NL8201349A - REAGENT FOR THE DETECTION AND DETERMINATION OF CHOLESTEROL AND CHOLESTEROL ESTERS. - Google Patents

Description

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Reagent for detecting and determining cholesterol and cholesterol esters.

The invention relates to a reagent for the detection of cholesterol and cholesterol esters.

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The determination of cholesterol is of great importance for medical diagnostics.

In clinical chemistry, there is an increasing need today for rapid assays for detecting substances in bodily fluids, which often do not yield very precise results, but allow faster and cheaper results for routine and serial investigations.

The diagnostic agents used for the rapid determination are either wicking vehicles or waterproof films containing all the reagents required for the reaction. When these diagnostics are brought into contact with the body fluids to be examined, color reactions are obtained, which can be assessed either on the basis of comparative colors or with simple reflection photometers.

. Tests for preparing rapid cholesterol determinants by impregnating absorbent paper with cholesterol oxidase, peroxidase, an oxidation indicator 20 and a buffer did not produce the desired result, since the test papers obtained did not react with the cholesterol-containing serum of usual concentration.

It has now been found that useful test papers or films are obtained which react stepwise with cholesterol-containing serum when additionally impregnating the support with a surfactant.

The reagent of the invention for detecting and determining cholesterol and cholesterol ester in body fluids therefore consists of a carrier or a plastic film, that with cholesterol oxidase, a system for detecting ^02 buffer and an interface active compound with lipophilic and 8201349 *. * 2 - hydrophilic properties are impregnated or embedded.

The interface active compound is advantageously present in a concentration of 2 to 30%, preferably 10 to 20% of the solid reagents.

Non-ionic detergent substances containing at least 1 OH group in the molecule are preferably used as the interface active compound with lipophilic and hydrophilic properties. Particular preference is given to polyoxyethylene derivatives of alkyl, aryl and aralkyl alcohols. Examples of the like 10, the particular preferred interfacial active compounds are polyoxyethylene alkyl ethers, polyoxyethylene-alkylcarbonzuuresters, polyoxyethyleensorbitanalkylcarbonzuur esters, polyoxyethyleenglycerolalkylcarbonzuuresters, polyoxy-ethyleenalkylaminen, polyoxyethyleenpolyoxypropyleenblokpolymeren, 15 polyoxyethyleenalkylthioethers and polyoxyethylene alkylaryl ethers.

Special examples are hydroxy polyethoxy dodecane, ethylene oxide addition products of alkyl phenols, polyethoxyethylene derivatives of sorbitan anhydrides, and the like. Also mercaptan-modified polyoxyethylene derivatives were found to be about -20 as well. Preferably used surfactant compounds when using said catagory, as a rule 0.005-0.1% by weight is sufficient.

There are whole series of these catagory compounds on the market, differing in the number of oxyethylene groups. The best are the water-soluble types with a medium number, oxyethylene groups (5-20). Lower ethoxylated types are only dispersible and therefore less useful. Higher ethoxylated compounds (more than 25) are soluble in water, but so hydrophilic that their activity is reduced.

Among the anionic surfactants, the salts of bile acids, such as cholic acid, taurocholic acid and deoxycholic acid, are particularly suitable, as well as fatty acid salts and fatty acid sarcosides. Of particular importance are sulfuric acid derivatives, which, as is known, can additionally stabilize the colored radical oxidation products of many indicators (o-tolidine, heterocyclic azines). These are, for example, the following categories: sulfosuccin esters, alkylaryisulfonates alkyl sulfates and alkyl polyoxyethylene sulfates.

Of the cationic surfactants, for example, alkyl pyridinium or trimethylammonium salts can be used, but also complex compounds, for example benethonium chloride.

Of the amphoteric surfactants, for example, imidazolium betaines can be used.

Special examples are sodium di (2-ethylhexyl) sulfosuccinate, sodium dodecyl sulfate, sodium oleate, benzalconium chloride and cetylpyridinium chloride.

Preferably, such ionic or amphoteric surfactants are used in combination with the above-mentioned nonionic surfactants. The appropriate amounts correspond to those of the nonionic surfactants.

Preference is given to the non-ionic border-surfactants with at least 1 OH group, because they provide an activation of about 5 to 10 times stronger and thus a corresponding increase in the reaction rate or shortening of the reaction time compared to the other useful ones. surfactants. They are also active in smaller amounts.

Alkyl groups are here understood to mean such groups having up to 20, in particular 12-18, carbon atoms.

Of the nonionic surfactant polyoxyethylene compounds, they are particularly useful which have a balanced ratio between the hydrophobic moiety and the polyoxyethylene chain and are water soluble.

Eexi's measure of the relationship between hydrophobic residue and polyoxyethylene chain is the so-called HLB value (see 35 W.C. Griffin in J.Soc.Cosmetic Chem. J_ (1950), 311 and 5 (1954), 8201349 / / 249.

