FI57783C - FOERFARANDE FOER BESTAEMNING AV KOLESTEROL - Google Patents

Description

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Patent- och regicteratyrelMn '' AmMcm utiagdoch utUkrifne pubMcwad 30.06.80 (32) (33) (31) P) 74 «t * y« * »oilw» «- Begird priority 28.03 * 73 - 03.0U.73 Federal Republic of Germany-Förbunds - Republic of Germany (DE) P 2315501.3, p 2316637.2 * (71) Boehringer Mannheim GmbH., Sandhofer Strasse 112-132, D-6800 Mannheim-

Waldhof, Federal Republic of Germany Förbundsrepubliken Tyskland (DE) (72) Klaus Beaucamp, Tutzing / Obb., Hans Mollering, Tutzing / Obb., Gunter Lang, Tutzing / Obb., Wolfgang Gruber, Tutzing / Obb., Peter Roeschlau, Tutzing / Obb., Federal Republic of Germany Förbundsrepubliken Tyskland (LE) (7U) Berggren Oy Ab (5U) Method for the determination of cholesterol - Förfarande för bestämning av cholesterol

The present invention relates to a method for the determination of cholesterol and thus to the determination of either total cholesterol or bound cholesterol.

Cholesterol is present in biological material, such as serum and the like, in a partially free form and in a partially bound form as an ester. In order to determine bound cholesterol or total cholesterol, it is necessary to first release the cholesterol in bound form. This has hitherto been done by saponification under temporal conditions using, for example, alcoholic potassium liquor. After saponification, the released cholesterol can be determined by any known method, either chemically or enzymatically. The chemical assay can be performed, e.g., by the Liebermann-Burchard method, and the enzymatic assay can be performed using cholesterol oxidase, cholesterol dehydrogenase, or cholesterol dehydrase. Since it is known that individual cholesterol esters as well as free cholesterol have different extinction coefficients in chemical assays, it is necessary that the cholesterol ester be converted to free cholesterol by alkaline hydrolysis.

57783

In any case, alkaline saponification of bound cholesterol is a disruptive and time consuming step in the process. In addition, it must be borne in mind that the relatively strong reagents used can cause cholesterol to decompose. To prevent such degradation and erroneous analytical results, the hydrolysis must be performed under relatively mild conditions, which in turn adversely prolongs the time required for the determination.

Alkaline release of cholesterol is very detrimental when more favorable enzymatic methods have to be used in the determination of cholesterol. Since the enzymes are known to become strongly inactive in an alkaline medium, the hydrolyzate must be brought to pH 5-8 by adding acid before performing the enzymatic assay. All of this causes the determination of total cholesterol and bound cholesterol to take longer and longer and requires a lot of work.

It is therefore an object of the present invention to provide a method for the determination of total cholesterol or bound cholesterol which avoids the above-mentioned disadvantages.

According to the invention, this can be carried out by a method for determining total cholesterol or bound cholesterol by releasing bound cholesterol and then determining the released cholesterol by known methods, which method is characterized in that bound cholesterol is released by cholesterol esterase obtained from a microorganism.

It has been found that cholesterol esterase can be used to rapidly and quantitatively saponify bound cholesterol. This method is very advantageous, especially when the determination of the released cholesterol is carried out enzymatically, e.g. using cholesterol oxidase or cholesterol dehydrase. In this case, the method according to the invention enables the full enzymatic determination of cholesterol and thus constitutes a decisive improvement in routine medical diagnostics as well as the easy application of the method for performing automated analyzes.

It has previously been known that cholesterol esterase activity occurs in the pancreas, liver, and Nocardia restrictus. However, it has not been possible to conclude from this that such an enzyme would be able to achieve rapid and complete saponification of the cholesterol ester in the quantitative analysis method, as the cleavage products found were not quantitative but reached up to 80% (Biochimica et Biophysica) ). Bound cholesterol is still present in biological material in the form of esters of very different acids. A prerequisite for the usefulness of the analytical method is that all the esters present are quantitatively degraded at approximately the same rate and with the same ease. Given the known properties of these enzymes, it is surprising that kdlesteroliesase is capable of quantitatively cleaving all cholesterol esters present in a very short time. This is also surprising, especially since it has been known that there have been considerable differences in the efficacy of known cholesterol esterases with respect to different cholesterol esters.

