DE2302721C2 - Method for the determination of creatine kinase - Google Patents

Description

The invention relates to a method for determining ao creatine kinase by converting creatine phosphate with ADP to creatine and ATP in the presence of an activating agent containing SH groups and Measurement of the quantitative change of a respondent.

Various diseases have characteristic changes in creatine kinase (CK) activity in the body fluids, e.g. B. in the serum, result. The CK determination is therefore of great importance for the detection of these diseases. In addition, the CK determination is also of considerable importance for research purposes.
Creatine kinase catalyzes the reaction (1)

Creatine phosphate I ADP = # = Creatine - {- ATP (1)

Various methods are known which determine the CK according to equation (1) Allow measurement of the quantitative change of one of the reactants according to the above equation. Processes that utilize the reverse reaction and have gained importance in this regard Creatine (R ο t t h a u w e et al, Klin. Wochenschrift 39, 1269 [1961]) or ATP (I.T. Oliver, J. biol. Chemistry 61, 116 [1955]) measure.

The activity of the enzyme is preferably measured by determining what is continuously produced ATP's. This is done in a known manner (I. T. Oliver, J. biol. Chem. 61, 116 [1955]) by reaction with Glucose under the catalysis of hexokinase.

ATP -I- glucose ^ ADP j glucose-6-phosphate (2)

Glucose-6-phosphate can easily be converted into gluconate-6-phosphate with NADP and G6P-DH. NADP is converted into NADPH, which strongly adsorbs light with a wavelength of 340 mm.

NADP -f G6P ^ NADPH (6-PG - \ H (3)

60

The activity of the CK can therefore very easily be determined by measuring the rate of NADPH formation be measured.

It has long been known that a method for determining creatine kinase Activating agent must be added, which is a compound containing SH groups. Various SH compounds have already been proposed for this purpose, such as cysteine, Glutathione (GSH), mercaptoethanol, mercaptoacetate, dithiothreitol, dithioerythritol and the like.

The previously proposed as activators SH group-containing Connections, however, have a wide variety of defects. So do cysteine solutions very easy to crystallize, freeze-dried preparations no longer dissolve completely after a while k! ar on. Mercaptoethanol, mercaptopropanol, mercaploacetate, thioglycolate, etc. can rarely used because of their intense unpleasant odor. Dithiothreitol, dithioerythritol, Cysteine methyl esters etc. are very easily oxidized by atmospheric oxygen and then lose their activating properties Properties. Aminoäthyüsothio-uronium-bromid has a strong protein-denaturing property, which makes it impossible to combine this substance with proteins, e.g. B. albumin, hexokinase. Lyophilize G6P-DH etc. / u. Other substances, like guanylcycysteine or carbamoylcysteine, are very good difficult to dissolve.

So until now, GSH has been the activation agent of choice. In 1970 and 1972 the German published Society for Clinical Chemistry (Z. Klin. Chem and Klin. Biochem. 8, 658-59 [1970] and 10, 185-86 [1972]) Recommendations on the standardization of the method for determining the activity of CK. In these publications recommend the use of GSH as a reactivator of the CK. Ls is relative. stable, almost odorless, the oxidation product GSSG is easily soluble and has no protein denaturants Properties.

However, it turned out that some of the serum samples examined had too low a CK activity is found when using GSH solutions or GSH lyophilisates that have been stored for a longer period of time will. The activity found no longer corresponds to the clinical picture. However, it is specific to a heart attack absolutely necessary that the exact activity as soon as possible after its onset the CK is determined. From the level of the increase in activation above the normal value of 50 U / l Inferred from the size of the infarct and the Ar / t can then take the necessary measures will.

The object of the invention is therefore to create a method for determining creatine kinase. in which an activator is used which does not have the disadvantages of the previously known activators having.

This task is solved according to crfuiki a method for determining the creatine kinase of the type mentioned, characterized in that the implementation is carried out in the presence of N-acetylcystcin as an activating agent.

With the found activating agent CK activity remains unchanged for very long periods of time, even if it is present in the form of solution, the activity remains unchanged after weeks of storage.

