FI70044B - FOERFARANDE OCH REAGENS FOER AKTIVERING AV KOLESTERINESTERAS - Google Patents

Description

ΓΒΐ / ιι \ ^ u u L UTU SJ U L K AIS U * 70044 ä§F $ ^ (11 * UTLÄGGNINGSSKRIFT / U U 4 4 C ,,,, Patents granted

Potent me Mel-1 12 CO 1336 ^ ^ '(51) Kv.lk//lnt.CI/ C 12 N 9/18 (21) Patent application - Patentansftkning 800743 (22) Application date - Ansöknlngsdag 1 1 .03.80 (F ») (23) Start date - Glltighetsdag 11 .03.80 (41) Become Public - Bllvit often tl ig 23.09 .80

National Board of Patents and Registration Date of Publication and Publication - 31 _ o 1.86

Patent and registration authorities' Ansftkan utlagd och utl.skrlften publlcerad (32) (33) (31) Requested privilege — Begärd prloritet 22.03.79 German Republic of the Federal Republic of Germany (DE) P 2911284.3 (71) Boehring Sandhofer Strasse 112-132, 6800 Mannheim - Waldhof, Federal Republic of Germany-Förbundsrepubliken Germany (DE) (72) Sigmar Klose, Berg, Herbert Buschek, Starnberg, Helmut Sch1umberger, Polling, Federal Republic of Germany-Förbundsrepubliken Berg (DE) (DE) Oy Ab (54) Method and reagent for activating cholesterol esterase -Förfarande och reagens för aktivering av cholesterinesteras

The present invention relates to a process and means for activating cholesterol esterase, in particular during the enzymatic reaction of esters in a reaction mixture. Such reaction mixtures are used e.g. in clinical chemical analysis and food chemical analysis.

Known problems in designing cholesterol ester experiments include saving expensive enzyme, providing a higher reaction rate, and the most free choice of quality and concentration of surfactants and buffers, taking into account the above considerations.

In a buffered medium that does not contain other ingredients, cholesterol esterase has very little or no activity relative to its substrates. As a result, it is necessary to find enzyme activating additives that result in increased ester cleavage. Some surfactants are known as such activating agents (U.S. Pat. No. 3,884,764, DE-OS 24 09 696, DE-OS 25 12 605, DE-OS 25 12 585, 70044 2 DE-PS 2 506 712, Biochim Biophys Acta 270 (1972) pp. 156-166, for example bile acids, Triton X-100, Thesit).

The higher salinity of the medium increases its activating effect. However, not all buffer systems are equally suitable. The same is true for surfactants. In this case, under certain conditions, there are those which have a better or worse effect up to those which have no effect at all. It is difficult to know in advance what kind of surfactant is suitable in this respect.

Common to the activated surfactants is that they develop optimum activity in a given concentration range in the indicated medium. Optimal values may be in too high concentration ranges. Outside the optimum values, the higher added amount of relatively expensive enzyme does not react optimally at the high reaction rate.

In addition, an increase in activity is achieved by optimizing the surfactant concentration and increasing the ionic concentration only within relatively narrow limits.

As the reaction mixtures in question also regularly contain other enzymes and substrates for subsequent and indicator reactions, it is not possible to use free choice when selecting buffers and detergents and when optimizing the amounts added in each case.

Conversely, the interaction of all the substances involved in the reaction must be taken into account when determining the reaction conditions, in particular the possible inhibition of other enzymes or an adverse effect on stability due to surfactants and / or high ionic strength.

Furthermore, galenic solubility limits and problems must be taken into account when designing test systems.

In the latter case, surfactants may be added rarely to the waxes to activate the 3 '· * ·' '.....

70044 in additional quantities to mixtures of solids which must be dry and flowable. This is especially important when it comes to addition reagents.

Finally, it should be noted that in most cases the surfactants in question are not specific compounds but mixtures of compounds. As a result, there are differences in quality, which are also reflected in the activating effect. As a result, it can be very detrimental if the activation is solely dependent on the surfactant.

As a result, there is a considerable need to activate cholesterol esterase using other and better methods and agents.

It is an object of the present invention to obviate the above drawbacks and to provide such a method and means by which cholesterol esterase can be saved, a higher reaction rate can be achieved under certain conditions, a lower ionic strength can be used, surfactants need only be used at very low concentrations, and also surfactants which, as such, have only a poor activating effect or no activating effect at all.