The cholesterol esterases produced by the microorganisms according to the invention have proven to be superior to a wide variety of cholesterol esters from elsewhere in terms of cleavage rate and efficiency.

It has further been found that various microorganisms contain highly active cholesterol esterases and can be used as such in the process of the invention without isolating and purifying the cholesterol esterases. In addition to very simple availability, this also has the advantage that by eliminating each separation and enrichment of cholesterol esterases, there is a risk that a mixture of cholesterol esterases specific for many different cholesterol esters would be subjected to separation measures that would complicate the quantification of all bound cholesterol.

In addition, it must be borne in mind that the purification of cholesterol esterases normally bound to lipid membranes is complicated and therefore results in a preparation which, because of its cost, is less suitable for conventional diagnostics than any microorganism preparation requiring any enzyme purification.

Very advantageous results have been obtained using the method according to the invention using cholesterol esterase obtained from Candida rugosa (also called Cylindracea) ATCC 14830 resp. WS 90031 and Aspergillus spec. WS 90030 strains. Both of these microorganisms can be added directly as such or in dissolved form, e.g. as acetone dry powder, within the scope of the invention. Of course, it is also possible to use an enriched cholesterol esterase preparation obtained from these microorganisms, with the particular advantage that a certain enrichment can be achieved very simply. Said Candida rugosa forms a microorganism produced on a large technical scale which is marketed. A useful commercial form is, for example, lactone-stabilized acetone dry powder, which has proven to be advantageous for use in the invention. Similar advantageous properties have been found in the following strains:

Actinomyces aureoverticillium WS 90002

Actinomyces cyaneofuscatus WS 90003

Actinomyces griseomycini WS 90004

Actinomyces longisporus-fl. WS 90005

Actinomyces malachiticus WS 90006

Actinomyces roseolus WS 90007

Actinomyces toxytricini WS 90008

Actinomyces variabilis WS 90009

Streptomyces spec. WS 90010

Streptomyces autotropicus WS 90011

Streptomyces canescens WS 90012

Streptomyces chartreusis WS 90013

Streptomyces michiganensis WS 90014

Streptomyces murinus WS 90015

Streptomyces hachijoensis WS 90016

Streptomyces caelestes WS 90017

Streptomyces tendae WS 90018

Nocardia rubra WS 90019

Candida mycoderma WS 90020

Candida albicans WS 90021

Candida albicans WS 90022

Candida albicans WS 90023

Candida spec. WS 90024

Cunninghamella elegans WS 90025

Mueor mucedo WS 90026

Rhizopus spec. WS 90027

Penicillium spec. WS 90028

Aspergillus spec. WS 90029 5 57783

However, in addition to the more preferred cholesterol esterases of microbiological origin, cholesterol esterases from elsewhere can also be used in many cases.

As mentioned above, an important advantage of the method of the present invention is that it becomes possible to perform a fully enzymatic total cholesterol assay. In this case, it is important that the use of the most advantageous cholesterol esterase preparations obtained from microorganisms makes it possible to release cholesterol rapidly and quantitatively from its esters. In particular, when using the most preferred microorganisms mentioned above, it is possible to achieve a quantitative release of cholesterol within a few minutes by adding them directly in very small amounts and maintaining the pH and temperature conditions as desired in the next enzymatic cholesterol assay. In this case, it has been found that other carbohydrate-based stabilizers used in connection with such microorganisms do not interfere with the determination of cholesterol in the context of such a full enzymatic method.

As already mentioned, isolated and enriched cholesterol esterase obtained from microorganisms can also be used in the method according to the invention. Suitable enrichment can be obtained starting from the acetone dry powder of the microorganism or similar biological material and subjected to dialysis treatment with a weakly basic anion exchanger and ammonium sulfate fractionation. In this case, 20- and up to 30-fold enrichment of cholesterol esterase can easily be performed. As a weakly basic anion exchanger, a carbohydrate-based preparation modified with diethylaminoethanol groups has been found to be very suitable. In the ammonium sulphate fractionation, a fraction between 1.8 and 2.2 mol of ammonium sulphate is preferably recovered. The enzyme fraction thus obtained is then chromatographed preferably using said ion exchange material.

