DD202601A5 - METHOD FOR DETERMINING LECITHIN AND REAGENT FOR IMPLEMENTING THIS PROCEDURE - Google Patents

Abstract

1. Process for the determination of lecithin in a sample, in which lecithin is split with the help of phospholipase-C and alkaline phosphatase to 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 according to known methods, characterised in that the splitting of the lecithin is carried out in the presence of a borate/borax buffer and of a non-ionic detergent based on a condensation product of polyethyleneglycol with isooctylphenol.

Description

Berlin, 7.12.1982

AP A 23 3/241 S79 / 7

2416797 ~ ^Λ ~ ^{61072 / ls}

Method for the determination of lecithin Field of application of the invention

The invention relates to a method for the determination of lecithin in a sample by enzymatic cleavage of lecithin using phospholipase C and alkaline phosphatase to choline, implementation of choline with the help of choline kinase in the presence of adenosine triphosphate to phosphorylcholine and adenosine diphosphate and subsequent detection of the resulting Adenosine diphosphate according to known methods and a reagent for carrying out the method *

A determination of the lecithin is carried out, for example, in the study of food. Lecithin is mainly found in egg yolks. The lecithin content of the yolk is subject to only small fluctuation margins, so that it can be seen as a measure of the egg content of a food sample,

Of particular importance is the determination of the lecithin content in the amniotic fluid. Thus, the fetal lung frost already can largely determine and assess the potential risk of respiratory distress syndrome of the newborn still represents birth. The Atemnotsyndroffl is

λ λ nr-T in Q 0 * Π λ R 9>')

16 7 9 7

one of the major complications that can occur in premature babies after birth. It is caused by a lack of surfactant, which in mature newborns as a thin film lining the lung alveoli and has the task to lower the surface tension at the air / liquid interface in the alveoli and thereby to ensure the stability of the alveoli. The surfactant consists of proteins, carbohydrates and lipids, with the phospholipids, such as dipalmitoyllecithin, phosphatidylglycerol and sphingomyelin, having the decisive surface-active properties.

The lecithin content in the amniotic fluid is fluctuating

During pregnancy for a long time between 1 and 2 mg / dl, increases to 5-10 mg / dl in the 35th and 37th week and is usually in the 40th Week at a value above 10 mg / dl. The steep rise ' _: the lecithin concentration between the 35th and the week of pregnancy can be used as a characteristic measure for the presence of fetal lung maturity.

² ^ Through the respiratory movements of the fetus there is a constant exchange of the alveolar fluid with the amniotic fluid, so that it is possible to make an impression of the surfactant content in the fetal alveoli by measuring the phospholipid content in the amniotic fluid and thus even before birth to get a statement about the maturity state of the lungs. The determination of lecithin in the amniotic fluid is therefore of crucial importance for the pregnancy diagnosis, especially with regard to an early termination of a risk pregnancy due to a threat of respiratory distress syndrome.

07/12/1982

w 1 r "* r * ^{Ap A 23 J / 241 579/7}

: 41b / 9-7 - 3 - ₆ i 072 / is

Characteristic of the known technical solutions'

For the determination of lecithin in the amniotic fluid numerous methods are available, which differ partly on the one hand by the different work expenditure and on the other hand by the accuracy of the prediction considerably.

From obstetrics and gynecology 39_ (1979) pp 849-356 an enzymatic determination of lecithin in amniotic fluid is known ι the easier to carry out than the non-enzymatic methods and these equal in accuracy and practicality, often even superior. The lecithin is cleaved in this 8estiramungsmethode using phospholipase-C in a solution containing glycine to phosphorylcholine, which is further degraded by alkaline phosphatase to choline.