This object is achieved according to the invention by a method for activating cholesterol esterase in ionic solutions by adding at least one surfactant based on a polyethoxy ether or ester and / or a bile acid compound and / or a salt of fatty acids having 6 to 10 carbon atoms, and the surface is characterized in that the active substance is added together with at least one synergistically active alcohol which is a straight-chain, branched or cyclic aliphatic alcohol having 5 to 12 carbon atoms, an optionally halogen-substituted aromatic alcohol or an aliphatic alcohol having 1 to 3 carbon atoms substituted by several halogen atoms.

70044 4

The invention is based on the surprising finding that said alcohols, which do not in themselves have any activating effect on cholesterol esterase, considerably synergistically enhance the known activating effect of certain surfactants, and when used in combination with non-activating surfactants per se, .

This means that such non-activated, commonly marketed surfactants can be used. Similarly, ionic surfactants which are hitherto unsuitable due to their defined molecular structure and solid consistency but which are hitherto unsuitable due to their inferior activating properties become useful. Furthermore, the enzyme can be saved, the maximum achievable reaction rate can be increased, the required ionic strength can be reduced and finally, due to the different possibilities of combination, the esterase activation system can be adapted to the conditions determined by the other components of the reaction mixture.

Examples of the present invention suitable polyetoksieettereistä include fatty alcohol polyglycol ether, such as polyoxyethylene, Sat-ryylieetteri and hydroksipolyetoksidodekaani, alkyyliaryylieet-ester and aryl alkyl ether, such as polyoksietyleeninonyylifenyy-ether and polyoxyethylene octylphenyl ether, fatty acid esters, such as polyoxyethylenesorbitan monolaurate and the like. Sappihappoyhdisteistä are preferred cholic acid, deoxycholic acid and their alkali metal salts. Of the fatty acid salts, alkali salts, most especially sodium salts, are preferred.

Typical examples of straight-chain, branched or cyclic aliphatic alcohols suitable for carrying out the invention are pentanol, tert-amyl alcohol, methylpentanol, hexanol, cyclohexanol, octanol, isooctanol, decanol and dodecanol. Examples of suitable aromatic alcohols are phenol and dihydroxynaphthol. Halogen-substituted phenols and naphthols such as mono-, di- and trichlorophenol are preferred. Among the halogen-substituted aliphatic alcohols, for example, dichloroethanol, trichloroethanol, dichloromethanol, 5,70044 trichloromethanol, trichloropropanol, tetrachloropropanol and the like can be used. Aromatic alcohols may also have one or two alkyl groups having 1 to 2 carbon atoms.

Which of these alcohols (hereinafter referred to as additives) is particularly suitable for use in a particular case can be readily determined by preliminary tests. Generally, conditions such as the quality and strength of the buffer system, the quality and concentration of the surfactant, and the shelf life and desired consistency of the reaction mixture will determine.

These conditions can be achieved by appropriate selection of additives and their use in appropriate amounts. In general, it has been found that concentrations of 1-300 mM additive / l give a good result.

The alcohols are preferably added in an amount between 0.01 and 5%, in the case of liquid alcohols in the case of volume% and in the case of solid alcohols in the case of weight%. It is highly preferred to use 0.05-3% amounts.

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

Surfactants are suitably added in an amount of from 0.05 to 5% by volume, i.e. by weight, in the case of solids. The most preferred range of use is 0.1-3%.

Another object of the invention is to provide a means for activating cholesterol esterase in an ionic solution containing at least one surfactant based on a polyethoxy ether or ester and / or a bile acid compound and / or a salt of fatty acids having 6 to 10 carbon atoms having characterized in that it contains a synergistically active alcohol which is a straight-chain, branched or cyclic aliphatic 6 7 CO 4 4 alcohol having 5 to 12 carbon atoms, an optionally halogen-substituted aromatic alcohol or a halogen-substituted aliphatic alcohol having 1 to 3 carbon atoms.

