IL46073A - Process for the activation of cholesterol oxidase and diagnostic agent for detection and determination of cholesterol - Google Patents

Description

«11 «ηη fHm» jns»3 tittnau onsi tawps non ia n¾mn¾> γ*&!% PROCESS FOR THE ACTIVATION OP CHOLESTEROL OXIDASE AMD DIAGHOSTIC AGEST FOR CASRYHTG OUT SAID PROCESS The present invention is concerned with a process ' for the activation of cholesterol oxidase and with a diagnostic agent for carrying out this process.

Israel 42289 G ormaii Patent Specification ¥10.2~, 224 , 02 describes a process for the determination of cholesterol in which cholesterol is incubated in an aqueous medium with cholesterol oxidase, followed by the determination either of the oxygen consumption or of the amount of hydrogen Israel peroxide or of cholestenone formed. This German Patent Specification also describes a reagent for the determination Of cholesterol which comprises cholesterol oxidase and a system for the determination of hydrogen peroxide or a system for the determination of cholestenone.

We have now found that cholesterol oxidase has an insufficient storage stability and is inactivated relatively quickly. In the course of the investigation of this instability, we have found that the inactivation is brought about by small amounts of detergent which are present as an accompanying substance and originate from the process used for the preparation of the enzyme. This preparation of the enzyme can be carried out, for example, by the process Israel 42292 described in frermtm Patent Specification No .-£-r¾4r+5+- in. which a micro-organism which metabolises cholesterol is digested by destruction of the cell walls with a non-ionic, surface-active agent present in a buffer solution and then extracted, whereafter the extract is centrifuged, the precipitate obtained is discarded and the supernatant liquid is applied to an anion exchanger, the enzyme then being eluted with a buffer solution containing the non-ionic surface-active agent and thereafter isolated from We have also found that, upon removal of these residual traces of detergent, a completely satisfactory storage stability of the enzyme can be achieved. However, we have also ascertained that the enzyme freed from detergent has a remarkably reduced activity. If, for example, the enzyme is used for the determination of cholesterol, then it is necessary either to use a comparatively freshly prepared preparation or considerably larger amounts of enzyme must be employed for the test or the time required for carrying out the determination must be considerably increased.

Consequently, it is an object of the present invention to provide a process for the activation of cholesterol oxidase. More particularl , it is an object of the present invention to provide such a process which, on the one hand, permits the use of cholesterol oxidase which is completely free from traces of detergent and, therefore, is sufficiently storage-stable and, on the other hand, provides an activity of the enzyme during use thereof which corresponds to that of the freshly prepared enzyme but which has not been subjected to the removal of small amounts of detergents.

Thus, according to the present invention, there is provided a process for the activation of cholesterol oxidase, wherein at least one surface-active compound with lipophilic and hydrophilic properties is added to the cholesterol oxidase before the use thereof.

As surface-active compounds with lipophilic and hydrophilic properties, there are preferably used non-ionic detergents which contain at least one liydroxyl group in the molecule. Polyox ethylene derivatives of alkyl, aryl and aralkyl alcohols are preferably employed. Examples of such preferred surface-active compounds include polyoxy-ethylene alkyl ethers, polyoxyethylene alkyl-carboxylic acid esters, polyoxyethylene-sorbitan alkyl carboxylic acid esters, polyoxyethylene-glycerol alkyl carboxylic acid esters, polyoxyethylene-alkylamines, polyoxyethylene-polyoxypropylene block polyraers, polyoxyethylene-alkyl thio-ethers and polyoxyethylene alkyl aryl ethers. Specific examples thereof include hydroxy-polyethoxydodecane, ethyleneoxy adducts of alkyl-phenols , polyethoxy-ethylene derivatives of sorbitan anhydrides and the like. Polyoxyethylene derivatives modified with mercaptans are also equally suitable.

According to the present invention, the above-mentioned surface-active compounds are preferably used in an amount of between about 0.005 and 0.1 wt.?', referred to the aqueous enzyme solution.

