascertained prior to birth and the possible risk of a respiratory failure syndrome assessed for a neonate. Respiratory failure syndrome is one of the most important complic-ations which can occur in premature neonates after birth.
It is brought about by a deficiency of surfactant ~ich, in the case of full term neonates, covers the lung alveoli in the form of a thin film and serves the purpose of lowering the surface tension on the boundar~
between air and fluid in the alveoli and thereby ensure~
the stability of the alveoli. The surfactant consists of proteins, carbohydrates and lipoids, the decisive surface-active properties being due to the phospho-lipids~ for example dipalmitoyllecithin, phosphatidyl-glycerol and sphingomyeline.
1 ~ 4~.~`d~
- 2 -The lecithin content in amniotic fluid varies during pregnancy for a long time between 1 and 2 mg./dl., increases in the 35th and 37th week of pregnancy to 5 to 10 mg./dl. and in the 40th week usually has a value of more than 10 mg./dl. The sharp increase of the lecithin concentration between the 35th and 37th week of pregnancy can be taken as being a characteristic value for the presence of foetal lung development.
Due to the respiratory movements of the foetus~
there results a continuous exchange of the alveolar fluids with the amniotic fluid so that the possibility exist~, by mea~urèment of the phospholipid content in the amniotic fluid, to make an assessment of the sur-factant content in the foetal alveoli and thereby, even prior to birth, to assess the state of development of the lungs. Therefore, the determination of lecithin in amniotic fluid is of decisive importance for pregnancy diagnosis, especially with regard to an early termination of a risky pregnancy because of the danger of a respiratory failure syndrome.
~umerous methods are available for the determin-ation of lecithin in amniotic fluid which differ, on the one hand, by the differing amount of work required and, on the other hand, by the e~actitude of the prediction, the degree of difference between the various available methods sometimes being considerable.
An enzymatic method for the determination of the .
Due to the respiratory movements of the foetus~
there results a continuous exchange of the alveolar fluids with the amniotic fluid so that the possibility exist~, by mea~urèment of the phospholipid content in the amniotic fluid, to make an assessment of the sur-factant content in the foetal alveoli and thereby, even prior to birth, to assess the state of development of the lungs. Therefore, the determination of lecithin in amniotic fluid is of decisive importance for pregnancy diagnosis, especially with regard to an early termination of a risky pregnancy because of the danger of a respiratory failure syndrome.
~umerous methods are available for the determin-ation of lecithin in amniotic fluid which differ, on the one hand, by the differing amount of work required and, on the other hand, by the e~actitude of the prediction, the degree of difference between the various available methods sometimes being considerable.
An enzymatic method for the determination of the .
3 --lecithin content of amniotic fluid is known, for example, from Geburtshilfe und Frauenheilkunde, 39, 849-856/1979, which method is easier to carry out than the non-enzymatic method~s and is of equal or even of better exactitude and practicability. In the case of this method of determination, the lecithin is split with the help of phospholipase-C ln a solution contain-ing glycine buffer to give phosphoryl-choline, which is further broken down by alkaline phosphatase to give choline. The latter is further reacted in the presence of adenosine triphosphate, with the help of choline kinase, with the formation of adenosine diphosphate.
The adenosine diphosphate formed is then measured photometrically via a known enzymatic reaction sequence.
~his detection process can be illustrated by the follow-ing reaction equations:
;
phospholipase-C
1) lecithin + H20 - ' 1,2-diglyceride + phosphorylcholine 2) phosphorylcholine + H20 ~ cholin~ + Po43 choline kinase 3) choline ~ ~TP - ~ phosphorylcholine ~ A~IP
The adenosine diphosphate formed is then measured photometrically via a known enzymatic reaction sequence.
~his detection process can be illustrated by the follow-ing reaction equations:
;
phospholipase-C
1) lecithin + H20 - ' 1,2-diglyceride + phosphorylcholine 2) phosphorylcholine + H20 ~ cholin~ + Po43 choline kinase 3) choline ~ ~TP - ~ phosphorylcholine ~ A~IP
4) ADP + PEP - ATP + pyruvate
5) pyruvat2 + ~ADH ~ H+ ~ lactate + NAD
The abbreviations used in the above equations have the following meanings:
AP = alkaline phosphatase ATP = adenosine-5'-triphosphate ADP = adenosine-5'-diphosphate PEP = phosphoenol pyruvate PK = pyruvate kinase ~ADH/~AD~ = nicotinamide-adenine-dinucleotide, reduced/
oxidised form LDH = lactate dehydrogenase The assessment of a threatened respiratory failure syndrome is admittedly substantially simplified by the enzymatic process for the determination of lecithin in amniotic fluid. However, it suffers from considerable disadvantages: in a considerable number o samples, disturbances arise in the course of the measurement due to a strong turbidity of the solution to be measured which, in some cases, even maXe a determination of the lecithin completely impossible. The reason for this is the high degree of turbidity of the amniotic fluid which is used in a cuvette with a relatively large sample volume and, therefore, in the test, too, can give rise to a strong and no longer measurable turbidity.
Too high an initial extinction is measured which results in a high error quote and in a deficient degree of precision of the measurement. Furthermore, in the ; case of the measurement of such samples, considerable - s -deviations are found from the linearity of the measure-ment.
It is an object of the present invention to provide a process which does not suffer from the above-mentioned disadvantages.
According to the present invention, this object is achieved by a process which differs from the prior art in that the splitting of the lecithin is not carried out in a glycine buffer but in a borate/borax buffer in the presence of a deter~ent, especially a non-ionic detergent based upon a condensation product of poly-ethyleneglycol with isooctylphenol.
In particular the splitting of the lecithin is carried out with phospholipase-C and alkaline phosphatase.