The superiority of the N-acetylcysteine (NAC) used according to the invention over the previous one preferred GSH are shown in Table I below, in which the results of activity determinations of CK are shown in 15 different sera, each with freshly prepared GSH or NAC solution and with GSH or NAC solution stored for 6 weeks with 4.

Table I.

serum With freshly made With 6 weeks at 4 ° With freshly made With 6 weeks at 4 " GSH solution U / l stored GSH solution U / l NAC solution U / l stored NAC solution U / l ι 30th 19th 32 30th 2 58 44 60 60 3 107 95 105 104 4th 124 109 130 125 5 230 219 235 2SO 6th 33 10 35 30th 7th 75 55 80 78 8th 132 115 130 128 9 190 170 185 180 10 239 220 245 240 11th 20th 0 24 20th 12th 49 2 52 47 13th 115 70 110 110 14th 180 140 185 179 15th 231 190 235 225

The above values show, especially when comparing sera nos. 1, 6, 7, 11, 12, 13 and 14, that with stored GSH solution very considerable incorrect determinations occur, while according to the invention only very minor deviations occur, mostly within the error limit of the determination method lie.

The superiority of the activating agent according to the invention probably related to its better shelf life compared to GSH. These Table II below shows improved stability both in solution and in lyophilized form.

Table Il

NAC solution Lyophilizate < solution JStI camp at 4 ³ at 33 ° at 4 ' duration mg / ml mg / bottle mg / ml Lyophilizate 102 102 100 at 33 Weeks 99 102.5 88 mg / bottle 0 98 101 80 105 1 87 98.7 68 100 2 81 97.0 55 95 3 77 97.5 45 92 4th 85 5 77

in a few bottles, through the production of reaction mixtures and through special types of packaging the determination should be quick and easy to carry out.

To carry out the method according to the invention, a reagent is required which consists of Creatine phosphate, ADP and a system for determining ATP or creatine and one Has content of N-acetylcysteine.

Such a reagent expediently consists of

The different stability is due to a different oxidation sensitivity of the SH group returned. The difference in stability found could not be foreseen.

The method of the present invention is preferred using the method illustrated by Equations 2 and 3.

As a result of the great importance of the determination of the CK activity in the serum in particular / ur detection of various diseases, reagent compositions are used by the industry for a certain number of tests are made available. These compilations contain the reagents necessary for the test in pre-weighed or measured amounts, and are in a Form presented, which allows an exact determination of the CK activity.

By combining the various necessary components that do not interfere with one another

1. 3.5 to 15 mmol imidazole,
0.8 to 3.5 mmol of glucose,

0.3 to 1.5 mmol magnesium acetate, 0.5 to 2.5 mmol alkali azide,

0.1 to 0.4 mmol EDTA (ethylenediaminotetraacetic acid) dissolved in (50 ml of water with a pH value of 6.5 to 7.0.

2. 0.04 to 2, OmMoI ADP,
0.02 to 1 mmol NADP-Na ₂ ,

0.3 to 1 mmol of AMP-Na, as a solution in 3 ml of water or lyophilisate.

3. 0.15 to 3 mmol of creatine phosphate in 5 ml of water or as a lyophilisate.

4. 30 to 350 U hexokinase,

30 to 350 U glucose-O-phosphate dehydrogenase (G6P-DH) in 1 ml glycerine / water 1: 1.

5. 0.3 to 3.0 mmol of N-acctylcystcin in 2 ml of water or as a lyophilisate or from an aliquot da \ on.

Ingredients I to 5 of this reagent are / wake in dry form or in the form of a Solution in five separate containers, / .. B. bottles, in front and used to determine the implementation mixed together in the manner indicated below.

