DK148658B - PROCEDURE AND METHOD FOR ACTIVATING CHOLESTERINE INTERESTASE - Google Patents

Description

in 148658

The invention relates to a method and means for activating cholesterol esterase by the enzymatic reaction of esters in reaction mixtures. Such reaction mixtures are used, inter alia, in the clinical-chemical analysis and in the food-chemical analysis.

In designing cholesterol ester-dependent tests, it is desirable to save expensive enzyme, achieve high rates of reaction and maximum freedom of choice in terms of nature and content of detergents and buffers according to overall views.

In a buffered medium containing no additional components, the cholesterol esterase develops against its substrates no or little activity at all. Therefore, it is necessary to find activating additions to the enzyme, thereby increasing the ester cleavage. As such activating additives, some detergents are known (U.S. Patent No. 3,884,753, DE publication no.

24 09 695, DE Publication No. 25 12 605, DE Publication No. 25 12 585, DE Patent 2,506,712, Biochim. Biophys. Acta, 270 (19721 156-166, e.g., bile acids, Triton X-100, Thesitl.

A higher salt content in the medium supports its activating effect. Not all buffer systems, however, are equally suitable. The same is true for the detergents. Here, under certain conditions, these are found to have good or less good effect equally to those which show no activating effect at all. For the most part, which detergents are suitable for this purpose cannot be predicted.

It is common for activating detergents that, in a present medium at a given concentration, they provide their optimum activating effect. This optimum may be in unfavorably high concentration ranges. Outside of this optimum, a greater amount of the relatively expensive enzyme is not optimally reacted at high reaction rates.

In addition, activity increases by optimizing detergent content and increasing ionic strength can only be achieved within relatively narrow limits.

As the reaction mixtures which are considered, as a rule, also contain additional enzymes and substrates for follow-on and indicator reactions, there is no choice in the choice of buffers and detergents and in the optimization of the quantities to be added.

Furthermore, when determining the reaction conditions, the interactions of all the reaction components must always be taken into account, especially possible inhibitions or adverse stability effects of other enzymes by means of detergents and / or high ionic strengths.

In developing a test system, consideration must also be given to solubility limits and galenic problems.

In the latter case, the frequent liquid to waxy detergents can rarely be introduced into the amount needed for activation in solid mixtures which must, of course, be dry and rice-proof. This is particularly important in mixing reagents.

Finally, it must be taken into account that most of the detergents involved do not exist as defined compounds but as substance mixtures. Accordingly, there are differences from portion to portion which are also shown by the activating effect. Therefore, it can be very unfortunate when the activation alone is dependent on a detergent.

As a result, there is a great need for a better agent as well as a method for activating carbon sterinsterase.

The object of the invention is to remove the above-mentioned disadvantages and to provide a method and a means of said species in which cholesterine esterase is saved, under certain conditions higher reaction rates can be obtained, work with poorer ionic strength, detergents are only required to the minimum possible concentration, and which can also use such detergents which alone show little sign of any activating effect at all.

French Patent Specification No. 2,263,516 discloses agents comprising cholesterinesterase, surfactant and phenol. However, it is not clear how to activate the enzyme cholesterol esterase. Phenol in unsubstituted form is mentioned exclusively as a component of a color reagent.

German Patent Specification No. 2,612,725 also discloses agents comprising cholesterinesterase, surfactant and phenol. However, it cannot be deduced here how to activate cholesterol esterase. As in the aforementioned French presentation, phenol is used solely to achieve a color reaction. In addition, the reagents mentioned in DE Specification No. 2,612,725 contain lower alkanols, which, however, are intended only to prevent blur in the serum.

The object of the invention is achieved by a method for activating cholesterol esterase in ionic solutions by adding at least one surfactant, characterized in that a) as a surfactant is added a polyethoxy ether or ester and / or a bile acid compound and / or salts of fatty acids of 6-10 carbon atoms in combination with b) a synergistically acting alcohol selected from the straight-chain, branched or cyclic aliphatic alcohols of 5-12 carbon atoms, halogen substituted, aromatic alcohols and those having several halogen atoms, aliphatic alcohols having 1-3 carbon atoms.