In principle, a clarification of the solution to be measured can be achieved with many different detergents.
~owever, most of the detergents display a more or less ~ strongly marked inhibition action on the en~ymatic process, i which gives rise to Ealse measurement results. On the other hand, en~ymatic reactions proceed in a satisfactory manner only within a relatively narrow pH range. In the case of the determination of lecithin in a sample, the pH
range of 7.0 to 8.5 is optimal. Furthermore, it has been found that for the com~lex system of the enzymatic reactions which are necessary for the lecithin deter~ination, only a few buffer systems can be considered.
Surprisingly, we have now found that optimum measurement conditions for a disturbance-iree determin-a, ~
-- 6 ~ation of lecithin can be achieved in a sample solution when, in the case of splitting the lecithin, use is made of a borate/borax buffer and of a deter~ent, especially a non-ionic detergent based upon a condensation product of polyethyleneglycol with isooctylphenol.
Thus in accordance with the invention there is provided in a process for the determination of lecithin in a fluid sample in which the lecithin in the sample is split in the presence of a buffer to provide a product capable of being measured as a measure of lecithin in the sample, the improvement wherein said buffer is a borate/borax buffer and the splitting is carried out in the presence of a detergent.
It will be understood that the product which is measured as a measure of the lecithin in the sample need not be formed directly in the splitting of the lecithin, but may result-from further reaction on the product of the lecithin splitting.
It will be further understood that in the ! 20 splitting of lecithin any detergent may be used which does not interfere with or inhibit the splitting process. In i the determination of lecithin by the process of the invention in which the splitting of lecithin represents one step, a detergent is employed which is a non-inter-fering detergent, by which is to be understood a detergent which does not exhibit an inhibition action on any of the process steps, particularly the enzymatic process steps, such as would result in false measurements. The non-interfering detergent should additionally be one which is effect:ive to clarify the fluid sample, at least at the measurement stage when the measurement is photo-metric in nature.
The invention particularly contemplates a process for the determination of lecithin in a sample in which lecithin is first split with the help of phospholipase-C and alkaline phosphatase to give choline, thereafter the resultant choline is further reacted in the presence of adenosine triphosphate with the help of choline kinase again to give phosphoryl choline and the adenosine diphosphate thereby formed is measured in Xnown manner, wherein the splitting of the lecithin is carried out in the presence of a borate/
borax buffer and in the presence of a non-ionic deter-gent based upon a condensation product of polyethylene-glycol with isooctylphenol.
According to a preferred embodiment of the process of the invention, the sample, for example amniotic fluid, optionally diluted with water, is first mixed with the borate~borax buffer, a magnesium salt, the detergent, phospholipase-C and alkaline phosphatase and incubated at ambient temperature or at a slightly elevated temperature. The incubation period is, depending upon the selected temperature, preferably 10 to 60 minutes. Thereafter, heating is carried out for a short time to 95 to 100C., for example in a water-bath. After cooling the mixture, it is centrifuged and the supernatant is mixed with NADH, ATP, phosphoenol pyruvate, glucose, pyruvate kinase and lactate dehydrogenase. After a short waiting period of about 5 minutes, the extlnction El is measured. Subsequently, a choline kinase solution is added thereto in order to initiate the reaction sequence for the detection of the choline liberated from the lecithin. When the reaction is concluded, the extinction E2 is measured. The lecithin content of the sample can be determined from the extinction difference. In addition to the actual determination of the sample, it is recommended to ; ~ measure a reagent blank.
It is preferable to combine the enz~nes and adjuvants necessary for the process according to the present invention to give definite solutions or suspensions.
Thus, the borate/borax buffer i5 preferably used together with the detergent and the magnesium salt in the form of a "buffer solution". It is preferable to use a borate/borax buffer with a pH value of 7.0 to 8.5 in a concentration of 0.05 to 0.5 mole/litre of buffer solution and preferably of 0.07 to 0.25 mole/litre of buffer solution ~specially preferred is a borate/borax buffer with a pH value of 708 to 8.2, which is used in a concentration of 0.1 to 0.2 mole/litre of buffer j! J
_9_ solution in the case of the determination. The non-ionic detergent based on a condensation product of polyethyleneglycol with isooctylphenol is added to the buffer solution in a concentration of 0.5 to S g./
litre and preferably of 1.0 to 3.0 g./litre. The commercially available product Tinoveti~ JU of Ciba-Geigy has proved to be especially useful as the detergent according to the present invention.
The phospholipase-C and the alkaline phosphatase are used together as "enzyme suspension A", the con-centration of phospholipase-C being 40 - 200 U/ml.
and preferably 100 Ujml. and the concentration of the alkaline phosphatase being 20 to 200 U/ml. and prefer-ably 80 UJml.
The NADH, ATP, phosphoenolpyruvate and glucose are advantageously combined as "coenzyme solution".
l'he individual concentrations in this solution are 2.5 to 6.0 mmole/litre and preferably 4.0 mmole/litre of NADH, 10 to 30 mmole/litre and preferably 20 mmole/
litre of ATP, 3 to 10 mmole/litre and preferably 7 mmole/
litre of phosphoenol pyruvate and 30 to 100 mmole/litre and preferably 50 to 60 mmole/litre of glucose.
Pyruvate kinase and lactate dehydrogenase are added together as "enzyme suspension B", the concen-trations of pyruvate kinase and of lactate dehydrogenase being, in each case, 150 to 1000 U~ml. and preferably 300 U/ml.
* trade mark Choline kinase is preferably used in the form of a separate solution with a content of 2 to 10 U/ml.
and preferably of 2.5 U/ml.