The invention is useful in connection with all measurements and reactions in which cholesterol esterase is used in the reaction mixture. Such reaction mixtures are well known to those skilled in the art and have been described when used for analytical purposes, e.g., H.U. In Bergmeyer's book "Methoden der enzymatischen Analyse",

Verlag Chemie Weinheim, Bergstrasse. A more detailed explanation of such reaction mixtures is therefore omitted in this context. The invention makes it possible to save the enzyme, to obtain a higher reaction rate under the conditions shown, to add a buffer with a lower ionic strength, to use surfactants at a lower concentration and also to use surfactants which have only insufficient or no activating effect. The invention is suitable for use in the treatment of cholesterol esterases derived from microorganisms or other sources such as the pancreas or liver.

The following examples further illustrate the invention with reference to the accompanying drawing. In this drawing:

Figure 1 is a diagrammatically used apparatus for determining cholesterol esterase activity, Figure 2 is a graphical indication of the extinction change in a unit of time used to indicate the rate of ester cleavage.

The following table shows the composition of the surfactants identified by the trademarks: 7 70044 "Brij 35" Polyoxyethylene lauryl ether "Genapol OX-100" Fatty alcohol polyglycol ether "Tergitol NPX" Polyoxyethylene nonyl phenyl ether (about 10.5 oxyethylene groups) "Hydroxypolyethoxydodecane" Triton X-100 "Polyethoxyoctylphenyl ether (9-10 oxyethylene groups)" Tween 20 "Polyoxyethylene sorbitan monolaurate.

To investigate the activating effect of the synergistic combination under study according to the invention on cholesterol esterase, the apparatus shown in Figure 1 was used.

From the annealing reaction vessel 1 (25 ° C), in which the cleavage of the ester takes place, 0.32 ml of the mixture per minute is continuously removed by means of a hose pump 2 via an outlet hose 3 of a defined cross-section. The same pump also continuously supplies 1.0 ml of indicator solution per minute from the storage vessel 5 via the second hose 4, after the pump 2 is connected to an air flow 6 with a constant rate of 0.6 ml air / minute, and thus sedimentation is carried out, and then combined with the above reaction mixture stream. The combined stream then passes through two glass tubes 7, providing mixing of the liquids and the necessary reaction time of the indicator reaction up to the end point. By removing a flow of 1.2 ml / minute immediately before the dish through the hose 8 vertically upwards, the air bubbles are again removed. The sedimented waste stream is passed to a photometric measurement (546 nm) in a flow-through dish (1 cm thick layer) and the measured values are recorded by means of a printer 10.

Due to the standard conditions, as far as the indicator reaction is concerned, the heights of the measurement signals and their course depend only on the events of the reaction vessel. If cleavage of the ester occurs, its kinetic occurrence will affect the growth of the measurement signal, which is recorded in the printer 10 as a positive growth. The steepness of growth is a measure of the rate of the reaction and also a measure of the cholesterol esterase activity in the reaction mixture.

70044 8

Due to the separate occurrence of ester cleavage and indicator reaction, they cannot interact. Of course, other indicator systems, such as oxygen electrodes, can also be used.

Indicator solution: 0.1 m potassium phosphate buffer, pH 7.0 1.5 mM / 1 4-amino-antipyrine 5 mM / 1 phenol 3% hydroxypolyethoxydodecane 8 U / ml peroxidase 1 U / ml cholesterol oxidase

The indicator solution first converts free cholesterol to cholestenone and H 2 O. The H 2 O formed is reacted with phenol and 4-amino-antipyrine to give a red dye, the absorption of which is measured photometrically. The concentration of the resulting dye is proportional to the amount of free cholesterol. The concentration of free cholesterol depends on the amount of cholesterol already present in the substrate as well as the amount of cholesterol released by the esterase.

Content of reaction vessel: 0.5 ml of substrate (reference serum 360 mg total cholesterol / dl)

Surfactant, depending on the test Additives, depending on the test Buffer, up to 5 ml.

Initially, 50 ul of esterase stock solution is used, followed by 0.003 U / ml of esterase. This amount was varied in the activity test.

After the start of the experiment and mixing, the pump hose 3 is immersed in the solution in the reaction vessel 1 for about 10 minutes while the pump 2 is running. Air and doubly distilled water are then drawn alternately from about 10 segments in each case to achieve better separation of the signals. The following configuration can then take place.

9 70044

The examples below show the saving of the enzyme (a) made possible by the invention, the achievement of higher reaction rates under certain conditions (b), the use of a buffer of lower ionic strength (c), the addition of surfactants at a lower concentration (d), and also the use of such surfactants. the use of active substances which, under certain conditions, have only an insufficient activating effect or no such effect at all.

a) Cholesterol esterase saving.