Considerable numbers of the above-mentioned surface-active materials are commercially available and differ from one another by the number of oxyethylene groups present therein. The most useful ones are those which are water-soluble and which contain, on average, 5 to 20 oxyethylene groups per molecule. Lower oxyethylated types of compounds are usually only dispersible and are, therefore, less useful. Higher ethoxylated compounds (greater than 25) are admittedly water-soluble but are so hydrophilic that their effectiveness is reduced.

Similarly good results are also obtained with physiological surface-active substances which satisfy the above-given definition, for example, desoxycholates . fl^ In addition, other surface-active substances with lipophilic and hydrophilic properties which contain at least one hydroxyl group, can also be used. Examples of such compounds include ethanol, butyl-diglycol and hexylene-glycol. However, lower mono- and dialcohols of this type must be added in relatively large amounts of 10 to 20 vol. for the achievement of the desired activation and the suitability thereof must be ascertained experimentally in each case. Thus, for example, no activation is achieved with methanol, ethylene glycol, polyethylene glycol, cyclohexanol, glycerol, lecithin and saponin. Surface-active compounds vrtiich have lipophilic and hydrophilic properties but which do not contain a hydroxyl group in the molecule can also be used.

Of the anionic wetting agents, the salts of bile acids, such as cholic acid, taurocholic acid and desoxy-cholic acid, are especially preferred, as well as fatty acid salts and fatty acid sarcosides. Of especial importance are the sulphuric acid derivatives which, as is known, have the additional property of stabilising the coloured radical oxidation products of some indicators, such as o-tolidine and heterocyclic aaines. These include, for example, the following classes of compounds: sulphosuccinic acid esters, alkyl aryl sulphonates, allcyl sulphates and alkyl polyoxy-ethylene sulphates.

Of the cationic wetting agents, there can be used, for example, alkyl pyridinium and trimethyl ammonium salts, as well as more complex compounds, for example, benzethonium chloride .

Of the amphoteric wetting agents, there can be used, | for example, the imidazolium betaines.

Particular examples of surface-active compounds which can be used according to the present invention include sodium (2-ethyl-hexyl)-sulphosuccinate, sodium dodecyl sulphate, sodium oleate, benzalkonium chloride and cetyl-pyridinium chloride.

The ionic or amphoteric detergents are preferably used in admixture with the above-mentioned non-ionic detergents, the appropriate amounts thereof corresponding to those of the non-ionic detergent.

The non-ionic, surface-active detergents containing at least one hydroxyl group in the molecule are preferred because they provide an approximately 5 to 10 times greater activation and thus a corresponding increase of the reaction velocity or shortening of the period of reaction in comparison with the other surface-active substances which can be employed. They are also effective in smaller amounts.

By alkyl radicals, there are here to be understood those containing up to about 20 carbon atoms and especially those containing 12 to 18 carbon atoms.

The inactivation of cholesterol oxidase in the presence of traces of detergent is especiall marked when the enzyme is present in ammonium sulphate solution. The following Table I shows the storage stability of the enzyme in 1H ammonium sulphate solution at 33°C. in "the presence of differing amounts of detergent. The experiments were carried out with an octyl-phenol-ethylene oxide adduct.

T A B L 1 Activity of cholesterol _oxidas_e in referred to freshly prepared enzyme The effective amount of surface-active compound used in the process according to the present invention depends upon its molecular weight and upon the degree of its hydrophobic and hydrophilic properties. Too snail an amount does not bring about an activation but too large an amount leads to the result generally known for surface- active substances of denaturing the enzyme. The appropriate concentration range, as well as the optimum concentration, can be experimentally determined for all appropriate surface-active agents. Such ranges are, for example, 0.15 to 0.6 wt.$ for hydroxypolyethoxydodecane , 0.05 to 0.1 wt.?o for octylphenol-ethylene oxide adducts, 0.15 to 0.6 wt.?S for polyoxyethylene derivatives of sorbitan anhydrides, 0.03 to 0.05 wt.jS for sodium desoxycholate and 0.005 to 0.02 wt.jS for mercaptan-modified alkyl-phenol ethylene oxide adducts (for example Sterox SE) .