In another aspect of the invention 'chere i s provided in a reagent for the determination of leci thin in a fluid sample comprisiny a buffer and an agent effective to split lecithin and to provide a product capable of being measured as a measure of lecithin in the sample, the improvement wherein said buffer is a borate/borax buffer and additionally including a detergent .
The present invention also contemplates a reagent for the determination Qf lecithin which comprises the following cornponents:
a ) a buf f er solution wi th 0.05 to 0.5 rr.ol/litre borate/borax buffer, pH 7.0 - 8.5 2 to 20 mmole~litre of magnesium ions 0.5 to 5.0 g./litre of detergent 1~) an enzyme suspension ~ with 40 to 200 U/ml. phospholipase-C
20 to 200 U/ml. alkaline phosphatase c) a coenzyme solution with 2.5 to 6.0 mrnole/litre N~
10 to 30 mmole/litre ATP
3 to 10 mmole~litre phosphoenol pyruvate 30 to 100 mmole~litre glucose d) an enzyme suspension B with f~
150 to 1000 U/ml. pyruvate kinase 150 to 1000 U/ml. lactate dehydrogenase I e) a solution with j 2 to 10 U/ml. choline kinase.
Quite especially preferred is a reagent com-~ prising the following components:
¦ a) a buffer solution with 0.2 mol/litre borate/borax buffer, pH 7.8 - 8.2 10 mmole/litre magnesium sulphate 2.5 g./litre Tinovetin JU~
b) an enzyme suspension A with 80 U/ml. phospholipase-C
100 U/ml. alkaline phosphatase c) a coenzyme solution with 4.0 mmole/litre ~ADH
; , 20 mmole/litre ATP
7 mmole/litre phosphoenol pyruvate , 56 mmole/litre glucose d)` an enzyme suspension B with ~ 300 U/ml. pyruvate kinase ; 300 U/ml. lactate dehydrogenase ` e) a solution with 2.5 U/ml. choline kinase.
!
i * trade mark In yet another aspect of the invention ther~
is provided in a reagent for splitting lecithin com-prising phospholipase-C, alkaline phosphatase and a buffer, the improvement wherein said buffer is a borate/borax buffer and further including a detergent.
The present invention particularly contemplates a reagent for splitting lecithin, which comprises:
~ to 10 U/ml. phospholipase-C
1 to 10 U/ml. alkaline phosphatase 0.05 to 0.5 mole/litre borate/borax buffer, pH 7.0 - 8.5 2 to 20 mmole/litre magnesium ions 0.5 to 5.0 g./litre detergent.
For splitting lecithin, a reagent composition is quite especially preferred which contains the follow-ing individual components in the given concentrations:
.
`p~
4 U/ml. phospholipase-C
5 U/ml. alkaline phosphatase 0.2 mole/litre borate/borax buffer, pH 7.8 - 8.2 10 mmole/litre magnesium sulphate 2~5 g./litre Tinovetin JU.
The reagent according to the present invention for splittlng lecithin is preferably prepared by mixing 1.0 ml. of the above-described buffer solution with 50 ~1~ of the above-described enz~ne suspension A. The reagent thus obtained is preferably added to the sample ; ; to be measured in the ratio of 1:1.
The following Examples are given for the purpose of illustrating the present invention:
Exam~e 1.
Determination of lecithin in amniotic fluid.
1 ml. of a sample solution which contains about ~` 0.1 mg. of lecithin is mixed with 1 m~. of a solution which contains 0.2 mole/litre borate/borax buffer ~pH
7.8 - 8.2), 10 mmole/litre magnesium sulphate and 2.5 g./litre Tinovetin ~U~ To this mixture is added 0.05 ml. of an enzyme suspension of 80 U/ml. phospho-lipase-C and 100 U/ml~ alkaline phos~hatase. The mixture is left to stand at 37C. for 20 minutes, whereafter it is heated for 5 minutes to 95 to 100C.
After cooling to ambient temperature, 0~1 ml. of a solution of 4~0 mmole/litre NADH, 20 mmole/litre ATP, 7 mmole/litre phosphoenol pyruvate and 56 mmole/litre * trade mark ..~
-glucose, as well as 0.02 ml. of an enzyme mixture of 300 U/ml. pyruvate kinase and 300 U/ml. lactate dehydrogenase are added thereto. The solution obtained is mixed and left to stand for 10 minutes at ambient temperature. The sample is introduced into a cuvette and the extinction El is measured. The enzymatic reaction is initiated by the addition of 0.05 ml. of a choline kinase solution with a content thereof of 2.5 U/ml. After leaving to stand for 30 ~inutes at ambient temperature, the extinction E2 is measured.
From the extinction difference El-E2, there is determined the extinction decrease ~E, from which is obtained the lecithin content of the sample according to the follow~ng equation:
lecithin (mg./100 ml.) = ~E x 47.86.
For each determination of lecithin in a sample solution, there is determined a reagent blank value which is obtained when, in the case of the above-described determination process, instead of the sample solution, there is used 1 ml. of distilled waterO The reagent blank value is deducted from the found extinction decrease ~E before calculation of the leclthin content according to the above-given equation.
ExamDle 2.
Determination of lecithin in mayonnaise .
5 g. of mayonnaise are shaken for about 5 minutes in a separating funnel with 30 ml. of ethanol/benzene (1:1 v/v) and 50 ml~ of water. The organic phase is separated off and the aqueous phase is shaken out twice with about 20 mlO of the ethanol/benzene mixture.
The collected organic phase is evaporated almost to dryness in a rotary evaporator~ The residue is dissolved in 5 ml. tert.-butanol, made up to 50 ml.
with water and vigorously mixed up. 2 ml. of the filtered and substantially clear solution are used for the lecithin determination, which is carried out in the manner described in Example 1.
Exam~le 3.
__.
Determinat on of lecithin in eqq liqueur.