Highly advantageous results have been obtained within the scope of the invention by the use of microorganisms cultured using a cholesterol ester-containing medium. In this case, the cholesterol ester or cholesterol ester mixture can be added as the sole carbon starting material during cultivation or used in combination with another carbon starting material. It is highly preferred to use microorganisms obtained by a multi-step culture method, in which case they are cultured in a first step using a suitable carbon donor such as glycerin and in a second step using a cholesterol ester. A suitable cultivation method is described, for example, in German Application Publication Nos. 2,224,133 and 2,307,518.

The cholesterol esterase from Candida rugosa ATCC 14830, preferably used according to the invention, has a very good stability in the weakly acidic range, between pH 5 and 6.5. The optimum pH of the enzyme is 7 »5 · The special feature of the enzyme is that the very good course of the catalytic reaction takes place at a relatively high salt content of the reaction medium. As a result, work is preferably carried out in a 0.2-0.8-m, especially 0.4-0.6-m, buffer solution. The pH value may be in the range between 4.5 and 7.5 and is preferably in the above-mentioned range between pH 5-6.5. · The efficiency of cholesterol esterase is preferably increased by the addition of surfactants. The addition of hydroxypolyethoxydodecane is very preferred.

As mentioned above, the process according to the invention is very preferably carried out fully enzymatically, i.e. the subsequent cholesterol assay is also performed enzymatically using preferably cholesterol oxidase. However, cholesterol dehydrase or cholesterol dehydrogenase can also be used.

The determination by means of cholesterol oxidase is described, for example, in German Patent Application No. 2,224,132. The method described therein can be advantageously combined with the method according to the invention. In principle, oxygen consumption, Og formation or cholestenone formation can then be measured.

Oxygen consumption can be measured, for example, chromatographically, polarometrically or by a polarization method. These ordering methods are known per se. The hydrogen peroxide formed can be determined by potentiometric, polarographic and colorimetric titration as well as enzymatically. An enzymatic assay is most preferred using a catalase or peroxidase, especially an assay using catalase in the presence of β-diketones such as acetylacetone, lower alcohols and ammonium ion-containing buffer, or using a peroxidase assay using a chromogen such as 2,2'-aminobenzthiazoline sulfone acid. Cholestenone is determined using keto reagents using, for example, 2,4-dinitrophenylhydrazine, or photometrically at 240 nm.

In the case of a fully enzymatic determination of total cholesterol or bound cholesterol by cholesterol oxidase, it is preferred to use one obtained, for example, from strains Nocardia erythropolis ATCC 17895 5 Nocardia erythro-Polis ATCC 4277 * Nocardia Formica ATCC 14811 or Practinomyces eryth

Another object of the invention is a reagent for determining cholesterol, which consists of cholesterol esterase, and a reagent for determining free cholesterol. Such a reagent is preferably a cholesterol esterase of microbiological origin, a cholesterol oxidase, a system for determining cholesterol or a system for determining cholestenone. Very particular preference is given to a reagent which uses as cholesterol esterase one obtained from one of the above-mentioned microorganisms, in particular in the form of acetone dry powder, or a protein fraction obtained therefrom having cholesterol esterase activity.

In a particular and preferred embodiment, such a reagent is formed from cholesterol oxidase, a cholesterol esterase preparation obtained from one of the above microorganisms, catalase, acetylacetone, methanol and ammonium ion-containing buffer, used either alone or in admixture. According to another preferred embodiment, the reagent is formed from cholesterol oxidase, a preparation of said microorganism having cholesterol esterase activity, a peroxidase, a chromogen and a buffer, either separately or as a mixture. 2,2'-Aminobenzthiazolinesulfonic acid is preferably used as the chromogen.