The invention is based on the surprising finding that said alcohols, which in themselves alone do not exhibit an activating effect on cholesterol esterase, synergistically clearly amplify the known activating effect of certain detergents, and by certain in themselves alone. non-activating detergents cause an activation.

This means that such non-activating, widely used detergents can be used. Similarly, because of their defined molecular structure and solid consistency, desirable but because of poorly activating properties, hitherto unsuitable ionic detergents can be used. Furthermore, enzyme, the maximum achievable reaction, can be saved. The rate of ionization is increased, the required ionic strength is reduced, and finally by the many combination possibilities the esterase activating system is adapted to the conditions dictated by additional components of the reaction mixture.

Examples of suitable polyethoxy ethers are fatty alcohol polyglycol ethers such as polyoxyethylene lauryl ether and hydroxypolyethoxy dodecane, alkyl aryl ethers and arylalkyl ethers such as polyoxyethylenonylphenyl ether and polyoxyethylene octylphenyl ether, fatty acid esters such as polyoxyethylene ether In the bile acid compounds, cholic acid, de-oxycholic acid and their alkali salts are preferred. Also, among the fatty acid salts, the alkali metal salts, especially the sodium salts, are preferred.

Typical examples of suitable straight-chain, branched or cyclic aliphatic alcohols are pentanol, tert-amyl alcohol, methylpentanol, hexanol, cyclohexanol, octanol, isooctanol, decanol and dodecanol. Examples of suitable halogen substituted aromatic alcohols are the halogen substituted phenols and naphthols such as the mono-, di- and trichlorophenols.

As polyhalogen-substituted aliphatic alcohols, e.g. dichloromethanol, trichloroethanol, dichloromethanol, trichloromethanol, trichloropropanol, tetrachloropropanol, etc. are used.

As halogen substituents, the aromatic and aliphatic alcohols take into account chlorine, bromine and fluorine. Chlorine is preferred. The aromatic alcohols may also have one or two alkyl groups of 1-2 carbon atoms.

Which of these alcohols (hereinafter referred to as adjuvant}, which are particularly useful in certain cases, can easily be determined by prior experiments. Most of the conditions are such as the nature and strength of the buffer systems, the nature and concentration of the detergent, and the durability and desired These conditions can then be met by selecting the appropriate adjuvant and adjusting the amount added. In general, concentrations between 1 and 300 mm adjuvant / 1 were found to give good results.

The alcohols are preferably used in amounts between 0.01 and 5%, volume percent by liquid alcohols, weight percent by solid alcohols. 0.05-3% is especially preferred.

In the polyethoxy derivatives, the alkyl groups generally contain from 8 to 22 carbon atoms. The aralkyl and alkaryl groups preferably contain a phenyl residue. The glycol residue of these detergents generally has 3-25 oxyethylene groups.

The detergents are suitably used in an amount between Q, 05 and 5% by volume, respectively, by weight of solids.

0.1-3% is preferred.

148658 6

The invention further relates to an agent for carrying out the above process containing at least one surfactant based on a polyethoxy ether or ester and / or a bile acid compound and / or salts of fatty acids having 6-10 carbon atoms, characterized by a content of a syn. ergonomically acting alcohol selected from straight-chain, branched or cyclic aliphatic alcohols having 5-12 carbon atoms, halogen substituted aromatic alcohols and halogen substituted aliphatic alcohols having 1-3 carbon atoms.

The invention can be used in conjunction with all kinds of measurements and reactions where cholesterol esterase is used in reaction mixtures. Such reaction mixtures are already known to those skilled in the art and are described insofar as they are intended for analytical purposes, e.g. in "Methods of Enzymatic Analysis", H.U. Bergmeyer, Verlag Chemie Weinheim, Bergstrasse. A detailed description of such reaction mixtures is therefore not needed here. The invention is suitable for cholesterol esterase from microorganisms or of other origin, such as pancreas or liver.

The following examples further illustrate the invention in connection with the following drawings. In the drawing, FIG. 1 is a schematic representation of the apparatus used for the determination of cholesterol esterase activity; FIG. 2 is a graphical representation of the extinction change occurring by the method used per time unit used to determine the ester cleavage rate.