Of the non-ionic surface-active polyoxy-ethylene compounds, those are especially useful which have a balance ratio of hydrophobic residues to polyoxyethylene chain and are water-soluble. A measurement value for the ratio of hydrophobic residue and polyoxyethylene chain is the so- called HLB value (cf. W.C. Griffin, J. Soc. Cosmetic. Ghem.^ 1, 311/1950 and 249/1954).

Those surface-active materials with HLB values between about 10 and 17 are especially useful. These values are, however, only to be regarded as being optimum standard values since the effectiveness also depends upon the nature of the hydrophobic residue.

The present invention is also concerned with a diagnostic agent for the detection and determination of cholesterol and of cholesterol esters in body fluids which can be used for carrying out the above-described process.

The determination of cholesterol is of considerable importance in medical diagnosis.

In clinical chemistry, rapid tests are being used more and more for the detection of substances in body fluids. Although they frequently do not provide sufficiently accurate results, nevertheless, they do permit a quicker and cheaper indication for routine and large-scale investigations. The diagnostic agents employed for rapid tests are either absorbent carriers or water-stable films which contain all of the reagents needed for the reaction. When these diagnostic agents are brought into contact with the body fluids to be tested, then colour reactions are obtained which can be evaluated either on the basis of comparative colours or with the use of simple reflection photometers.

Experiments for the prodiction of agents for cholesterol rapid tests by impregnation of absorbent paper with cholesterol oxidase, peroxidase, an oxidation indicator and a buffer do not give satisfactory results because the test papers thus obtained do not react with cholesterol- containing serum of the usual concentration.

We have now found that useful test papers or test films are obtained, which react in graduated stages with cholesterol-containing serum when the carrier additionally contains at least one of the above-described surface-active agents .

Thus, according to the present invention, there is also provided a diagnostic agent for the detection and determination of cholesterol and of cholesterol esters in body fluids which comprises a carrier or a synthetic resin film which is impregnated with or has embedded therein cholesterol oxidase, a system for the detection of hydrogen peroxide, a buffer and at least one of the above-described surface-active agents. The surface-active agent is preferably present therein in a concentration of 2 to 30$¾ and more preferably of 10 to 20S, referred to the solid reagents.

A preferred system for the detection of hydrogen peroxide comprises peroxidase and an oxidation indicator, optionally together with a swelling agent and/or a stabiliser.

For the detection of cholesterol in serum, test papers are outstandingly useful which have been obtained by the impregnation of absorbent papers with the necessary reagents. If, however, it is desired to detect cholesterol in whole blood, then the test papers are preferably rendered hydrophobic, for e ample, in the manner described in German Patent Specification No.1,598,048 or are coated with a semi-permeable membrane of cellulose esters. However, for the detection of cholesterol in whole blood, it is espec- ^ ially preferred to use test films, such as are obtainable according to German Patent Specification ITo .1 , 598, 153 , containing the necessary reagents.

For the preparation of such test films, the reagents, for example, cholesterol oxidase, peroxidase, buffer, indicator and optionally a swelling agent, are stirred, together with the surface-active materials to be used according to the present invention, into an aqueous dispersion of a film-forming polymer. This dispersion is then coated as a thin film and left to dry. When cholesterol-containing blood is dropped on to such a reagent-containing film and then wiped off after about one minute, colorations are also obtained, the colour depth of which depends upon the amount of cholesterol present in the blood. These colorations are particularly suitable for a quantitative evaluation with simple remission photometers.

The diagnostic agent according to the present invention can be used for the detection and determination of free cholesterol. If, however, cholesterol esters are also present, then the cholesterol must first be liberated therefrom. This can be carried out in known manner, for example, by saponification with aqueous alkali. However, it is particularly advantageous to split the ester with cholesterol esterase, which is preferably isolated from micro-organisms, since it is then possible to work under very mild conditions.