According to another preferred embodiment, the reagent according to the invention is formed from cholesterol oxidase, a cholesterol esterase preparation of one of said microorganisms and a hydrazine derivative which reacts with the keto group to form hydrazone and, if appropriate, a buffer. As the hydrazine derivative, 2,4-dinitrophenylhydrazine can be preferably used.

In addition to the mandatory ingredients set forth above, the above-mentioned preferred reagent combinations may also contain conventional solvents, stabilizers and / or surfactants. All of these additives are known to those skilled in the art and are conventional in hydrogen peroxide or cholestenone delivery systems.

The above reagent combinations preferably contain their main ingredients in the following proportions: 1. 13-150 U cholesterol oxidase, 0.05-0.5 mg microorganism cholesterol esterase, 2 x 10 ^ - 5 x 10 ^ U catalase, 0.05-0 , 287573 ml of acetylacetone and 2-10 ml of methanol in 100 ml of ammonium-containing buffer, pH 5-7, and, if appropriate, 0.02-0.3 ml of surfactant, preferably hydroxypolyethoxydodecane.

2. 3-40 U of cholesterol oxidase, 0.05-0.5 mg of microorganism cholesterol esterase, and 2 x 10 ^ - 1 x 10 ^ U of peroxidase, 50-200 mg of 2,2'-aminobenzthiazolinesulfonic acid, and optionally 0 .05-0.5 ml of surfactant, preferably hydroxypolyethoxydodecane, in 100 ml of buffer, pH 6-8.

3 · 0.1-1 U cholesterol oxidase *, 0.05-0.5 mg microorganism cholesterol esterase, 1-5 ml 1-mimo. 2,4-dinitrophenylhydrazine solution and, if appropriate, 0.005-0.1 ml of surfactant in 10 ml of buffer, pH 6-8.

4. 2-100 U of cholesterol oxidase, 0.05-0.5 mg of microorganism cholesterol esterase, and, if appropriate, 0.1-2.0 ml of surfactant (preferably hydroxypolyethoxydodecane) in 50 ml of buffer, pH 5 -9, preferably 0.5 M sodium phosphate buffer, pH 7.5.

Using the method and reagent of the invention, very rapid and complete saponification of bound cholesterol can be performed. For example, under cholesterol quantification conditions, the use of cholesterol oxidase according to the invention provides quantitative cleavage of bound cholesterol within 1-3 minutes upon addition of Candida rugosa ATCC 14830 or Aspergillus sp. WS 90030 acetone dry powder in an amount between 0.1 and 0.3 mg.

The following examples further illustrate the invention.

Example 1

Serum was determined to be 63 mg # (63 mg per 100 ml) of free cholesterol using the method described in Example 1 of German Application No. 2,224,132. To determine bound cholesterol, a serum reference sample was treated with alcoholic potassium hydroxide at 70 ° C for 30 minutes. After neutralization and a new measurement of the cholesterol present, a total cholesterol concentration of 181 mg # was obtained. This shows that 118 mg cholesterol / 100 ml was present in bound form.

The procedure was repeated using untreated serum; however, at the beginning of the assay, 0.3 mg (based on protein) of Candida rugosa ATCC 14830 acetone dry powder in commercial form was added. After 3 minutes, a total cholesterol content of 183 mg # was obtained by polarographic order.

9 57783

Example 2

To increase cholesterol esterase activity, commercial Candida rugosa ATCC 14830 acetone dry powder was dissolved in potassium phosphate buffer, pH 6.0, and dialyzed using the same buffer. After removal of the lactose used as a stabilizer, the specific cholesterol esterase activity was 0.3 U / mg protein in the dialyzed solution.

The solution thus obtained was mixed with a dextran-based ion exchanger modified with diethylaminoethanol groups, the ion exchanger was separated and eluted with 0.2 M phosphate buffer, pH 6.0. The specific cholesterol esterase activity of the eluate was 1.2 U / mg.

The solution thus obtained was fractionated by ammonium sulfate method. The protein fractions obtained between 1.8 and 2,4-ammonium sulfate were separated. The value of specific cholesterol esterase activity was 2.5 U / mg.