The potential for cholesterol esterase savings is presented as follows:

First, staggering of enzyme activity is performed without the presence of additives. Comparative measurements are then made by adding additives with a cholesterol esterase addition of 0.003 U / ml. Based on the relationship between the activity obtained and the resulting rate, the increase in activity achieved is then reported.

Example 1 0.1 M Tris / Tartaric Acid Buffer, pH 8.0 5% "Triton X-100"

Added activity Achieved rate 0 U / ml 0 extra / 10 min 0.0012 0.012 0.0030 0.032 0.0045 0.052 0.0060 0.058 0.0089 0.094 1.1 Addition 2% by volume of tert-amyl alcohol

Added activity 0.0030 U / ml

Achieved speed 0.046 ext / 10 min

The enzyme addition required to achieve this rate without additives is 0.0044 U / ml

Activity obtained 147% 70044 10 1.2 Addition 0.4% by volume 4-methylethylpentanoyl (2)

Added activity 0.003 U / ml

Achieved speed 0.065 ext / 10 min

The enzyme addition required to achieve this rate without additives is 0.0063 U / ml

Activity obtained 210% 1.3 Addition of 1% by volume of cyclohexanol

Added activity 0.0030 U / ml

Achieved speed 0.077 ext / 10 min

The enzyme addition required to achieve this rate without additives is 0.0075 U / ml

Activity achieved 250%

Example 2 0.1 M potassium phosphate buffer, pH 7.0 1.5% "Tergitol NPX"

Added activity Achieved rate 0.0012 U / ml 0.019 ext / 10 min 0.0030 U / ml 0.030 ext / 10 min 0.0060 U / ml 0.055 ext / 10 min 0.0090 U / ml 0.091 ext / min 10 min 0.0120 U / ml 0.120 ext / 10 min 2.1 Addition of 0.4% v / v 2,2,2-trichloroethanol

Added activity 0.003 U / ml

Achieved speed 0.046 ext / 10 min

The enzyme addition required to achieve this rate without additives is 0.0046 U / ml

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

Added activity 0.003 U / ml

Achieved speed 0.053 ext./10 min

The enzyme addition required to achieve this rate without additives is 0.0053 U / ml

Activity obtained 177% 11 70044 2.3 Addition 1.4% by volume 4-methylpentanol (2)

Added activity 0.003 U / ml

Achieved speed of 0.059 ext./lo min

The enzyme addition required to achieve this rate without additives is 0.0059 U / ml

Activity obtained 197% 2.4 Addition 2% by volume of cyclohexanol

Added activity 0.003 U / ml

Achieved speed of 0.114 ext./10 min

The enzyme addition required to achieve this rate without additives is 0.0114 U / ml

Achieved activity 380%

Example 3 0.3 M Potassium Phosphate Buffer, pH 7.0 5% "Genapol 0X-100" / 5% Na Caprylate

Added activity Achieved rate 0.0012 U / ml 0.01 ext / 10 min 0.0030 U / ml 0.023 ext / 10 min 0.0060 U / ml 0.038 ext / 10 min 0.0090 U / ml 0.055 ext ./10 min 0.0150 U / ml 0.093 ext / 10 min 0.0240 U / ml 0.138 ext / 10 min 3.1 Addition of 4 mM / 1 3,4-dichlorophenol (0.065% by weight)

Added activity 0.0030 U / ml

Achieved speed 0.058 ext./lo min

The enzyme addition required to achieve this rate without additives is 0.0093 U / ml

Activity obtained 310% 3.2 Addition of 10 mM / l (about 0.09% by weight) phenol

Added activity 0.003 U / ml

Achieved speed 0.064 ext / 10 min

The enzyme addition required to achieve this rate without additives is 0.0104 U / ml

Activity obtained 347% 70044 12 3.3 Addition 1% by volume 4-methylpentanol (2)

Added activity 0.003 U / ml

Achieved speed of 0.069 ext / 10 min

Necessary enzyme addition to achieve this rate without additives 0.0112 U / ml

Activity obtained 373% 3.4 Addition of 2% by volume of cyclohexanol

Added activity 0.003 U / ml

Achieved speed of 0.080 ext./10 min

The enzyme addition required to achieve this rate without additives is 0.0129 U / ml

Activity obtained 430% 3.5 Addition 0.3% by volume 2,2,2-trichloroethanol

Added activity 0.003 U / ml

Achieved speed of 0.089 ext./10 min

Necessary enzyme addition to achieve this rate without additives 0.0144 U / ml

Activity obtained 480% b) Achieving a higher reaction rate under the conditions shown.