2 g. of egg liqueur are weighed into a 100 ml.
measuring flask and dissolved with about 10 ml. tert.
butanol. This is then made up to 100 ml~ with water and mixed. 0.2 ml. of this solution are used for the lecithin determination, which is carried out in the manner described in Example 1.
~.
Determination of lecithin in baker~ ~roducts.
2 g. of a ~akery product sample are comminuted and introduced into a 100 ml. separating funnel. 30 ml.
of ethanol/benzene (1:1 v/v) and 50 ml. water are intro-duced into the funnel. The mixture is ~haken vigorously for 5 minutes. -The organic phase is separated off.
The aqueous phase i5 shaken out twice ~ith 10 ml. amounts of the ethanol~benzene mixture~ The organic phases are combined, filtered and evaporated almost to dryness in a rotary evaporator, whereby the rotary evaporator may be heated to a temperature o-f at most 45C. The residue is dissolved in 10 ml. tert-butanol, made up to 50 ml~ with ~1ater and vigorously mixed. 1 ml. of the filtered solution is used for the lecithin deter-mination, which is carried out in the manner described in Example 1.
The magnesium ions employed in the reagents of the invention function to activate the pyruvate kinase, and also have a positive influence on the lactate dehydrogenase reaction.
1'he enzymes referred to in this specification have been assigned the following enzyme numbers:
Phospholipase C E.C.3.1.4.3.
Alkaline phosphatase E.C.3.1.3 1.
Choline kinase E.C.2.7.1.32 Pyruvate kinase E.C.2.7.1.40 Lactate dehydrogenase ~.C.1.1.1.27
The abbreviations used in the above equations have the following meanings:
AP = alkaline phosphatase ATP = adenosine-5'-triphosphate ADP = adenosine-5'-diphosphate PEP = phosphoenol pyruvate PK = pyruvate kinase ~ADH/~AD~ = nicotinamide-adenine-dinucleotide, reduced/
oxidised form LDH = lactate dehydrogenase The assessment of a threatened respiratory failure syndrome is admittedly substantially simplified by the enzymatic process for the determination of lecithin in amniotic fluid. However, it suffers from considerable disadvantages: in a considerable number o samples, disturbances arise in the course of the measurement due to a strong turbidity of the solution to be measured which, in some cases, even maXe a determination of the lecithin completely impossible. The reason for this is the high degree of turbidity of the amniotic fluid which is used in a cuvette with a relatively large sample volume and, therefore, in the test, too, can give rise to a strong and no longer measurable turbidity.
Too high an initial extinction is measured which results in a high error quote and in a deficient degree of precision of the measurement. Furthermore, in the ; case of the measurement of such samples, considerable - s -deviations are found from the linearity of the measure-ment.
It is an object of the present invention to provide a process which does not suffer from the above-mentioned disadvantages.
According to the present invention, this object is achieved by a process which differs from the prior art in that the splitting of the lecithin is not carried out in a glycine buffer but in a borate/borax buffer in the presence of a deter~ent, especially a non-ionic detergent based upon a condensation product of poly-ethyleneglycol with isooctylphenol.
In particular the splitting of the lecithin is carried out with phospholipase-C and alkaline phosphatase.
In principle, a clarification of the solution to be measured can be achieved with many different detergents.
~owever, most of the detergents display a more or less ~ strongly marked inhibition action on the en~ymatic process, i which gives rise to Ealse measurement results. On the other hand, en~ymatic reactions proceed in a satisfactory manner only within a relatively narrow pH range. In the case of the determination of lecithin in a sample, the pH
range of 7.0 to 8.5 is optimal. Furthermore, it has been found that for the com~lex system of the enzymatic reactions which are necessary for the lecithin deter~ination, only a few buffer systems can be considered.
Surprisingly, we have now found that optimum measurement conditions for a disturbance-iree determin-a, ~
-- 6 ~ation of lecithin can be achieved in a sample solution when, in the case of splitting the lecithin, use is made of a borate/borax buffer and of a deter~ent, especially a non-ionic detergent based upon a condensation product of polyethyleneglycol with isooctylphenol.
Thus in accordance with the invention there is provided in a process for the determination of lecithin in a fluid sample in which the lecithin in the sample is split in the presence of a buffer to provide a product capable of being measured as a measure of lecithin in the sample, the improvement wherein said buffer is a borate/borax buffer and the splitting is carried out in the presence of a detergent.
It will be understood that the product which is measured as a measure of the lecithin in the sample need not be formed directly in the splitting of the lecithin, but may result-from further reaction on the product of the lecithin splitting.
It will be further understood that in the ! 20 splitting of lecithin any detergent may be used which does not interfere with or inhibit the splitting process. In i the determination of lecithin by the process of the invention in which the splitting of lecithin represents one step, a detergent is employed which is a non-inter-fering detergent, by which is to be understood a detergent which does not exhibit an inhibition action on any of the process steps, particularly the enzymatic process steps, such as would result in false measurements. The non-interfering detergent should additionally be one which is effect:ive to clarify the fluid sample, at least at the measurement stage when the measurement is photo-metric in nature.
The invention particularly contemplates a process for the determination of lecithin in a sample in which lecithin is first split with the help of phospholipase-C and alkaline phosphatase to give choline, thereafter the resultant choline is further reacted in the presence of adenosine triphosphate with the help of choline kinase again to give phosphoryl choline and the adenosine diphosphate thereby formed is measured in Xnown manner, wherein the splitting of the lecithin is carried out in the presence of a borate/
borax buffer and in the presence of a non-ionic deter-gent based upon a condensation product of polyethylene-glycol with isooctylphenol.