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Particularly useful are boundary, surfactants with ILB values 10-17. These values should be regarded as guide numbers, since the activity also depends on the nature of the hydrophobic residue.

A preferred system for detecting consists of peroxidase and an oxidation indicator, optionally together with swelling agents and / or stabilizers.

Normal test papers obtained by impregnating absorbent papers with the reagents are particularly suitable for detecting serum cholesterol. However, if it is desired to detect cholesterol in pure blood, it is effective to make the test papers hydrophobic, for example, according to DT-OS 1,598,048, or to coat with a semipermeable membrane from cellulose esters and the like. Preferably, however, test films according to DBP 1,598,153 are used for detection in pure blood.

For their preparation, the reagents, such as cholesterol oxidase, peroxidase, buffer, indicator and optionally swelling agents, are stirred together with the surfactant in an aqueous dispersion of a film-forming polymer. This dispersion is spread into a film and dried. When cholesterol-containing blood is dripped onto such a reagent film and wiped after 1 minute, discolourations are also obtained, the intensity of which depends on the amount of cholesterol. These discolourations are particularly suitable for quantitative determination with simple remission photometers.

The reagent according to the invention is suitable for detecting and determining free cholesterol. However, when cholesterol esters are involved, the cholesterol must first be released. This can be done in the usual manner, such as by saponification with aqueous alkali. However, the ester cleavage with cholesterol esterase 35 (which is preferably isolated from microorganisms) is particularly favorable, * .....

8201349 - 5 - V i 'because this allows very careful work.

The cholesterol esterase can be added to the body fluid and iridubate for a period of time. This method can be carried out in such a way that the body fluid is drawn up in capillaries, the inner wall of which is coated in analogy with the DBP 2,240,672 with cholesterol esterase and optional auxiliary substances. After incubation in the capillaries, the liquid is then applied to the test strips as described. When free and esterified cholesterol were already present in the body fluid, the sum of both is of course demonstrated. However, the cholesterol esterase can advantageously also be incorporated in the test strips, so that reagents are obtained with which the sum of free and esterified cholesterol can be detected or determined in one step.

The systems which are suitable for detecting the hydrogen peroxide formed in the oxidation of cholesterol, preferably peroxidase, buffers, oxidation indicators and optionally swelling agents and the like, are already known, for example, from descriptions of rapid tests for the determination of glucose. Examples of useful components of this preferred system follow below:

The peroxidase is particularly suitable from horseradish, but lacquer peroxidase and the like can also be used.

The usual buffers, such as phosphate, citrate or borate buffer, can be used as a buffer. The pH they give to the diagnostic agent should be between 4 and 9, preferably between 5 and 8.

All kinds of compounds are suitable as oxidation indicators, namely benzidine derivatives, for example o-tolidine, o-vanillin derivatives according to DT-AS 1,598,133 or heterocyclic azines according to DT-AS 1,648,840.

Swelling agents such as sodium alginate, carboxymethylcellulose, and the like 8201349 'i * V * - 6 - and additionally stabilizers for the enzymes, for example dithioerythritol, can also be added to the recipes for the diagnostic agents, in particular for the test films.

Raw materials for test films are plastic dispersions of, for example, polyvinyl propionate or acetate. In this, all reagents are preferably dissolved, stirred, after which the films are streaked and dried.

Test papers are prepared by dissolving the reagents in water or mixtures of water and organic solvent, impregnating filter papers with the solution and drying. However, it is also possible to impregnate beforehand with the water-soluble reagents and then with the indicators in organic solution.

The following examples illustrate the invention.

Example I

Filter paper (Schleicher and Schuil 597 NF Ind.) Was impregnated with a solution of the following composition and dried at 40 ° C: 1 molar citrate buffer, pH 5.25 20 ml 20 cholesterol oxidase (60 U / mg) 0.1 grams peroxidase (70 E / mg). 0.05 grams of distilled water, 100 ml in total

This paper was impregnated with solutions of 0.2 grams of o-tolidine in 100 ml of methylene chloride, each further containing 1 gram of the following surfactants: a) polyoxyethylene tributyl phenol ether b) polyoxyethylene sorbitan monolaurate c) polyoxyethylene nonyl phenol ether d) polyoxyethylene lauryl ether 30 e) polyoxyethylene cetyl ether polyoxyethylene stearate g) polyoxyethylene dodecylthioether

After drying, test papers were obtained which reacted to cholesterol-containing sera with green colors. When the sera also contain cholesterol esters at the same time, ________ 8201349 - 7 -

stronger green discolorations obtained when some time of. a drop of cholesterol esterase solution has been added to the sera beforehand. A test paper which does not contain any of the surfactants mentioned above does not react with the above sera. 5 Example XI