The latter is reacted in the presence of adenosine triphosphate with the aid of choline kinase again to phosphorylcholine to form adenosine. Indiphosphate »The Adsnosindiphosphat formed is measured photometrically via a known enzymatic reaction chain. This detection method can be described by the following reaction equations:

(1) Lecithin ₊ HO ^ 1,2-diglyceride ₊

phosphorylcholine

AP "ζ

(2) Phosphorylcholine + H-O -, - ^ Choline + PO / "

(3) Choline '+ ATP _{Pho8phorylcholine + A} DP

(4) ADP + PEP £ - £ ^ ATP + pyruvate

(5) pyruvate + NADH + H ⁺ LDH , ^^ ...-

^v ' ^ϊ ιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιιι Lactate + NAD

7.12 * 1982 1 C * 7 A n AP A 23 α / 241 679/7

4h i H / Iw / ^ /. 4 - si 072/18

The abbreviations used meanings:.

AP = alkaline phosphatase

ATP = adenosine S'-triphosphate

ADP = adeno3in 5'-diphosphate

PEP a phosphoenol pyruvate

PK = pyruvate kinase

NADH / NAD ⁺ = nicotinamide adenine dinucleotide, reduced /

oxidized form LDH = lactate dehydrogenase

Although the assessment of a threatening respiratory distress syndrome has been considerably simplified by the enzymatic method for the determination of lecithin in amniotic fluid. However, it has a considerable drawback: in a considerable number of samples disturbances occur in the measurement, which result from a strong turbidity of the measurement solution and sometimes make a Laoithinbestimmung completely impossible. The reason lies in the high turbidity of the amniotic fluid, which is used with a relatively large sample volume in the cuvette and therefore can cause a strong, no longer measurable turbidity in the test. Too high initial extinctions are measured which lead to a high error rate and to a poor precision of the measurement. Further, in the measurement of such samples that substantial deviations from linearity are OCE measurement found

Aim of the invention

The aim of the invention is to provide an improved process for the determination of lecithin, which does not have the disadvantages of the known processes *

07/12/1982

ί * ~ ^ AP A 23 υ / 241 579/7

* 4 1 / ij J-Aa 61 072/13

Presentation of the invention of the invention

The invention has for its object to use other means of cleaving the lecithin.

This object was achieved by the method according to the invention, which differs from the method according to the prior art in that the cleavage

f * - ^ lf \ τ-} r

of the lecithin with phospholipase C and alkaline phosphatase is carried out not in a glycine buffer but in a borate / borax buffer in the presence of a nonionic detergent based on a condensation product of polyethylene glycol with isooctylphenol.

 Basically, a clarification of the solution to be measured by many detergents can be achieved. However, most detergents show a more or less pronounced inhibitory effect on the enzymatic processes that lead to incorrect measurement results. On the other hand, enzymatic reactions proceed satisfactorily only

In the case of lecithin determination in a sample, a pH range of 7.0-8.5 is optimal, and it has also been found that, for the complex system of enzymatic reactions, which are required for the lecithin determination, only a few buffer systems come into question.

It has surprisingly been found that optimal measurement conditions for trouble-free determination of lecithin in a sample solution can be achieved if, in the cleavage of the lecithin, a borate / borax buffer and a non-ionic detergent based on a condensation product of polyethylene glycol with isooctylphenol used become.

The invention therefore relates to a method for the determination of lecithin in a sample in which first lecithin using phospholipase C and alkaline phosphatase cleaved to choline, then the resulting choline in the presence of

! b / b /

Adenosine triphosphate reacted with the aid of choline kinase again to phosphorylcholine and the adenosine diphosphate thereby formed is measured by known methods, characterized in that the - cleavage of the lecithin in the presence of a borate / borax buffer and in the presence of a non-ionic detergent based on a Kondensationsprcduktes of polyethylene glycol is carried out with isooctylphenol. , '' '

According to a preferred embodiment of the method according to the invention, the sample .-. For example, amniotic fluid, optionally diluted with water - with the borate / borax buffer, 'a magnesium salt, the detergent mixed with phospholipase C and alkaline phosphatase and at room temperature or slightly elevated temperature incubated. The incubation time is preferably 10-60 minutes, depending on the chosen temperature. Then a short time 9S-IOO ⁰ C, for example, 'in a water bath heated. After cooling the mixture is centrifuged off and the supernatant with NADH, ATP, phosphoenolpyruvate, glucose, Pyruvatkinase and Lactatdehydrogenase added. After a short wait (about 5 minutes) the extinction E. is measured. Subsequently, a choline kinase solution is added to initiate the reaction chain for detection of lecithin-released choline. When the reaction is complete, absorbance E _{2 is} measured. From the extinction difference, the lecithin content of the sample can be determined. It is recommended to measure a reagent blank value in addition to the actual determination of the sample.