According to a preferred embodiment of the process of the invention, the sample, for example amniotic fluid, optionally diluted with water, is first mixed with the borate~borax buffer, a magnesium salt, the detergent, phospholipase-C and alkaline phosphatase and incubated at ambient temperature or at a slightly elevated temperature. The incubation period is, depending upon the selected temperature, preferably 10 to 60 minutes. Thereafter, heating is carried out for a short time to 95 to 100C., for example in a water-bath. After cooling the mixture, it is centrifuged and the supernatant is mixed with NADH, ATP, phosphoenol pyruvate, glucose, pyruvate kinase and lactate dehydrogenase. After a short waiting period of about 5 minutes, the extlnction El is measured. Subsequently, a choline kinase solution is added thereto in order to initiate the reaction sequence for the detection of the choline liberated from the lecithin. When the reaction is concluded, the extinction E2 is measured. The lecithin content of the sample can be determined from the extinction difference. In addition to the actual determination of the sample, it is recommended to ; ~ measure a reagent blank.
It is preferable to combine the enz~nes and adjuvants necessary for the process according to the present invention to give definite solutions or suspensions.
Thus, the borate/borax buffer i5 preferably used together with the detergent and the magnesium salt in the form of a "buffer solution". It is preferable to use a borate/borax buffer with a pH value of 7.0 to 8.5 in a concentration of 0.05 to 0.5 mole/litre of buffer solution and preferably of 0.07 to 0.25 mole/litre of buffer solution ~specially preferred is a borate/borax buffer with a pH value of 708 to 8.2, which is used in a concentration of 0.1 to 0.2 mole/litre of buffer j! J
_9_ solution in the case of the determination. The non-ionic detergent based on a condensation product of polyethyleneglycol with isooctylphenol is added to the buffer solution in a concentration of 0.5 to S g./
litre and preferably of 1.0 to 3.0 g./litre. The commercially available product Tinoveti~ JU of Ciba-Geigy has proved to be especially useful as the detergent according to the present invention.
The phospholipase-C and the alkaline phosphatase are used together as "enzyme suspension A", the con-centration of phospholipase-C being 40 - 200 U/ml.
and preferably 100 Ujml. and the concentration of the alkaline phosphatase being 20 to 200 U/ml. and prefer-ably 80 UJml.
The NADH, ATP, phosphoenolpyruvate and glucose are advantageously combined as "coenzyme solution".
l'he individual concentrations in this solution are 2.5 to 6.0 mmole/litre and preferably 4.0 mmole/litre of NADH, 10 to 30 mmole/litre and preferably 20 mmole/
litre of ATP, 3 to 10 mmole/litre and preferably 7 mmole/
litre of phosphoenol pyruvate and 30 to 100 mmole/litre and preferably 50 to 60 mmole/litre of glucose.
Pyruvate kinase and lactate dehydrogenase are added together as "enzyme suspension B", the concen-trations of pyruvate kinase and of lactate dehydrogenase being, in each case, 150 to 1000 U~ml. and preferably 300 U/ml.
* trade mark Choline kinase is preferably used in the form of a separate solution with a content of 2 to 10 U/ml.
and preferably of 2.5 U/ml.
In another aspect of the invention 'chere i s provided in a reagent for the determination of leci thin in a fluid sample comprisiny a buffer and an agent effective to split lecithin and to provide a product capable of being measured as a measure of lecithin in the sample, the improvement wherein said buffer is a borate/borax buffer and additionally including a detergent .
The present invention also contemplates a reagent for the determination Qf lecithin which comprises the following cornponents:
a ) a buf f er solution wi th 0.05 to 0.5 rr.ol/litre borate/borax buffer, pH 7.0 - 8.5 2 to 20 mmole~litre of magnesium ions 0.5 to 5.0 g./litre of detergent 1~) an enzyme suspension ~ with 40 to 200 U/ml. phospholipase-C
20 to 200 U/ml. alkaline phosphatase c) a coenzyme solution with 2.5 to 6.0 mrnole/litre N~
10 to 30 mmole/litre ATP
3 to 10 mmole~litre phosphoenol pyruvate 30 to 100 mmole~litre glucose d) an enzyme suspension B with f~
150 to 1000 U/ml. pyruvate kinase 150 to 1000 U/ml. lactate dehydrogenase I e) a solution with j 2 to 10 U/ml. choline kinase.
Quite especially preferred is a reagent com-~ prising the following components:
¦ a) a buffer solution with 0.2 mol/litre borate/borax buffer, pH 7.8 - 8.2 10 mmole/litre magnesium sulphate 2.5 g./litre Tinovetin JU~
b) an enzyme suspension A with 80 U/ml. phospholipase-C
100 U/ml. alkaline phosphatase c) a coenzyme solution with 4.0 mmole/litre ~ADH
; , 20 mmole/litre ATP
7 mmole/litre phosphoenol pyruvate , 56 mmole/litre glucose d)` an enzyme suspension B with ~ 300 U/ml. pyruvate kinase ; 300 U/ml. lactate dehydrogenase ` e) a solution with 2.5 U/ml. choline kinase.
!
i * trade mark In yet another aspect of the invention ther~
is provided in a reagent for splitting lecithin com-prising phospholipase-C, alkaline phosphatase and a buffer, the improvement wherein said buffer is a borate/borax buffer and further including a detergent.
The present invention particularly contemplates a reagent for splitting lecithin, which comprises:
~ to 10 U/ml. phospholipase-C
1 to 10 U/ml. alkaline phosphatase 0.05 to 0.5 mole/litre borate/borax buffer, pH 7.0 - 8.5 2 to 20 mmole/litre magnesium ions 0.5 to 5.0 g./litre detergent.
For splitting lecithin, a reagent composition is quite especially preferred which contains the follow-ing individual components in the given concentrations:
.