The following table shows the chemical composition indicated by trade names: 148658 7

Melt 35 Polyoxyethylene lauryl ether

Genapol OX-IOQ Fat Alcohol Polyglycol Ether

Tergitol NPX Polyoxyethylenonylphenyl ether (approx. 1Q, 5 oxyethylene groups)

Thes it He droxypropyle thoxydodecan

Triton X-100 Polyethoxyoctylphenyl ether (9-10 oxyethylene = groups}

Tween 2Q Polyoxyethylene sorbitan monolaurate.

For the detection of the activating effect of the synergistic combination of the invention to be investigated on cholesterol esterase, it was shown in FIG. 1.

In a temperable reaction vessel 1 (25 ° C) in which the ester cleavage takes place, via a hose pump 2 through a feed hose 3 of defined cross-section, 0.32 ml of mixture per continuous is continuously withdrawn. mine. Via the same pump, through another hose 4 from a storage container 5, 1.0 ml of indicator solution per continuous is also continuously fed. mine. behind the pump 2 together with an air flow 6 of constant 0.6 ml of air per mine. and is then segmented and then combined with the previously described reaction mixture stream. Bone total flow then passes through 2 glass tubes 7, ensuring a mixing of the fluids and the required reaction time for the indicator reaction until the end point. When taking a flow of 1.2 ml / min. immediately before the container through hose line 8 vertically upwards, the air bladders are removed again. The non-segmented residual current is conducted for photometric measurement (at 546 nm) in a through-flow vessel (1 cm layer thickness) and the measurement values are recorded by the printer 10.

Due to the unchanged conditions with respect to the indicator reaction, changes in altitude and the course of the measurement signal depend solely on the course of the reaction vessel.

If an ester cleavage occurs here, the kinetic process causes an increase in the measurement signal, which is registered as a positive increase with the printer 10. The slope of the increase is a measure of the reaction rate and thus of the cholesterol esterase activity in the reaction mixture.

Due to the separate course of the ester cleavage and the indicator reaction, a mutual influence is excluded. Of course, other indicator systems including the oxygen electrode could also be used.

Indicator solution: 0.1 m potassium phosphate buffer, pH 7.0 1.5 itM / 1 4-amino-antipyrin 5 nM / 1 phenol 3% o Hydroxypolyethoxydodecan 8 U / ml Peroxidase 1 ϋ / ml cholesterol oxidase.

The indicator solution first converts free cholesterol to cholesterone and 'H2®2 * The resulting reaction is reacted with phenol and 4-amino-antipyrin to a red dye whose absorption is measured photometrically. The amount of dye formed is proportional to the concentration of free cholesterol. The concentration of free cholesterol consists of the amount of free cholesterol already present in the substrate and of the cholesterol released by the esterase.

Composition in the reaction vessel: Q, 5 ml of substrate (control serum; 360 mg cholesterol total / dl) Detergent according to test Adjuvant according to test Buffer to 5 ml.

Start with 5Q μΐ of an esterase stock solution containing 0.0Q3 U / ml esterase in the mixture. At activity changes, this amount varied.

9 1Λ 8 6 5 8

After starting and mixing, the feed hose 3, by running pump 2, is dipped into the solution in the reaction vessel 1 for approx. 10 min. For better separation of the signals, air and bite style are then alternated. water sucked in every approx.

10 segments. Then the next determination can take place.

The following examples show the enabling of enzyme (a) saving in the invention, the attainment of higher reaction rates under given conditions (ia the use of lower ionic strength buffers (c); the addition of inferior concentration detergents (dl and the use of such detergents which, under certain conditions, produce only an insufficient or no activating effect (s).

a) Cholesterol esterase saving.

The possibility of the cholesterol esterase saving is demonstrated as follows: First, a stepwise enzymatic activity change is made in the absence of adjuvants. The comparative measurements are then made with the addition of adjuvants at a cholesterol esterase addition of 0.0.03 U / ml. On the basis of the shown relationship between employed activity and the resulting speed, the activity increase obtained is then determined.

Example 1 0.1 M Tris / Tartaric Acid Buffer, pH 8.0 5% o Triton X-1Q0

Starting activity Resulting rate 0 U / ml 0 Ext / 10 minutes 0.0012 0.012 0.0030 0.032 0.0045 0.052 0.0060 Q, 058 0.0089 0.094 148658 10 1.1 Addition of 2% by volume of tertiary amyl alcohol

Starting activity Q, Q030 U / ml

Achieved speed 0.046 Ext / 10 min.