AP A 23 0/241 579/7

.6 7 9 7 - ⁷ - ^{5α072 / 18}

It is expedient to combine the enzymes and auxiliaries required for the process according to the invention into specific solutions or suspensions.

Thus, preferably the borate / borax buffer is used together with the detergent and the magnesium salt in the form of a "buffer solution". Advantageously, a borate / borax buffer with pH 7.0 to 8.5 in a concentration of 0.05 to 0.5 mol / 1 buffer solution, preferably from 0.07 to 0.25 mol / 1 buffer solution used. Very particularly preferred is a borate / borax buffer of pH 7.8 to 3.2, which is used in a determination of 0.1 to 0.2 mol / 1 buffer solution in the determination. The nonionic detergent based on a condensation product of polyethyleneglycol with isooctylphenol is added to the buffer solution in a concentration of 0.5 to 5 g / l, preferably 1.0 to 3.0 g / l.

The phospholipase C and the alkaline phosphatase are used together as "enzyme suspension A", wherein the concentration of phospholipase C 40 to 200 U / ml, preferably 100.U / ml, and the concentration of alkaline phosphatase 20 to 200 U / ml , preferably 80 U / ml.

NADH, ATP, phosphoenolpyruvate and glucose are advantageously combined as "coenzyme solution". The individual concentrations in this solution are 2.5 to 6.0 mmol / l, preferably 4.0 mmol / l NADH, 10 to 30 mmol / l, preferably 20 mmol / l ATP, 3 to 10 mmol / l, preferably 7 mmol / 1 phosphoenolpyruvate and 30 to 100 mmol / l, preferably 50 to 60 mmol / l glucose.

/.a 7

Pyruvate kinase and lactate dehydrogenase are added together as "enzyme suspension B", the concentrations of pyruvate kinase and lactate dehydrogenase being 150-1000 U / ml each, preferably 300 U / ml.

Choline kinase is preferably used in the form of a separate solution containing 2-10 U / ml, preferably 2.5 U / ml.

The invention further provides a reagent for lecithin determination, which consists of the following constituents:

a) a buffer solution with

0.05-0.5 mol / 1 borate / borax buffer, pH 7.0-8.5 2-20 mmol / l magnesium ion 0.5-5.0 g / l detergent

b) an enzyme suspension A with

40-200 U / ml phospholipase C ^ ⁰ 20-200 U / ml alkaline phosphatase

c) a coenzyme solution with

2.5-6.0 mmol / l NADH 10-30 mmol / l ATP • 3-10 mmol / l phosphoenol pyruvate ₇ r 30-100 mmol / l 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.

Very particular preference is given to a reagent having the following constituents:

C 7 Q η " -

a) a buffer solution with _;

* 0.2 mol / 1 borate / borax buffer, 'pH 7.8-8,' 2 10 mmol / 1 magnesium sulfate 2.5 g / L linovetin JU

r b) an enzyme suspension A with

80 U / ml phospholipase-C

100 U / ml alkaline phosphatase

c) a coenzyme solution with

4.0 mmol / 1 NADH '20 mmol / 1 ATP

7 mmol / 1 Phosphoejiolpyruvat

56 mmol / 1 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

Another object of the invention is a reagent for the cleavage of lecithin, characterized by a content of.