`p~
4 U/ml. phospholipase-C
5 U/ml. alkaline phosphatase 0.2 mole/litre borate/borax buffer, pH 7.8 - 8.2 10 mmole/litre magnesium sulphate 2~5 g./litre Tinovetin JU.
The reagent according to the present invention for splittlng lecithin is preferably prepared by mixing 1.0 ml. of the above-described buffer solution with 50 ~1~ of the above-described enz~ne suspension A. The reagent thus obtained is preferably added to the sample ; ; to be measured in the ratio of 1:1.
The following Examples are given for the purpose of illustrating the present invention:
Exam~e 1.
Determination of lecithin in amniotic fluid.
1 ml. of a sample solution which contains about ~` 0.1 mg. of lecithin is mixed with 1 m~. of a solution which contains 0.2 mole/litre borate/borax buffer ~pH
7.8 - 8.2), 10 mmole/litre magnesium sulphate and 2.5 g./litre Tinovetin ~U~ To this mixture is added 0.05 ml. of an enzyme suspension of 80 U/ml. phospho-lipase-C and 100 U/ml~ alkaline phos~hatase. The mixture is left to stand at 37C. for 20 minutes, whereafter it is heated for 5 minutes to 95 to 100C.
After cooling to ambient temperature, 0~1 ml. of a solution of 4~0 mmole/litre NADH, 20 mmole/litre ATP, 7 mmole/litre phosphoenol pyruvate and 56 mmole/litre * trade mark ..~
-glucose, as well as 0.02 ml. of an enzyme mixture of 300 U/ml. pyruvate kinase and 300 U/ml. lactate dehydrogenase are added thereto. The solution obtained is mixed and left to stand for 10 minutes at ambient temperature. The sample is introduced into a cuvette and the extinction El is measured. The enzymatic reaction is initiated by the addition of 0.05 ml. of a choline kinase solution with a content thereof of 2.5 U/ml. After leaving to stand for 30 ~inutes at ambient temperature, the extinction E2 is measured.
From the extinction difference El-E2, there is determined the extinction decrease ~E, from which is obtained the lecithin content of the sample according to the follow~ng equation:
lecithin (mg./100 ml.) = ~E x 47.86.
For each determination of lecithin in a sample solution, there is determined a reagent blank value which is obtained when, in the case of the above-described determination process, instead of the sample solution, there is used 1 ml. of distilled waterO The reagent blank value is deducted from the found extinction decrease ~E before calculation of the leclthin content according to the above-given equation.
ExamDle 2.
Determination of lecithin in mayonnaise .
5 g. of mayonnaise are shaken for about 5 minutes in a separating funnel with 30 ml. of ethanol/benzene (1:1 v/v) and 50 ml~ of water. The organic phase is separated off and the aqueous phase is shaken out twice with about 20 mlO of the ethanol/benzene mixture.
The collected organic phase is evaporated almost to dryness in a rotary evaporator~ The residue is dissolved in 5 ml. tert.-butanol, made up to 50 ml.
with water and vigorously mixed up. 2 ml. of the filtered and substantially clear solution are used for the lecithin determination, which is carried out in the manner described in Example 1.
Exam~le 3.
__.
Determinat on of lecithin in eqq liqueur.
2 g. of egg liqueur are weighed into a 100 ml.
measuring flask and dissolved with about 10 ml. tert.
butanol. This is then made up to 100 ml~ with water and mixed. 0.2 ml. of this solution are used for the lecithin determination, which is carried out in the manner described in Example 1.
~.
Determination of lecithin in baker~ ~roducts.
2 g. of a ~akery product sample are comminuted and introduced into a 100 ml. separating funnel. 30 ml.
of ethanol/benzene (1:1 v/v) and 50 ml. water are intro-duced into the funnel. The mixture is ~haken vigorously for 5 minutes. -The organic phase is separated off.
The aqueous phase i5 shaken out twice ~ith 10 ml. amounts of the ethanol~benzene mixture~ The organic phases are combined, filtered and evaporated almost to dryness in a rotary evaporator, whereby the rotary evaporator may be heated to a temperature o-f at most 45C. The residue is dissolved in 10 ml. tert-butanol, made up to 50 ml~ with ~1ater and vigorously mixed. 1 ml. of the filtered solution is used for the lecithin deter-mination, which is carried out in the manner described in Example 1.
The magnesium ions employed in the reagents of the invention function to activate the pyruvate kinase, and also have a positive influence on the lactate dehydrogenase reaction.
1'he enzymes referred to in this specification have been assigned the following enzyme numbers:
Phospholipase C E.C.3.1.4.3.
Alkaline phosphatase E.C.3.1.3 1.
Choline kinase E.C.2.7.1.32 Pyruvate kinase E.C.2.7.1.40 Lactate dehydrogenase ~.C.1.1.1.27
Claims (30)
1. A process for the determination of lecithin in a fluid sample, comprising:
splitting lecithin with phospholipase-C and alkaline phosphatase to give choline in the presence of a borate/borax buffer and of a non-ionic detergent based on a condensation product of polyethyleneglycol with isooctylphenol;
reacting the formed choline in the presence of adenosine triphosphate and choline kinase to give phosphoryl-choline and adenosine diphosphate, and measuring the adenosine diphosphate pro-duction as a measure of the lecithin in said sample.
splitting lecithin with phospholipase-C and alkaline phosphatase to give choline in the presence of a borate/borax buffer and of a non-ionic detergent based on a condensation product of polyethyleneglycol with isooctylphenol;
reacting the formed choline in the presence of adenosine triphosphate and choline kinase to give phosphoryl-choline and adenosine diphosphate, and measuring the adenosine diphosphate pro-duction as a measure of the lecithin in said sample.