The resulting product was redissolved in phosphate buffer, pH 6.0, dialyzed against the same buffer to give it free of salt, and then chromatographed on a column packed with the same anion exchanger as described above. Elution was again with 0.2 M phosphate buffer, pH 6.0. In the fraction with cholesterol ester activity, the value of the specific activity was 7 U / mg protein.

The enriched cholesterol esterase preparation thus obtained was treated as described in Example 1 in a cholesterol assay. However, the amount added was only 0.001 mg based on protein. The result corresponded to the result obtained in Example 1.

Cholesterol esterase from Candida rugosa can be purified by conventional enzyme fine purification methods. Instead of the enrichment steps mentioned above, other biochemical purification measures can also be used, such as precipitation or fractionation with polyethyleneimine or with organic solvents or salt formation, chromatography using molecular sieves or weak anion exchangers with protamate sulfates, etc., other than diethylamine .

Example 3 To 0.5 ml of serum or cholesterol standard is added 1.0 ml of 0.05 M potassium phosphate buffer, pH 7.5, containing 0.5% hydroxypolyethoxydodecane and 2.5 U cholesterol esterase, e.g. 2. This reaction mixture is incubated for 40 minutes at 37 ° C. 0.25 ml of this solution is then added to 3 ml of cholesterol reagent containing 2 parts of acetic acid, 3 parts of acetic anhydride and 1 part of sulfuric acid (Liebermann-Burchard reagent).

Using the standard result as a reference, the total cholesterol content of the conventional sample was 170 mg #. A control assay by saponification of the cholesterol ester with an alcoholic potassium salt gave 165 mg #.

Example 4 To 0.02 ml of serum is added 10 ml of 0.5 M potassium phosphate buffer containing 0.4% hydroxypolyethoxydodecane and 0.2 U cholesterol esterase according to Example 2. The reaction solution is incubated for 60 min. at 37 ° C. The extinction (Eβ) at 240 nm is then determined in a suitable spectral photometer and the reaction is started with 0.1 U sterol dehydrase from a Brevibacterium sterolicum strain. After 15 minutes, the extinction (E2) is determined again. The concentration of Δ ^ -cholestenone formed and thus the amount of cholesterol is obtained from the first to second order 4. . ...

taking into account the value of the molar extinction coefficient of Δ -cholestenone at 240 nm. The total cholesterol value of a typical sample was 183 mg #.

A control assay using cholesterol oxidase (obtained from Nocardia erythropolis strain) instead of sterol dehydrase gave a total cholesterol of 181 mg #.

Example 5 ~ 10 g of diammonium hydrogen phosphate are dissolved in 100 ml of water and this is adjusted to pH 7.0 with 85% phosphoric acid. Then 10 μl of catalase is added. The solution thus obtained is added to a mixture of 0.2 ml of acetylacetone, 10 ml of methanol and 0.1 g of hydroxypolyethoxydodecane up to a volume of 100 m. To this solution is added 2.5 U cholesterol esterase obtained from the strain Rhizopus spec. (WS 90027) · 5.0 ml of the solution thus obtained are mixed with 0.02 ml of serum, resp. With 0.02 ml of a standard cholesterol solution containing 200 mg of #cholesterol. In equal volumes of serum and standard cholesterol, 0.1 U cholesterol oxidase is added to each and incubated for 60 min at 37 ° C. The dye formed is then measured photometrically at 405 nm, taking into account the initial value of the sample.

11 57783

The serum-containing sample had a cholesterol content of 154 mg% total cholesterol when used as a standard reference. Reference assay using cholesterol esterase obtained from Candida rugosa strain ATCC 14830 instead of cholesteroliesase derived from Rhizopus spec. (WS 90027), gave the same value.

Example 6

The activity of the microorganism cholesterol esterases suitable for the method according to the invention was determined, the results of which are given in Table 1.

Table 1.