In many cases, certain conditions, such as buffer and ionic strength and the quality of the surfactant in the reaction mixture, are predetermined for the reasons set forth above. Such reasons may be, for example, the higher sensitivity of another enzyme still required in the mixture. In these cases, when using standard cholesterol esterase addition, the maximum possible rate of ester cleavage is limited due to the dependence of esterase activation on the quality of the surfactant and its concentration. This dependence usually has an optimum value in the lower concentration range of the surfactant. The speed achieved at this stage cannot be exceeded under the above conditions and sets limits for the system in this regard.

13 70044

The compounds of the invention described in the following examples are intended to exceed these limits.

Example 4

Mixture ingredients: 0.1 M Tris / tartaric acid buffer, pH 8.0 "Triton X-100" 0.003 U / ml cholesterol esterase% "Triton X-100" in the mixture Achieved rate 0% 0 extra / 10 min 1% 0.003 extra ./10 min 3% 0.028 ext / 10 min 5% 0.037 ext / 10 min 7% 0.037 ext / 10 min 10% 0.037 ext / 10 min 15% 0.029 ext / 10 min

Optimi System limits 5% 0.037 ext./10 min

Additives are added at 5% "Triton X-100".

4.1 Addition of 2% by volume of tert-amyl alcohol

Achieved speed of 0.046 ext / 10 min

Increase to 124% 4.2 Addition 0.4% by volume 4-methylpentanol (2)

Achieved speed 0.065 ext / 10 min

Increase to 176% 4.3 Addition of 1% v / v cyclohexanol

Achieved speed of 0.077 ext./10 min

208% increase in value

Example 5 Mixture used: 0.3 M potassium phosphate buffer, pH 7.0 "Tergitol NPX" 0.003 U / ml cholesterol esterase 700 4 4% "Tergitol NPX" in the mixture Achieved rate O% O extra / min min 2% O extra / 10 min 5% 0.029 ext / min min 10% 0.042 ext / min 15% 0.049 ext / 10 min 20% 0.039 ext / 10 min

Optimi System limits 15% 0.049 ext./10 min

Additives are added using 15% "Tergitol NPX" 5.1 Addition with 2 mM / 1 3,4-dichlorophenol and 1% v / v cyclohexanol

Achieved speed 0.058 ext./10 min

Increase to 118% 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 / 10 min

Increase to 241% c) Use of a buffer with a lower ionic strength As already mentioned, a buffer with a higher ionic strength increases the activating effect of surfactants on cholesterol esterase. However, within the range of possible free conditions used, it is desirable that the solution does not contain excessive levels of salt. The following example shows that according to the invention the salt contents can be significantly reduced.

Comparison: 0.3 M potassium phosphate buffer pH 7.0 0.1 M potassium phosphate buffer pH 7.0 70044 15

By adding suitable additives to the 0.1 m buffer system, the speeds can be allowed to increase so that they reach and in some cases clearly exceed the higher reference value of the 0.3 M buffer system without additives.

Example 6 15% "Tergitol NPX" 0.003 U / ml cholesterol esterase 6.1 Addition 2% v / v tert-amyl alcohol System Speed 0.1 m buffer without addition 0.030 ext / 10 min 0.3 m buffer without addition 0.049 ext / 10 min 0.1 m buffer with addition 0.053 ext./10 min

The reference value is exceeded.

6.2 Addition 1.4% by volume 4-methylpentanol (2) 0.1 m buffer without addition 0.030 ext / min min 0.3 m buffer without addition 0.049 ext / 10 min 0.1 m buffer with addition 0.059 ext ./10 min

The reference value is exceeded.

6.3 Addition 2% v / v cyclohexanol 0.1 m buffer without addition 0.030 ext / 10 min 0.3 m buffer without addition 0.049 ext / 10 min 0.1 m buffer with addition 0.114 ext / 10 min

The reference value is clearly exceeded.