Necessary enzyme addition at this rate without adjuvant 0.0044 U / ml

Activity achieved 147% 1.2 Addition of 0.4 volume% 4-methylpentanol- (21

Starting activity 0.003 U / ml

Achieved speed 0.065 Ext / 10 min.

Required enzyme addition to this rate without adjuvant 0.0063 U / ml

Activity achieved 210% 1.3 Addition of 1 volume% cyclohexanol

Starting activity 0.0030 U / ml

Achieved speed 0.077 Ext / 10 min.

Necessary enzyme addition to this rate without adjuvant 0.0075

Achieved 250% activity

Example 2

In 0.1 M potassium phosphate buffer, pH 7.0 1.5% Tergitol NPX

Starting activity Resulting speed 0.0012 U / ml 0.019 Ext / 10 min.

0.0030U / ml 0.030 Ext / 10 min.

0.0060 U / ml 0.055 Ext / 10 min.

0.0090 U / ml 0.091 Ext / 10 min.

Q, 012Q U / ml 0.120 Ext / 10 min.

2.1 Addition of 0.4% by volume of 2,2,2-trichloroethanol

Starting activity 0.003 U / ml

Achieved speed 0.046 Ext / 10 min.

Required enzyme addition to this rate without adjuvant 0.046 Ext / 10 min.

Activity achieved 153% 2.2 Addition of 2% by volume of tert-amyl alcohol

Starting activity 0.003 U / ml

Achieved speed 0.053 Ext / 10 min.

Necessary enzyme addition to this rate without adjuvant 0.0053 U / ml

Activity achieved 177% 2.3 Addition of 1.4% by volume of 4-methylpentanol- (2)

Starting activity 0.003 U / ml

Achieved speed 0.059 Ext / 10 min.

Necessary enzyme addition at this rate without adjuvant 0.0059 U / ml

Activity achieved 197% 2.4 Addition of 2 volume% cyclohexanol

Starting activity 0.003 U / ml

Achieved speed 0.114 Ect / 10 min.

Necessary enzyme addition to this rate without adjuvant 0.114 U / ml

Activity achieved 380%

Example 3 In 0.3 M Potassium Phosphate Buffer, pH 7.0 5% o Genapol OX-100/5% o Sodium Caprylate 12 U8658

Starting activity Resulting speed 0.0012 -U / ral 0.01 Ext / 10 min.

0.00.30 U / ml 0.023 Ext / 10 min.

0.0060 U / ml 0.038 Ext / IQ min.

0.0Q90 U / ml 0.055 Ext / 10 min.

0.0150 U / ml Q, Q93 Ext / 10 min.

0.0240 U / ml 0.138 Ext / 10 min.

3.1 Addition of 4 mM / l 3,4-dichlorophenol (0.065 wt% l

Starting activity 0.0030 U / ml

Achieved speed 0.058 Ext / 10 min.

Required enzyme addition at this rate without adjuvant 0.0093 U / ml

Activity achieved 310% 3.2 Addition of 1 volume% 4-methylpentanol- (2)

Starting activity 0.003 U / ml

Achieved speed 0.069 Ext / 10 min.

Required enzyme addition to this rate without adjuvant 0.0112 U / ml

Activity achieved 373% 3.3 Addition of 2 volume% cyclohexanol

Starting activity 0.003 U / ml

Achieved speed 0.080 Ext / 10 min.

Required enzyme addition to this rate without adjuvant 0.0129 U / ml

Activity achieved 430% 3.4 Addition of 0.3% by volume 2,2,2-trichloroethanol

Starting activity 0.003 U / ml

Obtained speed 0.089 Ext / 10. mine.

Required enzyme addition to this rate without adjuvant 0.0144 U / ml

Obtained activity 480% 148658 13 bl Achieving higher reaction rates under given conditions. In many cases, certain conditions such as buffer and ionic strength as well as the nature of the detergent in reaction mixtures have been given in advance because of overall views. Such reasons may e.g. be given due to greater sensitivity of another enzyme needed in the mixture. In this case, the constant rate of ester cleavage attained by constant cholesterol esterase addition is characterized and limited by dependence on the esterase activation of the nature of the detergent - and detergent concentration. This dependency generally shows an optimum in the lower concentration range of detergent.