2. A process according to claim 1, wherein said borate/borax buffer is effective to establish a pH
of 7.0 to 8.5.
of 7.0 to 8.5.
3. A process according to claim 1, wherein said borate/borax buffer is effective to establish a pH
of 7.8 to 8.2.
of 7.8 to 8.2.
4. A process according to claim 1, 2 or 3, wherein said borate-borax buffer and said non-ionic detergent are added together as a buffer solution, said borate/borax buffer having a concentration of 0.05 to 0.5 mole/litre of buffer solution and said detergent in a concentration of 0.5 to 5.0 g./litre of buffer solution.
5. A process according to claim 1, 2 or 3, wherein said borate/borax buffer and said non-ionic detergent are added together as a buffer solution, said borate/-borax buffer having a concentration of 0.07 to 0.25 mole/litre of buffer solution and said detergent having a concentration of 1.0 to 3.0 g./litre of buffer solution.
6. A process according to claim 1, 2 or 3, wherein said detergent is Tinovetin JU (trade mark).
7. A process according to claim 1, 2 or 3, wherein said sample is amniotic fluid.
8. In a process for the determination of lecithin in a fluid sample in which the lecithin in the sample is split in the presence of a buffer to provide a product cap-able of being measured as a measure of lecithin in the sample, the improvement wherein said buffer is a borate/borax buffer and the splitting is carried out in the presence of a detergent.
9. A process according to claim 8, wherein said detergent is a non-ionic detergent based on a condensation product of polyethyleneglycol with iso-octylphenol.
10. A process according to claim 8 or 9, wherein said buffer is effective to establish a pH of 7.0 to 8.5.
11. A process according to claim 8 or 9, wherein said buffer is effective to establish a pH of 7.8 to 8.2.
12. A process according to claim 8 or 9, wherein said buffer and detergent are employed as a buffer solution, said buffer having a concentration of 0.05 to 0.5 mole/litre of buffer solution, and said deter-gent having a concentration of 0.5 to 5.0 g./litre of buffer solution.
13. A process according to claim 8 or 9, wherein said buffer and detergent are employed as a buffer solution, said buffer having a concentration of 0.07 to 0.25 mole/litre of buffer solution, and said detergent having a concentration of 1.0 to 3.0 g./litre of buffer solution.
14. In a reagent for the determination of lecithin in a fluid sample comprising a buffer and an agent effective to split lecithin and to provide a product capable of being measured as a measure of lecithin in the sample, the improvement wherein said buffer is a borate/borax buffer and additionally including a detergent.
15. A reagent according to claim 14, wherein said detergent is based on a condensation product of poly-ethyleneglycol with isooctylphenol.
16. A reagent according to claim 14 or 15, wherein said buffer is effective to establish a pH of 7.0 to 8.5.
17. A reagent according to claim 14 or 15, wherein said buffer is effective to establish a pH of 7.8 to 8.2.
18. A reagent according to claim 14 or 15, wherein said buffer and detergent are present as a buffer solution having a concentration of said borate/borax buffer of 0.05 to 0.5 mole/litre of buffer solution, and a concentration of said deter-gent of 0.5 to 5.0 g./litre of buffer solution.
19. A reagent according to claim 14 or 15, wherein said buffer and detergent are present as a buffer solution having a concentration of said borate/borax buffer of 0.07 to 0.25 mole/litre of buffer solution, and a concentration of said detergent of 1.0 to 3.0 g./litre of buffer solution.
20. A reagent for the determination of lecithin in a sample comprising the following components:
a) a buffer solution with 0.05 - 0.5 mole/litre borate/borax buffer effective to establish a pH
of 7.0 - 8.5 2 - 20 mmole/litre magnesium ions 0.5 - 5.0 g./litre detergent b) an enzyme suspension A with 40 - 200 U/ml. phospholipase-C
20 - 200 U/ml. alkaline phosphatase c) a coenzyme solution with 2.5 - 6.0 mmole/litre NADH
10 - 30 mmole/litre ATP
3 - 10 mmole/litre phosphoenol pyruvate 30 - 100 mmole/litre glucose d) an enzyme suspension B with 150 - 1000 U/ml. pyruvate kinase 150 - 1000 U/ml. lactate dehydrogenase e) a solution with 2-10 U/ml. choline kinase.
a) a buffer solution with 0.05 - 0.5 mole/litre borate/borax buffer effective to establish a pH
of 7.0 - 8.5 2 - 20 mmole/litre magnesium ions 0.5 - 5.0 g./litre detergent b) an enzyme suspension A with 40 - 200 U/ml. phospholipase-C
20 - 200 U/ml. alkaline phosphatase c) a coenzyme solution with 2.5 - 6.0 mmole/litre NADH
10 - 30 mmole/litre ATP
3 - 10 mmole/litre phosphoenol pyruvate 30 - 100 mmole/litre glucose d) an enzyme suspension B with 150 - 1000 U/ml. pyruvate kinase 150 - 1000 U/ml. lactate dehydrogenase e) a solution with 2-10 U/ml. choline kinase.
21. A reagent according to claim 20, comprising the following components:
a) a buffer solution with 0.2 mole/litre borate/borax buffer, effective to establish a pH of 7.8 to 8.2 10 mmole/litre magensium sulphate 2.5 g./litre Tinovetin JU (trade mark) b) an enzyme suspension A with 80 U/ml. phospholipase-C
100 U/ml. alkaline phosphatase c) a coenzyme solution with 4.0 mmole/litre NADH
20 mmole/litre ATP
7 mmole/litre phosphoenol pyruvate 56 mmole/litre glucose d) an enzyme suspension B with 300 U/ml. pyruvate kinase 300 U/ml. lactate dehydrogenase e) a solution with 2.5 U/ml. choline kinase.
a) a buffer solution with 0.2 mole/litre borate/borax buffer, effective to establish a pH of 7.8 to 8.2 10 mmole/litre magensium sulphate 2.5 g./litre Tinovetin JU (trade mark) b) an enzyme suspension A with 80 U/ml. phospholipase-C
100 U/ml. alkaline phosphatase c) a coenzyme solution with 4.0 mmole/litre NADH
20 mmole/litre ATP
7 mmole/litre phosphoenol pyruvate 56 mmole/litre glucose d) an enzyme suspension B with 300 U/ml. pyruvate kinase 300 U/ml. lactate dehydrogenase e) a solution with 2.5 U/ml. choline kinase.