The cholesterol esterase can be added to the body fluid, followed by incubation. This process can be carried carried out, for example, by drawing the body fluid into capillaries, the inner walls of which are coated, in a manner analogous to that described in German Patent Specification Wo .2 , 240 , 672 , with cholesterol esterase and optionally with adjuvant materials. After incubation in the capillaries, the body fluid is then applied to the test strips in the above-de cribed manner. If free and esterified cholesterol were both present in the body fluid, then, of course, the sum of the two is detected. However, the cholesterol esterase can also be incorporated especially advantageously into the test strips to give diagnostic agents with which, in one step, the sum of the free and esterified cholesterol can be detected and determined.

Systems which can be used for the detection of the hydrogen peroxide formed by the oxidation of the cholesterol, for example peroxidase, buffer, oxidation indicator and optionally a swelling agent and the like, are known, for example, from the descriptions of rapid tests for glucose. Examples of components for this preferred system are given in the following: Of the peroxidases, that from horseradish is especially preferred but lactoperoxidase and the like can also be employed.

As buffers, there can be used those which are conventional, for example, phosphate, citrate and borate buffers. The pH value which they give on the diagnostic agent should be between 4 and 9 and preferably between 5 and 8.

As oxidation indicators, there can be used various classes of compounds, namely, benzidene derivatives, for example o-tolidine and o-vanillin derivatives according to^ German Patent Specification No.1 ,598 , 133 , or heterocyclic azines according to German Patent Specification ilo.1 ,648,840.

The formulations for the diagnostic agents and especially those for test films, can also contain a conventional swelling agent, such as sodium alginate, carboxy-methyl-cellulose or the like, as well as a stabilising agent for the enzyme, for example dithioerythritol.

Substrate materials for test films are synthetic resin dispersions of, for example, polyvinyl propionate or acetate. Into these are stirred all the necessary reagents, preferably in dissolved form, whereafter the mixture obtained is then coated into thin films and dried.

Test papers can be produced by dissolving the reagents in water or in a mixture of water and organic solvents with which filter papers are impregnated and then dried. However, the paper can first be impregnated with the water-soluble reagents and then impregnated with, for example, the indicators in organic solution.

The following Examples are given for the purpose of illustrating the present invention: -Example 1. 0.05 ml. of serum are added to 10 ml. 0.5M potassium phosphate buffer of pH 7.5 whic contains 0.4^ hydroxy-polyethoxydodecane. The extinction ( ^ ) is read off at 240 mu in a suitable spectrophotome er and the reaction started with 0.02 ml. (0.1 U) of storage-stable cholesterol oxidase, freed from traces of detergent, in 1M aqueous ammonium sulphate solution. After 3 minutes, the extinction (E2) is read off. The concentration of the cholestenone formed and thus of the cholesterol is given by the difference between the first and second readings, having regard to the molar extinction coefficients for cholesterol at 240 nm. The measurement of a typical sample gave 62 mg. '/ free cholesterol and 167 mg.'ό total cholesterol (after saponification) .

A comparative determination but without the addition of the surface-active agent reqtiired a reaction time of 15 minutes.

The separation of traces of detergent for the improvement of the storage stability of the enzyme is preferably carried out with the use of hydrophobic adsorption resins. Especially preferred for this purpose are, for example, the material which is commercially available as "Bio-Beads" from Biorad, as well as the products obtainable from Rohm & Haas under the designation XAJD-resins.

Example 2. 0.05 ml. serum are added to 10 ml. 0.5H potassium phosphate buffer of pli 7.5 which contains 0.02^ hydroxy-polyethoxy-dodecane and 0.035^ sodium desoxycholate . The extinction ( ^ ) is read off at 240 nm in a suitable spectro photometer and the reaction is started with 0.02 ml. (= 0.1 U) storage-stable cholesterol oxidase, freed from traces of detergent, in 1H aqueous ammonium sulphate solution. ... .