The speed obtained at this point cannot be exceeded in accordance with the conditions set out above and in this respect constitutes a limit to the system.

The combinations of the invention described in the following example are suitable for exceeding such limits.

Example 4

Preset: 0.1 M tris / tartaric acid buffer, pH 8.0 Triton X-1Q0, Q, Q03 U / ml cholesterol esterase% o Triton X-100 in the mixture Resulting rate 0% o Q Ext / 10 min.

1% o 0.003 Ext / 1Q min.

3% o 0.028 Ext / 10 min.

5% o 0.037 Ext / 10 min.

7% o 0.037 Ext / 10 min.

10% o 0.037 Ext / 10 min.

15% o Q, 029 Ext / 10 min.

Optimum System Limit 5% o 0.037 Ext / 10 min.

148658 14

The adjuvant addition occurs at 5% o Triton X-10Q.

4-1 Addition of 2% by volume of tert-amyl alcohol

Achieved speed 0, Q46 Ext / 10 min.

Increase to 124% 4.2 Addition of Q, 4 volume% 4-methylpentanol- (2)

Speed achieved. 0.065 Ext / 10 min.

Increase to 176% 4.3 Addition of 1 volume% cyclohexanol

Achieved speed 0.077 Ext / 10 min.

Increase to 208%

Example 5

Preset: 0.3 M potassium phosphate buffer pH 7.0

Tergitol NPX, 0., 0Q3 U / ml cholesterol esterase% o Tergitol NPX in the mixture Resulting rate 0% o 0 Ext / 10 min.

2% o 0 Ect / 10 min.

5% o 0.029 Ext / 10 min.

10% o 0.042 Ext / 10 min.

15% o 0.049 Ext / 10 min.

20% o 0.039 Ext / 10 min.

Optimum System Limit 15% o 0.049 Ext / 10 min

Addition of adjuvants occurs at 15% o Tergitol NPX.

5.1 Addition of 2 itM / 1 3,4-dichlorophenol and 1 volume% cyclohexanone

Achieved speed 0.058 Ext / 10 min.

Increase to 118% 15 148650 5.2 Addition of 2% by volume of tert-amyl alcohol

Achieved speed 0.066 Ext / 10 min.

Increase to 135% 5.3 Addition of 1.4% by volume of cyclohexanol

Achieved speed 0.118 Ext / 1Q min.

Rise to. 241% c) Use of low ionic strength buffers.

As already mentioned, buffers of higher ionic strength support the activation of the effect of detergents on cholesterol esterase. However, within the limits of the required maximum freedom of choice of the conditions, it is desirable not to be forced to high salt content in the solution. The following example shows that the salt content of the invention can be substantially reduced.

Comparison: 0.3 M potassium phosphate buffer, pH 7.0 0.1 M potassium phosphate buffer, pH 7.0

By adding suitable adjuvants to the 0.1 m buffer system, speeds can be increased such that the higher comparative value of the 0.3 M buffer system without adjuvant can be achieved and partially clearly exceeded.

148658

Example 6 16 15%% o Tergitol NPX, O, QQ3 ϋ / ml cholesterol esterase 6.1 Addition of 2% by volume of tert-amyl alcohol

System Speed 0.1 m buffer without addition 0.030 Ext / 10. mine.

0.3 m Buffer Q, 049 Ext / 10 min.

0.1 m buffer with addition 0.053 Ext / 10 min.

The comparison value is surpassed.

6.2 Addition of 1.4 volume% 4-methylpentanol (2) 0.1 m buffer without addition 0.030 Ext / 10 min.

0.3 m Buffer 0.049 Ext / 10 min.

0.1 m buffer with addition 0.059 Ext / 10 min.

The comparison value is surpassed.

6.3 Addition of 2 volume% cyclohexanol 0.1 m buffer without addition 0.030 Ext / 10 min.

0.3 m Buffer 0.049 Ext / 10 min.

0.1 m buffer with addition 0.114 Ext / 10 min.

The comparison value is clearly surpassed.