No. Strain WS ΔΕ / h 1 Actinomyces aureoverticillium 90 002 0.600 2 Actinomyces griseomycini 90 004 0.600 3 Streptomyces spec. 90 010 0,064 4 Streptomyces autotrophicus 90 011 0,500 5 Streptomyces caelestes 90 017 0,600 6 Streptomyces tendae 90 018 0,550 7 Candida mycoderma 90 020 0,080 8 Candida albicans 90 021 0,082 9 Candida albicans 90 022 0,050 10 Candida albicans 90 023 0,080 11 Candida 90 024 0,070 12 Mucor mucedo 90 026 0,150 13 Rhizopus spec. 90 027 0.130 14 Penicillium spec. 90 028 0.130 15 Aspergillus spec. 90 029 0.130 16 Candida rugosa 90 031 0.020 ΔΕ is obtained by the following incubation test: 3.00 ml potassium phosphate buffer 0.5 M; pH 7.5 (0.33 ml Thesit ad 100 ml), bubble with CO2 gas 0.05 ml cholesterol oleate (10 mg / ml) 0.1 ml crude extract (concentrated 1:10 biomass) incubate for 5 ~ 17 hours At ° C start: by adding 0.07 ml of cholesterol oxidase

Claims (3)

57783 12 1. A compound for the treatment of total cholesterol or cholesterol in the genome of cholesterol and the use of cholesterol in the cholesterol form of cholesterol. 2. A method according to claim 1, comprising cholesterol ester of Candida rugosa ATCC 14830, Rhizopus spec. WS 90027 or Aspergillus spec. WS 90030 användes. A method for the determination of total cholesterol or bound cholesterol by releasing bound cholesterol and then determining the released cholesterol by known methods, characterized in that the bound cholesterol is released by means of cholesterol esterase obtained from a microorganism. Process according to Claim 1, characterized in that a cholesterol esterase obtained from the strain Candida rugosa ATCC 14830, Rhizopus spec. WS 90027 or Aspergillus spec. WS 90030. Process according to Claim 1, characterized in that a cholesterol esterase obtained from a strain is used: Actinomyces aureoverticillium WS 90002 Actinomyces griseomycini WS 90004 Streptomyces spec. WS 90010 Streptomyces autotrophicus WS 90011 Streptomyces caelestes WS 90017 Streptomyces tendae WS 90018 Candida mycoderma WS 90020 Candida albicans WS 90021 Candida albicans WS 90022 Candida albicans WS 90023 Candida spec. WS 90024 Mucor mucedo WS 90026 Penicillium spec. WS 90028 Aspergillus spec. WS 90029 Method according to one of Claims 1 to 3, characterized in that cholesterol esterase obtained from a microorganism which has been cultured using a cholesterol ester-containing nutrient is used. Reagent for carrying out the method according to claim 1, characterized in that it consists of cholesterol esterase obtained from microorganisms and a previously known system for determining free cholesterol. Reagent according to Claim 5, characterized in that it consists of cholesterol esterase of microbiological origin, cholesterol oxidase, a system for determining HgCl 2 or a system for determining cholestenone. 13 57783 Reagent according to Claim 6, characterized in that it contains a cholesterol esterase obtained from a microorganism according to Claim 2 or 3. Reagent according to Claim 5, characterized in that it consists of cholesterol oxidase, a cholesterol esterase obtained from a microorganism according to claim 2 or 3, catalase, acetylacetone, methanol and an ammonium ion-containing buffer. Reagent according to Claim 7, characterized in that it contains peroxidase, chromogen and buffer as a system for determining H 2 Cl 2. Reagent according to Claim 7, characterized in that it contains, as a system for determining cholestenone with keto groups, a hydrazone to form a reactive hydrazine derivative. Reagent according to one of Claims 5 to 1.0, characterized in that it contains a surfactant. 3. A patent according to claim 1, comprising cholesterol esters, comprising: Actinomyces aureoverticillium WS 90002 Actinomyces griseomycini WS 90004 Streptomyces spec. WS 90010 Streptomyces autotrophicus WS 90011 Streptomyces caelestes WS 90017 Streptomyces tendae WS 90018 Candida mycudivrma WS 90020 Candida albicans WS 90021 Candida albicans WS 90022 Candida albicans WS 90023