After 3 minutes, the extinction (E2) is again read off. The concentration of the cholestenone fonaed and thus of the cholesterol is given from the difference between the first and second readings, having regard to the molar extinction coefficients for cholestenone at 240 nm (£ = 15.5 cm // iaol) .

The measurement of a typical sample gave 65 mg.j'-free cholesterol and 170 mg.?S total cholesterol (after saponification) .

Example 3 ♦ 0.05 ml. serum are added to 10 ml. 0.5M potassium phosphate buffer of pH 7.5 which contains 0.02^ hydroxy-polyethoxy-dodecane and 0. ?J secondary alkyl sulphate.

The extinction (E^ ) is measured at 240 nm in a suitable spectrophotometer and the reaction is started with 0.02 ml. (= 0.1 U) storage-stable cholesterol oxidase, freed from traces of detergent, in 1LI aqueous ammonium sulphate solution.

After 3 minutes, the extinction (E2) is again read off. The concentration of the cholestenone formed and thus of the cholesterol is give by the difference between the first and second readings, having regard to the molar extinction coefficients of cholestenone at 240 nm.

The measurement of a typical sample gave 62 mg.% free cholesterol and 167 mg.$ total cholesterol (after saponification) .

Example 4.

Filter paper (Schleicher & Schull No. 97 Ϊ ind.) is impregnated with a solution of the following composition and dried at 40°C: 1 M citrate buffer, pH 5.25 20 ml. cholesterol oxidase (60 U/mg.) 0.1 g. peroxidase (70 U/mg.) 0.05 g. .distilled water ad 100 ml.

This paper is then impregnated with solutions of 0.2 g. o-tolidine in 100 ml. methylene chloride which, in addition, each contain 1 g. of the following surface-active materials: a) polyoxyethylene tributyl-phenol ether b) polyoxyethylene sorbitan monolaurate c) polyoxyethylene-nonyl-phenol ether d) polyoxyethylene lauryl ether e) polyoxyethylene cetyl ether f) polyoxyethylene 3tearate g) polyoxyethylene dodecyl thioether.

After drying, test papers are obtained which react with cholesterol-containing sera with a green colour. If the sera also contain cholesterol esters, then stronger green colorations are obtained if the sera have previously been mixed with a drop of cholesterol esterase solution. A test paper which does not contain one of the above-mentioned surface-active materials does not react with the sera.

E ample 5 « Filter paper (Schleicher & Schill Ko.597 Ki1 Ind.) is impregnated with a solution of the following composition and dried at 40°C: 1 M citrate buffer, pH 7 20 ml. cholesterol oxidase (60 U/mg.) 0.1 g. cholesterol esterase (18 U/mg.) 0.25 g. peroxidase (70 U/mg.) 0.05 g. distilled water 100 ml.

This paper is impregnated with solutions of 0.2 g. o-tolidine in 100 ml. methylene chloride which, in addition, each contain 0.5 g. of one of the following surface-active materials: a) polyoxyethylene cocoate b) polyoxyethylene oleate c) polyoxyethylene polypropylene glycol d) polyoxyethylene stearylamine e) polyoxyethylene glycerol monolaurate.

After drying, test papers are obtained which react with a green colour with those sera which contain cholesterol cholesterol and/or g'holoo orol esters. Test papers without surface-active agent show no reaction.

Practically the same behaviour is shorn by test papers which contain the same amount of citrate buffer with a pH of 5.25 or 6.

Example 6.

Paper pre-impregnated in the manner described in Example 4 is subsequently impregnated with solutions of 0·3 g* o-tolidine in 100 ml. acetone which contain 1.5 g. of one of the following surface-active materials: a) dioctyl sodium sulphosuccinate b) sodiurn dodecyl-benzene-sulphonate c) sodium lauryl pol gl col ether sulphate d) sodium lairyl sarcosinate e) sodium laurate f) cholic acid s) desoxycholic acid h) sodium taurocholate These test papers possess practically the same properties as the test papers produced according to Example 4 but the reaction colours are stable for a longer Test papers with a phosphate buffer of pH 7 possess similar properties.