Example 7 10% o Genapol OX-10Q / 10% o Na-caprylate 0.003 U / ml cholesterol esterase 7.1 Addition of 1 volume% 2,2,2-trichloroethanol 0.1 m buffer without addition 0.017 Ext / 1Q min.

0.3 m buffer "" Q, 053 Ext / 1Q min.

0.1 m buffer with addition 0.063 Ext / 10 min,

The comparison value is surpassed.

7.2 Addition of 2 volume% cyclohexanol 0.1 m buffer without addition 0.017 Ext / 10 min.

0.3 m buffer "0.053 Ext / 10 min.

0.1 m buffer with addition 0.072 Ext / 10 min.

The comparison value is surpassed.

d) The use of detergents at a lower concentration.

If a detergent is capable of activating the cholesterol esterase, in order to fully utilize this property, it must be present in the reaction mixture at a particular concentration. This detergent optimum is substantially dependent on the nature of the detergent and may in many cases lie in an unfavorably high concentration range. This can, among other things, inhibit enzymes needed for follow-up reactions and also cause difficulties due to solubility and galenics. During the detergent optimization, only insufficient activation is detected. However, in order to have as wide a choice of conditions as possible, it is desirable to be able to use as many different detergents as possible, including those which have a higher concentration concentration.

The following example shows that according to the invention it is possible to obtain and exceed the reaction rates of the concentration optimizer with less detergent, thereby saving detergent.

148658

Example 8 18 0., 3 m Potassium Phosphate Buffer, pH 7.0 Tergitol NPX, Q, OQ3 U / ml cholesterol esterase

So Tergitol NPX in the mixture Resulting rate 0% o 0 Ext / 10 min.

«2% o 0 Ext / 10 min.

5% o 0.029 Ext / 10 min.

10% o 0.042 Ext / 10 min.

15% o 0.049 Ext / 10 min.

20% o 0.039 Ext / 10 min.

Optimum Comparative Value 15% o 0.049 Ext / 10 min.

8.1 Addition of 0.1 volume% 2,2,2-trichloroethanol

System Speed 15% o Tergitol without addition>. 0.049 Ext / 10 min.

5% o Tergitol 0.029 Ext / 10 min.

5% o Tergitol with addition 0.059 Ext / 10 min.

The comparison value is exceeded, · 8.2 Addition of 2 volume of tert-amyl alcohol 15% o Tergitol without addition 0.049 Ext / 10 min.

5% o Tergitol 0.029 Ext / 10 min.

5 So Tergitol with addition 0.075 Ext / 1Q min.

The comparison value is clearly exceeded.

8.3 Addition of 1 volume cyclohexanol

System Speed 15 So Tergitol without addition 0.049 Ext / 10 min.

5 Tergitol 0.029 Ext / 10 min.

5 So Tergitol with addition 0.124 Ext / 10 min.

The comparison value is clearly surpassed.

Example · 9 0.3 m potassium phosphate buffer, pH 7.0 buffer

Genapol OX-100 / Sodium Caprylate, Q, QQ3 U / ml cholesterol esterase.

% o Genapol CK-100 / Na-caprylate Speed 0% o 0 Ext / 10 min.

5 Sun 0.020 Ext / 10 min.

10% o 0.053 Ext / 10 min.

15% o 0.062 Ext / 10 min.

20% o 0.066 Ext / 10 min.

30% o 0.068 Ext / 10 min, 40% o 0.068 Ext / 10 min.

Optimum Comparative Value 30% o 0.068 Ext / 10 min.

9.1 Addition of 1 volume% 4-methylpentanol- (2)

System Speed 30% o Genapol / caprylate without addition 0.068 Ext / 10 min.

5% o Genapol / caprylate without addition 0.020 Ext / 10 min.

5 So Genapol / caprylate with addition 0.069 Ext / 10 min.

The comparison value is obtained.

9.2 Addition of 2% by volume of cyclohexanol

System Speed 30% o Genapol / caprylate without addition 0.068 Ext / 10 min.

5% o Genapol / caprylate without addition 0.020 Ext / 10 min.

5 So Genapol / caprylate with addition 0.080 Ext / 1Q min.

The comparison value is surpassed.