The reagent can also consist of just two different mixtures, of which

Mixture 1 of 3.5 to 15 mmol of imidazole, 0.8 to 3.5 mmol of glucose,

0.3 to 1.5 mmol of magnesium acetal, 0.5 to 2.5 mmol of alkali azide in 60 ml Dissolved water at pH 6.5 to 7.0 and

Mixture 2 of 0.2 to 0.6 mmol ADP, 1.0 to 5.0 mmol AMP, 0.05 to 0.5 mmol NADP, 5.0 to 20.0 mmol creatine phosphate, 2.0 to 5.0 mmol N-acetylcysteine, 100 to 300 U hexokinase, 100 to 1500U G6P-DH, 100 mg serum albumin in 100 ml water at pH 6.5 to 7.0 or as a lyophilisate or an aliquot thereof.

In this embodiment of the reagent, a certain amount of the reagent is present in the form of a solution Mixture 1 is used to dissolve a certain part of the mixture 2 present in solid form or mixed with an appropriate proportion of the solution from Mixture 2.

The invention enables a marked increase in the reliability of the creatine kinase determination by providing a superior activating agent. This activating agent NAC does not have a protein denaturing effect, has practically no odor, is very soluble in water and costs only about a tenth of the previously preferred activator GSH. _a5

The following examples explain the process according to the invention.

example 1

Solution!: 7.0 mmol imidazo !,

1.6 mmol glucose,

0.73 mmol magnesium aretate,

1.07 mmol sodium azide,

0.22 mmol EDTA

are redistilled with 50 ml. Dissolved water, adjusted to pH 6.8 with 2N acetic acid and made up to 60 ml with water.
Solution 2: 0.06 mmol ADP,

0.037 mmol NADP-Na ₂ ,

0.46 mmol AMP-Na ₁

Are dissolved with 3 ml of water and freeze-dried in a suitable bottle.
Solution 3: 2.1OmMoI creatine phosphate in 5 m! Dissolve water and lyophilize it in a suitable hash.
Solution 4: 175 U hexokinase,

175 U G 6 P-DH

»Earth in 1 ml 50-jiroz. Glycerine dissolved. Solution 5: 0.7 mMul N-acetylcysteine are dissolved in 2 ml water and in a suitable Lyophilized bottle.

This is the form in which the reagent mixtures are Stable for at least 1 year at 4 ° C and 4 weeks at 33 ° C.

To prepare a reaction mixture, the contents of bottles 2, 3 and 5 are dissolved with solution 1. This solution can be kept at -t 4 C for at least 6 weeks.

The determination of the CIC activity is now like carried out as follows:

2.50 ml reaction mixture and
0.10 ml of serum

are pipetted into a cuvette, mixed and incubated at 25 ° C for 5 min. After adding

0.02 ml of solution 4

is in a suitable photometer, which the exact Isolation of wavelengths 340 nm, 334 nm or 366 nm, the absorbance is measured and the increase in erosion at an interval of exactly 1 min Followed for 5 min. From Jen extinction differeii7cn per min results, multiplied by an invoice, the CK. activity present in the serum.

Example 2

Solution 1: 7.0 mmol imidazole,

1.6 mmol glucose,

0.73 mmoles of magnesium acetate.

1.07 mmol Nairi.'smazid,
0.22 mmol EDTA

are redistilled with 50 ml. Dissolved water, adjusted to pH 6.8 with acetic acid and made up to 60 ml with water.
Solution 2: 0.25 mmol ADP,

2.50 mmol AMP,

0.15 mmol NADP,

8.75 mmol creatine phosphate,

2.5 mmoles of N-acetylcysteine.

700 U hexokinase,

35OU G6P-DH,

100 mg serum albumin

are redistilled in 80 ml. Dissolved water, diluted with Sodium hydroxide solution or acetic acid adjusted to pH 6.8, with double dist. Water made up to 100 ml, mixed and filled into suitable bottles of 1.0 ml each. The solution is lyophilized.

The content of a 2.50 ml bottle is used for the test Solution 1 gelösi, 0.1 ml of serum added and the Determination of the CK. Activity as in the example! described carried out.

Claims (1)

Claim: Method for the determination of creatine kinase by reacting Creaünphosphat with ADP on creatine and ATP in the presence of an activating agent containing SH groups and measurement the quantitative change of a reaction participant, characterized in that, that the reaction is carried out in the presence of N-acetylcysteine as an activating agent.