Example 7 10% "Genapol 0X-100" / 10% Na-caprylate 0.003 U / ml cholesterol esterase 7.1 Addition 40 mM (about 0.35% by weight) phenol 70044 System Speed 0.1 m buffer without addition 0.017 ext./10 min 0.3 m buffer without addition 0.053 ext / 10 min 0.1 m buffer with addition 0.053 ext / 10 min

The reference value is reached.

7.2 Addition 1% v / v 2,2,2-trichloroethanol 0.1 m buffer without addition 0.017 extra / 10 min 0.3 m buffer without addition 0.053 extra / 10 min 0.1 m buffer with addition 0.063 extra / 10 min

Benchmarks are exceeded.

7.3 Addition 2% v / v cyclohexanol 0.1 m buffer without addition 0.017 extra / 10 min 0.3 m buffer without addition 0.053 extra / 10 min 0.1 m buffer with addition 0.072 extra / 10 min

The reference value is exceeded.

(d) Use of surfactants at lower concentrations If the surfactant is capable of activating cholesterol esterase, it must be present in the reaction mixture in order to take full advantage of this property at the specified concentration. This optimum of surfactant depends considerably on the quality of the surfactant and can in many cases be in an unfavorably high concentration range. As a result, other enzymes required for further reactions can cause a barrier reaction and difficulties can also arise due to solubility and galenic phenomena. Below the detergent optimum, only unsatisfactory activation can be achieved. However, for the widest possible choice of conditions, it is desirable to be able to use as many different surfactants as possible, including those with a high optimum concentration.

The example below shows that according to the invention it is possible to achieve and even exceed the optimum concentration rate by using a lower amount of surfactant, whereby the amount of surfactant can be saved.

Example 8 0.3 m potassium phosphate buffer, pH 7.0 "Tergitol NPX" 0.003 U / ml cholesterol esterase 17 70044% in "Tergitol NPX" mixture Achieved rate O% 0 extra / 10 min 2% O extra / 10 min 5% 0.029 ext / 10 min 10% 0.042 ext / 10 min 15% 0.049 ext / 10 min 20% 0.039 ext / 10 min

Optimum Reference value 15% 0.049 ext./10 min 8.1 Addition 0.1% v / v 2,2,2-trichloroethanol System Speed 15% "Tergitol" without addition 0.049 ext./10 min 5% "Tergitol" without addition 0.029 ext. / 10 min with the addition of 5% "Tergitol" 0.059 ext./10 min

The reference value is exceeded.

8.2 Addition 2% v / v tert-amyl alcohol 15% "Tergitol" without addition 0.049 extra / 10 min 5% "Tergitol" without addition 0.029 extra / 10 min 5% "Tergitol" with addition 0.075 extra / 10 min

The reference value is clearly exceeded.

8.3 Addition of 1% v / v cyclohexanol System Speed 15% "Tergitol" without addition 0.049 extra / 10 min 5% "Tergitol" without addition 0.029 extra / 10 min 5% "Tergitol" with addition 0.124 extra / 10 min

The reference value is clearly exceeded.

70044 18

Example 9 0.3 m potassium phosphate buffer, pH 7.0 "Genapol 0X-100" / sodium caprylate 0.003 U / ml cholesterol esterase% "Genapol OX-100" / Na caprylate Rate 0% 0 extra / 10 min 5% 0.020 extra / 10 min 10% 0.053 extra / 10 then 15% 0.062 extra / 10 min 20% 0.066 extra / 10 min 30% 0.068 extra / 10 min 40% 0.068 extra / 10 min

Optimum Reference value 30% 0.068 ext./10 min 9.1 Addition 1% v / v 4-methylpentanol- (2) System Speed 30% "Genapol" / caprylate without addition 0.068 ext./10 min 5% "Genapol" / caprylate without addition 0.020 extra / 10 min 5% "Genapol" / caprylate with addition 0.069 extra / 10 min

The reference value is reached.

9.2 Addition 2% v / v cyclohexanol System Nooeus 30% "Genapol" / caprylate without addition 0.068 extra / 10 min 5% "Genapol" / caprylate without addition 0.020 extra / 10 min 5% "Genapol" / caprylate with addition 0.080 extra ./10 min

The reference value is exceeded.

9.3 Addition 0.3% v / v 2,2,2-trichloroethanol System Speed 30% "Genapol" / caprylate without addition 0.068 ext./10 min 5% "Genapol" / caprylate without addition 0.020 ext./10 min 5% " Genapol "/ caprylate with addition of 0.089 ext./10 min 19 70044

The reference value is clearly exceeded.