9.3 Addition of 0.3% by volume of 2,2,2-trichloroethanol

System Speed 30% o Genapol / caprylate without addition 0.068_Ext / 10 min.

. 5% o Genapol / caprylate without addition Q, 020 Ext / 10 min.

5% o Genapol / caprylate with addition 0.089 Ext / 10 min.

The comparison value is clearly surpassed.

e). Use of detergents which, under certain conditions, have no or only insufficient activating effect.

Among the large number of detergents available, it has been found that only a few are capable of sufficiently activating cholesterol esterase. The inhibition of other reaction components, the stability of the reaction mixture, the consistency, solubility, and uniqueness of the compound or preparation are among the overarching reasons that the greatest possible choice of detergents is desired. The following example shows that detergents which, under the given conditions, have only little or no activating effect according to the invention.

Example 10 0.1 m phosphate buffer, pH 7.0, 0.003 U / ml cholesterol esterase.

Detergent: 6% o Thesit

Addition: 1 volume% n-amyl alcohol

System Speed

Detergent without addition 0.003 Ext / 10 min.

Addition 0. Ext / 10 min.

Detergent with addition 0.029 Ext / 1Q min.

148658

Example 11 21 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Detergent: 12% o Thesit

Addition: 2% by volume of cyclohexanol

System Speed

Detergent without addition 0.010 Ext / 10 min.

Addition 0.003 Ext / 10 min.

Detergent with addition Q, 1Q5 Ext / 10 min.

Example 12 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Detergent: 12% o Thesit

Addition: 1 volume% 2,2,2-trichloroethanol

System Speed

Detergent without addition 0.010 Ext / 1Q min.

Addition 0 Ext / 10 min.

Detergent with addition 0.081 Ext / 10 min.

Example 13 0.1 Tris / tartaric acid buffer, pH 8.0 0.003 U / ml cholesterol esterase

Detergents: 2 volume% o Thesit / 2 volume% o Tween 20

Addition: 0.4 volume% hexanol-1

System Speed

Detergents without addition Q, Q18 Ext / 10 min.

Addition 0 Ext / 10 min.

Detergents with addition Q, Q28 Ext / 10 min.

EXAMPLE 14 0.1 m phosphate buffer, pH 7, Q 0.003 U / ml cholesterol esterase

detergents; 5 volume% o Thesit / 5 volume% o Tween 20 Addition; 8 mM / l 3,5-dichlorophenol

System Speed

Detergents without addition 0.0Q5 Ext / 1Q min.

Addition Q Ext / 1Q min.

Dergents with addition Q, 033 Ext / 10 min.

Example 15 0.3m Phosphate Buffer, pH 7.0 O, Q03 U / ml Cholester Inesterase

Detergents: 5 volume% o Genapol OX-1Q0./5 wt% o sodium caprylate

Addition: 4 mM / L (about 0.06.5% by weight) of 3,4-dichlorophenol

System Speed

Detergents without addition 0.020 Ext / 10 min.

Addition Q Ext / 10 min.

Detergents with addition 0, Q58 Ext / 10 min.

Example 16 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Detergent: 10% by volume Brij 35 (30% solution)

Addition: 4 mM / L 3,4-dichlorophenol 23

System Speed

Detergents without addition Q Ext / 1Q min.

Addition Q Ext / IQ min.

Detergents with addition Q, Q1Q Ext / IQ min.

Example 17 0.1 m phosphate buffer, pH 7.0 0.00.3 U / ml koiesterine esterase

detergent; 10 volume * Brij 35 (30% solution) Addition; 1 volume% 2,2,2-trichloroethanol

System Speed

Detergent without addition 0 Ext / IQ min.

Addition 0 Ext / 10 min.

Detergent with addition 0.018 Ext / IQ min.

Example 18 0.1 m phosphate buffer, pH 7.0 0.003 U / ml koiesterine esterase

Detergent: 2 volumes * o Brij 35 (30% solution)

Addition: 2 volume * cyclohexanol

System Speed

Detergent without addition 0 Ext / 10 min.

Addition 0.003 Ext / IQ min.

Detergent with addition 0.027 Ext / IQ min.