Example ' 7.

Paper pr©-impregnated in the manner described in Example 5 is impregnated with solutions which contain 0.4 g. o-tolidine and 0.5 g. of one of the following-surface-active materials: a) lauryl pyridinium chloride b) benzethonium chloride c) cetyl trimethyl ammonium chloride d) 1 -hydroxyethyl-1 -carboxymethyl-2-alkyl-imidazolinium betaine .

The properties of these test papers correspond to those of Example 5.

Example 8.

Filter paper (Schleicher & Scbtill No. 597 NF Ind.) is impregnated with a solution of the following composition and dried at 40°G. : 1M phosphate buffer, pH 6 25 ml. cholesterol oxidase (60 U/mg. ) 0.1 g. peroxidase (70 U/mg.) 0.05 g. cholic acid 1.0 g. acetone 10 ml. distilled water ad 100 ml.

This paper is subsequently impregnated with solutions of oxidation indicators in 100 ml. acetone.

The amount of indicator used, the chemical name thereof and the colour reaction with cholesterol-containing sera are summarised i the following Table II: T A B L II Example 9» A mixture is prepared from the following components: polyvinyl propionate dispersion 45 g. sodium alginate, 1.85·$ in 0.5M 35 g. phosphate buffer, pH 5.5 cholesterol oxidase (60 U/mg. ) 0.5 g. peroxidase (70 U/mg.) 0.25 g. dioctyl sodium sulpho succinate 1 g. o-tolidine, dissolved in 6 ml. acetone 0.6 g. water 50 ml.

This mixture is either spread out to give a film with a wet film thickness of ahout 300 nm or is painted on to a solid carrier and dried at 35°C. When blood containing cholesterol is dropped on to the film and the blood wiped off after one minute, then, depending pon the cholesterol concentration, green colorations of ¾¾-. varying intensity are obtained.

If, in addition, 1.0 g. cholesterol esterase is added to the above-described formulation, then films are obtained with which it is also possible to determine increased cholesterol ester contents. j^. if

Claims (11)

1. What we claim is :- 1. A process for the activation of cholesterol oxidase, wherein at least one surface-active compound vrith lipophilic as hereinbefore defined/ and hydrophilic properties/is added to the cholesterol oxidase before use.

2. A process according to claim 1 , wherein the surface-active compound used is non-ionic and contains at least one hydro yl group.

3. A process according to claim 2, wherein the non-ionic compound is a polyox ethylene derivative of an alkyl, aryl or aralkyl alcohol.

4. A process according to any of the preceding claims, wherein the surface-active compound is used in an amount of between 0.005 and 0.1 wt.?¾, referred to the aqueous enzyme solution.

5. A process according to claim 1 for the activation of cholesterol oxidase, substantially a3 hereinbefore described and exemplified.

6. Diagnostic agent for the detection and determination of cholesterol and cholesterol esters in body fluids by oxidation of the cholesterol vrith oxygen in the presence of cholesterol oxidase to give cholestenone and hydrogen peroxide and detection of the hydrogen peroxide by peroxidase-catalysed oxidation of an oxidation indicator, which comprises an absorbent carrier or a synthetic resin film impregnated with or having embedded therein cholesterol oxidase, a system for the detection of hydrogen peroxide, buffer and at least one surface-active compound according to claim 1.

7. Diagnostic agent according to claim 6, wherein the surface-active agent is present therein in a concentration of 2 to 30$ΐ, referred to the solid reagents.

8. Diagnostic agent according to claim 7, wherein the surface-active agent is present therein in a concentration of 10 to 20S, referred to the solid reagents.

9. Diagnostic agent according to any of claims 6 to 8, wherein cholesterol esterase is also present.

10. Diagnostic agent according to any of claims 6 to 9> wherein a swelling agent and/or a stabilising agent is also presen .

11. Diagnostic agent according to claim 6, substantially as hereinbefore described and exemplified.