2-10 U / ml phospholipase C 1-1.0 U / ml alkaline phosphatase 0.05-0.5 mol / 1 borate / borax buffer, pH 7.0-8.5 2-20 mmol / 1 magnesium ion 0, 5-5.0 g / l detergent. 5

For the cleavage of the lecithin, a reagent composition which contains the individual constituents in the following concentrations is very particularly preferred:

ö ^v

4 U / ml phospholipase-C

5 U / ml alkaline phosphatase

0.2 mol / l borate / borax buffer, pH 7.8-8.2 10 mmol / l magnesium sulfate 2.5 g / l Tinovetin JU.

7 -

07/12/1982

AP A 23 Ö / 241 679/7 10 - 61 072/18

The reagent according to the invention for the cleavage of the lecithin is conveniently prepared by dissolving 1.0 ml of the buffer solution described above with 50 pl of the above mentioned enzyme suspension A will be added. The reagent thus obtained is preferably added to the sample to be measured in a ratio of 1: 1.

embodiment

The invention is explained in more detail by the following examples:

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Example 1 'ν'

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Determination of lecithin in amniotic fluid

1 ml of a sample solution containing approximately 0.1 mg of lecithin is treated with 1 ml of a solution containing 0.2 mol / 1 borate / borax buffer, pH 7.8-8.2, 10 mmol / 1 magnesium sulfate and Contains 2.5 g / l Tinovetin JU, added. To this mixture are added 0.05 ml of an enzyme suspension of 80 U / ml phospholipase-C and 100 U / ml alkaline phosphatase. The mixture is allowed to stand at 37 ^° C. for 20 minutes. Thereafter, it is heated to 95-100 C for 5 minutes. After cooling to room temperature, 0.1 ml of a solution of 4.0 mmol / 1 NADH, 20 mmol / 1 ATP, 7 mmol / 1 phosphoenolpyruvate and 56 mmol / l glucose and 0.02 ml of an enzyme mixture of 300 U / ml Pyruvate kinase and 3 00 U / ml lactate dehydrogenase added. The resulting solution is mixed and allowed to stand at room temperature for 10 minutes. The sample is transferred to a cuvette and the absorbance (E,) is measured. The enzymatic reaction is started by adding 0.05 ml of a choline kinase solution with 2.5 U / ml. After standing at room temperature for 30 minutes, the absorbance (E2) is measured. From the extinction difference E ..- E2 the extinction decrease ΔΕ is calculated, from which the lecithin content of the sample according to the formula:

Lecithin (mg / 100ml) = ΔΕ. · 47.86

results.

b / a 7

For each determination of lecithin in a sample solution, a reagent blank value is obtained which results when 1 ml of distilled water is used in place of the sample solution in the determination method described above. The reagent blank value is calculated before the calculation of the lecithin content after o.a. Formula subtracted from found extinction Δ.Ε.

Example 2 Determination of lecithin in mayonnaise

5 g of mayonnaise are mixed with 30 ml of ethanol / benzene solution (1 + 1, v / v) and 50 ml of water in a separating funnel. Shaken for 5 minutes. The organic phase is separated, the aqueous phase is extracted by shaking twice with about 20 ml of ethanol / benzene solution. The collected organic phase is evaporated in a rotary evaporator until almost to dryness. The residue is tert with 5 ml. Butanol dissolved, made up to 50 ml with water and mixed vigorously. 2 ml of the filtered, substantially clear solution are used for lecithin determination, which is carried out as described in Example 1.

Example 3 o

Determination of lecithin in eggnog

2 g of eggnog are weighed into a 100 ml volumetric flask and tert with 10 ml of tert. Butanol dissolved. Make up to the mark with TOO ml of water and mix.

0.2 ml of this solution are used for lecithin determination, which, as indicated in Example 1, is performed.

- ¹⁵ -

Example 4 Determination of lecithin in bakery products

2 g of a bakery sample are comminuted and transferred to a 100 ml separating funnel. There are 30 ml of ethanol / benzene solution (1 + 1, v / v) and 50 ml

• - Added water. The mixture is shaken vigorously for 5 min. The organic phase is separated off.