Example 19 0.1 m phosphate buffer, pH 7.0 0.003 U / ml koiesterine esterase

Detergent: 6 weight o cholic acid

Addition: 1 volume * 2,2,2-trichloroethanol 24658 24

System Speed

Detergent without addition O Ext / 1Q min.

Addition Q Ext / 10 min.

Detergent with addition 0.029 Ext / 1Q min.

Eksempel2O

0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Detergent: 6% by weight o cholic acid

Addition: 16 rtM / 1 3.5 dichlorophenol

System Speed

Detergent without addition 0 Ext / 1Q min.

Addition Q Ext / 10 min.

Detergent with addition 0.036 / 10 min.

Example 21 Q, in m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Detergent: 1% by weight o cholic acid

Addition: 4 mM / 1 3,5-dichlorophenol, 1 volume! cyclohexanol

System Speed

Detergent without additives 0 Ext / 10 min.

Additions 0.010 Ext / 10 min.

Additive Detergent 0.103 Ext / 10 min.

Example 22 0.1 m Tris / tartaric acid buffer, pH 8.0 Q, 003 U / ml cholesterol esterase

Detergent: 3% by weight o sodium deoxycholate

Addition: 0.2 volume% hexanol-1

System Speed

Detergent without addition Q Ext / 10 min.

Addition 0 Ext / 1Q min.

Detergent with addition 0.056 Ext / 10 min.

Example 23 0.1 m Tris / tartaric acid buffer, pH 8.0 0.003 U / ml cholesterol esterase

Detergent: 3 wt sodium desoxycholate

Addition: 1 volume! 2,2,2-trichloroethanol

System Speed

Detergent without addition 0 Ext / 10 min.

Addition 0 Ext / 10 min.

Detergent with addition 0.069 Ext / 10 min.

Example 24 0.1 m phosphate buffer, pH 7.0 0.003 D / ml cholesterol esterase

Detergent: 6 volumes! o Genapol OX-100

Addition: 0.4 volume! 2,2,2-trichloroethanol

System Speed

Detergent without addition 0 Ext / 10 min.

Addition Q Ext / 10 min.

Detergent with addition 0.059 Ext / 10 min.

Example 25 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Detergent: 20% by volume o Tween 20

Addition: 1 volume% 2,2,2-trichloroethanol

System Speed

Detergent without addition Q., 0Q5 Ext / 1Q min.

Addition Q Ext / 1Q min.

Detergent with addition Q, 067 Ext / 1Q min.

Method of use Example 26

Reagent for Determination of Cholesterol in Serum

Prescription: 4.5 g / l tartaric acid 7.5 g / l tris 8.7 g / l sodium sulfate 2Q0 mg / fnl 4-amino-antipyrin 282 mg / l phenol 5000 ϋ / l peroxidase 430 U / l cholesterol esterase 310 U / in cholesterol oxidase

Activator: 2.2 g / l Thesit 1.3 g / l sodium deoxycholate 0.815 g / l 3,4-dichlorophenol

The choice of additions above allows the activation of cholesterol esterase under the conditions set by the properties of the other enzymes. At the same time, it is possible to realize the storage form of the reaction mixture as a solid mixture reagent.

Claims (6)

148658 A process for activating cholesterol esterase in ionic solution by adding at least one surfactant, characterized in that a polyethoxy ether or ester and / or a bile acid compound and / or salts of fatty acids of 6-10 are added as a surfactant. . carbon atoms in combination with b) a synergistically acting alcohol selected from the straight-chain, branched or cyclic aliphatic alcohols of 5-12 carbon atoms, halogen-substituted aromatic alcohols, and the multiple halogen atoms substituted aliphatic alcohols having 1-3 carbon atoms. Process according to claim 1, characterized in that cholic acid, deoxy = cholic acid or an alkali metal salt thereof is used as the bile acid compound. Process according to claim 1, characterized in that an alkali metal salt is used as fatty acid salt. Process according to any one of the preceding claims, characterized in that 0.05-5% surfactant is used. Process according to any one of the preceding claims, characterized in that 0.01-5% alcohol is used. An agent for carrying out the process according to claim 1 containing at least one surfactant based on a polyethoxy ether or ester and / or a bile acid compound and / or salts of fatty acids of 6-10 carbon atoms, characterized by a content of a synergistic acting alcohol, that is chosen from among the equal-