Filter paper (Schleicher and Schuil 597 NF, Ind.) Was impregnated with a solution of the following composition and dried at 40 ° C: 1 molar citrate buffer 20 ml 10 cholesterol oxidase (60 U / mg) 0.1 grams cholesterol esterase (18 U (mg)) 0.25 grams of peroxidase (70 U / mg) 0.05 grams of distilled water, up to 100 ml

This paper was impregnated with solutions of 0.2 grams of o-tolidine in 100 ml of methylene chloride, each of which additionally also contained 0.5 grams of the following surfactants: a) polyoxyethylene cocoate b) polyoxyethylene oleate 20 e) polyoxyethylene polypropylene glycol d) polyoxyethylene stearylamine e) polyoxyethylene glycerine monolaurate

After drying, test papers were obtained, which reacted green with such sera. When these contain cholesterol and / or 25 cholesterol ester. Test papers without surfactants show no reaction.

Practically the same behavior show test papers, which contain the same amount of citrate buffer with pH 5.25 or 6. Example III

Pre-impregnated paper according to Example I was then impregnated with solutions of 0.3 grams of o-tolidine in 100 ml of acetone containing the following surfactants in concentrations of 1.5 grams, a) dioctyl sodium sulfosuccinate 35 b) dodecylbenzenesulfonic acid , sodium salt

8 2 0 1 3 M

* -v - 8 - c) lauryl polyglycol ether sulfate, sodium salt.

d) sodium aluryl sarcosinate e) sodium laurate f) cholic acid 5 g) deoxycholic acid h) sodium taurocholate

These test papers had practically the same properties as the test papers according to example I, except that the reaction colors were stable for longer.

Example IV

Pre-impregnated paper according to Example II was impregnated with solutions containing 0.4 grams of o-tolidine and each 0.5 grams of the following surfactants: 15 a) lauryl pyridinium chloride b) benzethonium chloride c) cetyl trimethyl ammonium chloride d) 1-hydroxyethyl-1 -carboxymethyl-2-alkylimidazolinium betaine

The properties of the test papers corresponded to those of Example II.

Example V

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Filter paper (Schleicher and Schuil 597 NF, Ind.) Was impregnated with a solution of the following composition and dried at 40 ° C: 25 1 molar phosphate buffer (pH 6) 25 ml cholesterol oxidase (60 U / mg) 0.1 gram peroxydase (70 U / mg) 0.05 gram of cholic acid 1.0 gram of acetone 10 ml of distilled water, up to 100 ml

This paper was then impregnated with solutions of oxidation indicators in 100 ml of acetone.

The amount of indicator, their chemical structure and their color reaction with sera containing cholesterol are shown in Table 35 A: 8201349 *. - = - 9 -

Table A

Amount of 'Indicator' 'coloration 0.3 g o-vanillylidene.-p-van.il-violet loylhydrazone 5 0.2 g' azino-bis- (N-ethylbenzthia green zolon-2-sulfonic acid-5) -diammonium salt 10 0 , 1 g azino-bis- (N-alkylquinolone-blue-violet 2-sulfonic acid-6) -diammonium salt 0.1 g bis- (N-alkylquinolone-2) -azin violet 15 0.1 g (N-methylbenzthiazolone-2) - N- blue-green ethylquinolone-2) -azin 0.3 g (N-methylbenzthiazolone-2) - blue 1-phenyl-3,4-dimethyltri-20-azolone-5) -azine «

Example VI

A mixture was prepared from the following components: polyvinyl propionate dispersion 45 grams of sodium alginate, 1.85% in 0.5 molar phosphate buffer, pH 5.5 35 grams of cholesterol oxidase (60 U / mg) 0.5 grams of peroxydase (70 U / mg) 0 25 grams of 30 dioctyl sodium sulfosuccinate 1 gram of o-tolidine dissolved in 6 ml of acetone 0.6 grams of water 50 ml

From this mixture, a film about 300 nm thick was made wet or was coated on a solid support and dried at 35 ° C. When blood with cholesterol 82 0 1 3 4 9 - 10 - terol there. After dripping onto the film and wiping the blood after 1 minute, green discolourations of different intensity are obtained, depending on the cholesterol concentration.

When an additional 1.0 gram of cholesterol esterase is added to said recipe, films are obtained which can also be used to determine elevated cholesterol levels.

* 10 8201349

Claims (2)

1. Reagent for detecting and determining cholesterol and cholesterol requirement in liquids by oxidation of the cholesterol with oxygen in the presence of cholesterol oxidase to cholestenone and hydrogen peroxide and for detecting hydrogen peroxide by oxidation of an oxidation indicator catalyzed with peroxydase, characterized in that it consists of an aspirating support or a plastic film impregnated with cholesterol oxidase, a system for detecting 13 2, buffer and at least one interfacial compound having lipophilic and hydrophilic properties, or embedded. Reagent according to claim 1, characterized in that the interfacial active compound is present in a concentration of 10 to 20%. 20 y 8201349