22. In a reagent for splitting lecithin comprising phospholipase-C, alkaline phosphatase and a buffer, the improvement wherein said buffer is a borate/borax buffer and further including a detergent.
23. A reagent according to claim 22, wherein said detergent is a non-ionic detergent based on a condensation product of polyethylene glycol with iso-octylphenol.
24. A reagent according to claim 22 or 23, wherein said buffer is effective to establish a pH of 7.0 to 8.5.
25. A reagent according to claim 22 or 23, wherein said buffer is effective to establish a pH of 7.8 to 8.2.
26. A reagent for splitting lecithin, comprising the following components:
2 - 10 U/ml. phospholipase-C
1 - 10 U/ml. alkaline phosphatase 0.05 - 0.5 mole/litre borate/borax buffer, effective to establish a pH of 7.0 - 8.5 2 - 20 mmole/litre magnesium ions.
0.5 - 5.0 g./litre of detergent.
2 - 10 U/ml. phospholipase-C
1 - 10 U/ml. alkaline phosphatase 0.05 - 0.5 mole/litre borate/borax buffer, effective to establish a pH of 7.0 - 8.5 2 - 20 mmole/litre magnesium ions.
0.5 - 5.0 g./litre of detergent.
27. A reagent according to claim 26, wherein said detergent is a non-ionic detergent based on a condensation product of polyethyleneglycol with iso-octylphenol.
28. A reagent according to claim 26, comprising the following components:
4 U/ml. phospholipase-C
5 U/ml. alkaline phosphatase 0.2 mole/litre borate/borax buffer, effective to establish a pH of 7.8 to 8.2 10 mmole/litre mag-nesium sulphate 2.5 g./litre Tinovetin JU (trade mark).
4 U/ml. phospholipase-C
5 U/ml. alkaline phosphatase 0.2 mole/litre borate/borax buffer, effective to establish a pH of 7.8 to 8.2 10 mmole/litre mag-nesium sulphate 2.5 g./litre Tinovetin JU (trade mark).
29. In a process for splitting lecithin in which lecithin is split with phospholipase-C and alkaline phosphatase in the presence of a buffer, the improve-ment wherein the buffer is borate/borax buffer and further including a detergent.
30. A process according to claim 29, wherein said detergent is a non-ionic detergent based on a condensation product of polyethylene glycol with isooctylphenol.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19813128480 DE3128480A1 (en) | 1981-07-18 | 1981-07-18 | METHOD FOR DETERMINING LECITHIN IN FRUIT WATER AND REAGENT FOR CARRYING OUT THIS METHOD |
| DEP3128480.9 | 1981-07-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1184101A true CA1184101A (en) | 1985-03-19 |
Family
ID=6137239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000407494A Expired CA1184101A (en) | 1981-07-18 | 1982-07-16 | Determination of lecithin |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0071076B1 (en) |
| JP (1) | JPS5816698A (en) |
| AT (1) | ATE13074T1 (en) |
| CA (1) | CA1184101A (en) |
| DD (1) | DD202601A5 (en) |
| DE (2) | DE3128480A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59193356A (en) * | 1983-03-17 | 1984-11-01 | バイオウィッテッカー・インコーポレーテッド | Fluorescent luminous intensity analysis for total ige concentration and reagent therefor |
| JPH061252B2 (en) * | 1984-08-31 | 1994-01-05 | 株式会社島津製作所 | Oxidase electrode |
| JPS6351626A (en) * | 1986-08-20 | 1988-03-04 | Nec Corp | Semiconductor manufacturing device |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS50142090A (en) * | 1974-04-30 | 1975-11-15 | ||
| JPS5324888A (en) * | 1976-08-19 | 1978-03-08 | Kyowa Hakko Kogyo Kk | Composition for quantitating fats containing phosphorus |
| GB1555173A (en) * | 1977-08-17 | 1979-11-07 | Kyowa Hakko Kogyo Kk | Method and reagent for quantitative determination of phospholipids |
-
1981
- 1981-07-18 DE DE19813128480 patent/DE3128480A1/en not_active Withdrawn
-
1982
- 1982-07-12 JP JP57119993A patent/JPS5816698A/en active Granted
- 1982-07-14 EP EP82106278A patent/EP0071076B1/en not_active Expired
- 1982-07-14 DE DE8282106278T patent/DE3263391D1/en not_active Expired
- 1982-07-14 AT AT82106278T patent/ATE13074T1/en active
- 1982-07-15 DD DD82241679A patent/DD202601A5/en unknown
- 1982-07-16 CA CA000407494A patent/CA1184101A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| ATE13074T1 (en) | 1985-05-15 |
| DE3263391D1 (en) | 1985-06-05 |
| EP0071076A2 (en) | 1983-02-09 |
| JPS5816698A (en) | 1983-01-31 |
| DE3128480A1 (en) | 1983-01-27 |
| DD202601A5 (en) | 1983-09-21 |
| EP0071076B1 (en) | 1985-05-02 |
| EP0071076A3 (en) | 1983-03-23 |
| JPS619839B2 (en) | 1986-03-26 |
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