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

Of the several possible surfactants, only a few are capable of activating cholesterol esterase in sufficient amounts. The inhibition of the reaction of the other substances involved in the reaction, the stability, consistency, solubility and uniformity of the preparation of the reaction mixture, support the above reasons that it is desirable to be able to select the surfactants as freely as possible. The example below shows that surfactants which, under the prevailing conditions, have little or no activating effect, are also useful according to the invention.

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

Surfactant 6% "Thesit"

Addition 1% v / v n-amyl alcohol System Rate

Surfactant without addition 0.003 ext / 10 min Add 0 ext / 10 min

Surfactant with addition 0.029 ext./10 min

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

Surfactant 6% "Thesit"

Addition of 10 mM / 1 (about 0.16% by weight) 1,7-dihydroxynaphthalene System Speed

Surfactant without addition 0.003 ext./10 min Addition 0.005 ext./10 min

Surfactant with addition 0.024 ext./10 min 20

Example 12 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase 700 44

Surfactant: 12% "Thesit"

Addition of 2% v / v cyclohexanol System Speed

Surfactant without addition 0.010 ext./10 min

Addition 0.003 ext / 10 min

Surfactant with addition 0.105 ext./l° min

Example 13 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Surfactants: 12% "Thesit"

Addition: 1% v / v 2,2,2-trichloroethanol System Speed

Surfactant without addition 0.010 ext./10 min

Addition O ext./10 min

Surfactant with addition 0.081 ext./10 min

Example 14 0.1 m Tris / tartaric acid buffer, pH 8.0 0.003 U / ml cholesterol esterase

Surfactants: 2% v / v "Thesit" / 2% v / v "Tween 20"

Addition: 0.4% v / v hexanol-1 System Rate

Surfactant without addition 0.018 ext / 10 min

Addition 0 ext./10 min

Surfactant with addition 0.028 ext./10 min

Example 15 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase 21 70044

Surfactants: 5% v / v "Thesit" / 5% v / v "Tween 20"

Addition: 8 mM / 1 3,5-dichlorophenol System Rate

Surfactant without addition 0.005 extra / 10 min

Addition O extra / 10 min

Surfactant with addition 0.033 ext./2.C> min

Example 16 0.3 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Surfactants: 5% by volume "Genapol 0X-100" / 5% by weight Na-caprylate

Addition: 10 mM / 1 phenol System Rate

Surfactant without addition 0.020 ext./10 min

Addition 0 ext./10 min

Surfactant with addition 0.064 ext / 10 min

Example 17 0.3 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Surfactants: 5% by volume "Genapol 0X-100" / 5% by weight Na-caprylate

Addition: 4 mM / l (about 0.065% by weight) 3,4-dichlorophenol System Speed

Surfactant without addition 0.020 ext./10 min

Addition 0 extra / 10 min

Surfactant with addition 0.058 ext./10 min

Example 18 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase 22 70044

Surfactant: 10% v / v "Brij 35" (30% solution) Addition: 4 mM / 1 3,4-dichlorophenol System Speed

Surfactant without addition O ext./10 min

Addition 0 ext./10 min

Surfactant with addition 0.010 ext./10 min

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

Surfactant: 10% v / v "Brij 35" (30% solution) Addition: 1% v / v 2,2,2-trichloroethanol System Speed

Surfactant without addition 0 ext / 10 min

Addition O ext./10 min

Surfactant with addition 0.018 ext./10 min

Example 20 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Surfactant: 2% v / v "Brij 35" (30% solution) Addition: 2% v / v cyclohexanol System Speed

Surfactant without addition 0 ext / 10 min Addition 0.003 ext / 10 min

Surfactant with addition 0.027 ext./10 min

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

Surfactant: 6% by weight of cholic acid

Addition: 1% v / v 2,2,2-trichloroethanol 23 7 0 0 4 4 System Speed

Surfactant without addition O ext./10 min

Addition O ext./10 min

Surfactant with addition 0.029 ext./10 min

Example 22 0.1 m phosphate buffer α, pH 7.0 0.003 U / ml cholesterol esterase

Surfactant: 6% by weight of cholic acid

Addition: 16 mM / 1 3,5-dichlorophenol System Speed

Surfactant without addition 0 ext / 10 min

Addition 0 extra / 10 min

Surfactant with addition 0.036 ext./10 min

Example 23 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Surfactant: 1% by weight of cholic acid