The aqueous phase is shaken twice with 10 ml of ethanol / benzene solution. The organic phases are combined, filtered and evaporated on a rotary evaporator almost to dryness, the rotary evaporator may be heated to at most 45-G.

The residue is tert in 10 ml. Butanol dissolved, made up to 50 ml with water and mixed vigorously 1 ml of the filtered solution are used for lecithin, the ', as described in Example 1, performed.

Claims (5)

.7.12.1982 AP A 23 D / 241 679/7 U \ Ο / ^. / - 14 - 61 072/18 1 to 10 U / ml alkaline phosphatase 0.05 to 0.5 raol / 1 borate / borax buffer, pH 7.0 to 8.5 1, Method for the determination of lecithin in a sample in which lecithin using phospholipase C and alkaline phosphatase cleaved to choline, this converted in the presence of adenosine triphosphate with the help of choline kinase again to phosphorylcholine and the resulting adenosine diphosphate is measured by known methods characterized in that the cleavage of the lecithin is carried out in the presence of a borate / borax buffer and a non-ionic detergent based on a condensation product of polyethylene glycol with isooctylphenol » / 1 Γ 7 Π Π Erfindunqsanspruch 2 to 20 ramol / 1 magnesium ion 0.5 to 5.0 g / l detergent. 8, reagent according to item 7 * characterized by a content of 2 to 10 U / ml phospholipase-C 2.5 U / ml choline kinase. 7. reagent for cleavage of lecithin, characterized by a content of 2 to 10 U / ml choline kinase * 5 «Reagent according to item 5, characterized by the following constituents: a) a buffer solution with 0.2% borate / borax buffer, pH 7.8 to 8.2 10 mmol / 1 magnesium sulfate 2.5 g / l Tinovetin GU b) an enzyme suspension A with 80 U / ml phospholipase-C 100 U / ml alkaline phosphatase c) a Coenzyra solution with 2.5 to 6.0 mmol / 1 NADH 10 to 30 mmol / 1 ATP 2 to 20 mmol / l magnesium ions 0.5 to 5.0 g / l of detergent 7,12.1982 AP A 23 D / 241 679/7 Q / 9 7 ^{"15" 072 S1} / ¹⁸ b) an enzyme suspension A with 40 to 200 U / ml phospholipase-C 20 to 200 U / ml alkaline phosphatase c) a coenzyme solution with 2. The method according to item 1, characterized in that a borate / borax buffer, pH 7.0 to 3.5, in a concentration of 0.05 to 0.5 mol / 1 buffer solution and the detergent in a concentration of 0 Add 5 to 5.0 g / l buffer solution. 3 to 10 mmol / l phosphoenolpyruvate 30 to 100 mmol / l glucose d) an enzyme suspension 8 with 150 to 1000 U / ml pyruvate kinase 150 to 1000 U / ml lactate dehydrogenase e) a solution with 3 »Method according to one of the points 1 and 2, characterized in that 0.07 to 0.25 mol / 1 buffer solution borate / borax buffer and 1.0 to 3.0 g / l buffer solution detergent are used. 4 U / ml phospholipase-C 4 1 D / ^ / - 16 - 61 072/18 d) an Enzyrasuspension B with 300 U / ml pyruvate kinase 300 U / ml lactate dehydrogenase e) a solution with 4.0 mmol / 1 NADH 20 mmol / 1 ATP 7 mmol / 1 phosphoenolpyruvate 56 mmol / 1 glucose 07/12/1982 O / 1 C 1 η η AP A 23 0/241 679/7 4, Method according to one of the items 1 to 3, characterized in that the detergent used is tinovetin, 3U, 5. Reagent for carrying out the process according to items 1 to 4, characterized by the following constituents: a) a buffer solution with 0.05 to 0.5 mol / 1 borate / borax buffer, pH 7.0 to 8.5 5 U / ml alkaline phosphatase C 0.2 mol / 1 borate / borax buffer, pH 7.8 to 8.2 10 mmol / 1 magnesium sulfate 2.5 g / l Tinovetin OU »