Addition: 4 mM / 1 3,5-dichlorophenol, 1% v / v cyclohexanol System Speed

Surfactant without addition 0 ext / 10 min

Addition 0.010 ext./10 min

Surfactant with addition 0.103 ext./10 min

Example 24 0.1 m Tris / tartaric acid buffer, pH 8.0 0.003 U / ml cholesterol esterase

Surfactant: 3% by weight of sodium deoxycholate Addition: 0.2% by volume of hexanol-1 70044 24 System Speed

Surfactant without addition O ext./10 min

Addition O ext./10 min

Surfactant with addition 0.056 ext./10 min

Example 25 0.1 m Tris / tartaric acid buffer, pH 8.0 0.003 U / ml cholesterol esterase

Surfactant: 3% by weight of sodium deoxycholate Addition: 1% by volume of 2,2,2-trichloroethanol System Speed

Surfactant without addition 0 ext / 10 min

Addition 0 ext./10 min

Surfactant with addition 0.069 ext./10 min

Example 26 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Surfactant: 6% by volume "Genapol 0X-100"

Addition: 0.4% v / v 2,2,2-trichloroethanol System Speed

Surfactant without addition of 0 extra / l so

Addition 0 ext./10 min

Surfactant with addition 0.059 ext./10 min

Example 27 0.1 m phosphate buffer, pH 7.0 0.003 U / ml cholesterol esterase

Surfactant: 20% by volume "Tween 20"

Addition: 1% v / v 2,2,2-trichloroethanol 70044 25 System Speed

Surfactant without addition 0.005 ext./10 min

Addition O ext./10 min

Surfactant with addition 0.067 ext./10 min Application Example 28

Reagent for the determination of serum cholesterol

Composition: 4.5 g / l tartaric acid 7.5 g / l tris 8.7 g / l sodium sulphate 200 mg / ml 4-amino-antipyrine 282 mg / l phenol 5000 U / l peroxidase 430 U / l cholesterol esterase 310 U / l l cholesterol oxidase

Activating agent: 2.2 g / l "Thesit" 1.3 g / l sodium deoxycholate 0.815 g / l 3,4-dichlorophenol

The above choice of additives allows the activation of cholesterol esterase under conditions provided by the properties of other enzymes. At the same time, it is possible to form the storage form of the reaction mixture into a solid addition reagent.

Claims (10)

26 7 0 0 4 4 A process for activating cholesterol esterase in an ionic solution by adding at least one surfactant based on a polyethoxy ether or ester and / or a bile acid compound and / or a salt of fatty acids having 6 to 10 carbon atoms, characterized in that the surfactant is added together with at least one synergistically active alcohol which is a straight-chain, branched or cyclic aliphatic alcohol having 5 to 12 carbon atoms, an optionally halogen-substituted aromatic alcohol, or an aliphatic alcohol having 1 to 3 carbon atoms substituted by several halogen atoms. Process according to Claim 1, characterized in that cholic acid, deoxycholic acid or an alkali salt thereof is used as the bile acid compound. Process according to Claim 1, characterized in that an alkali salt is used as the fatty acid salt. Process according to one of the preceding claims, characterized in that 0.05 to 5% of surfactant is used. Process according to one of the preceding claims, characterized in that 0.01 to 5% of alcohol is used. A device for activating cholesterol esterase in an ionic solution containing at least one surfactant based on a polyethoxy ether or ester and / or a bile acid compound and / or a salt of fatty acids having 6 to 10 carbon atoms, characterized in that it contains at least one synergistically active alcohol which is a straight-chain branched or cyclic aliphatic alcohol having 5 to 12 carbon atoms, an optionally halogen-substituted aromatic alcohol or a halogen-substituted aliphatic alcohol having 1 to 3 carbon atoms. 27 70044 Device according to Claim 6, characterized in that it contains a surfactant based on 0.05 to 5% of the enzyme solution. Device according to Claim 7, characterized in that it contains 0.1 to 3% of surfactant. Device according to Claims 6 to 8, characterized in that it contains 0.01 to 5% of alcohol. Device according to Claim 9, characterized in that it contains 0.05 to 3% of alcohol.