DK151396B - PROCEDURE AND REAGENT FOR DETERMINING CHOLESTERIN - Google Patents

Description

151596 i

The present invention relates to a method for determining cholesterol and, in particular, the total cholesterol content or the bound cholesterol content.

Cholesterin is present in biological material, e.g. serum and the like, partly in free form, partly in bound form as ester. In order to determine the bound cholesterol or total cholesterol level, it is necessary to first release the cholesterol present in the bound form. This was done so far by saponification under alkaline 1Q conditions, e.g. with alcoholic potash. After saponification, the released cholesterol can then be determined by one of the known methods, either chemical or enzymatic. The chemical assay may e.g. is done according to the Liebermann-Burchard method, the enzymatic determination can be by cholesterol oxidase, cholesterine dehydrogenase or cholesterine dehydrase. Since, as is known, the individual cholesterol esters and also the free cholesterol by the chemical determination methods have different extinction coefficients, it is necessary by alkaline hydrolysis to convert the cholesterol ester to free cholesterol.

However, in each case, the alkaline saponification of the bound cholesterol is a disruptive and time consuming process step. In addition, the relatively aggressive reagents used can lead to the degradation of the cholesterol. In order to prevent such degradation and thus a falsification of the assay results, there must be hydrolyzed under relatively mild conditions, which in turn undesirably increases the time required for the determination.

Particularly unfortunate is the alkaline release of the cholesterin when the determination of cholesterin is then to be carried out according to the preferred 30 enzymatic methods. As the enzymes are known to be inactivated in highly alkaline medium, the hydrolyzate must be brought to a pH of 5-8 by the addition of acid before the enzymatic assay can be started. All this results in the determination of the total amount of cholesterol or the bound cholesterol level still taking a long time and is too labor intensive.

The object of the invention is therefore to provide a method for determining the total cholesterol content or the content of bound cholesterin which removes the above disadvantages.

___ 151396 2

This succeeds according to the invention by a method for determining the total cholesterol or bound cholesterol content by releasing the bound cholesterol content and subsequently determining the released cholesterol content according to known methods, characterized in that the bound cholesterin is released with a chole sterinesterase of microorganisms.

It has now been found that with cholesterine esterase a rapid and quantitative release of the bound cholesterol can be achieved. This process is particularly advantageous when the subsequent determination of the released cholesterin also occurs enzymatically, e.g. with cholesterol oxidase or with cholesterol dehydrase. In this case, the method of the invention allows for the complete enzymatic determination of cholesterol in the mesh and thus a crucial improvement in medical routine diagnostics as well as an easy adaptation of the method to the implementation in assay machines. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Admittedly, it was already known that in Pancreas, liver 2 and in Nocardia restrictus, a cholesterol esterase activity occurs. However, it could not be concluded that such enzymes 4 should be suitable for the rapid completion of cleavage of 1 - 5 ester esters in the framework of a quantitative method of analysis, for the rates of cleavage found were not quantitative but only reached a maximum. 80% (Biochimica et Biophysica 8

Acta, 270 (1972) 156-166). In addition, bound chole 9 sterin is present in biological material, in the form of esters of very different 10 different acids. For a utility within the scope of an 11 assay method, it is a prerequisite that all occurring 12 esters at approximately the same rate and with the same reliability 13 be quantitatively cleaved. Due to the known properties of these enzymes, it is surprising that the cholesterol esterases are capable of quantitatively cleaving all the cholesterol ester occurring in a very short time. This is also particularly surprising, because it was known that in the known cholesteric esterases there were significant differences in activity with respect to various cholesterol esters.

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It has been found that cholesterol esterases from microorganisms are particularly suitable for the process of the invention. In terms of cleavage rate and activity against different cholesterol esters, they were found to be superior to cholesterol esterases of other origins and are therefore preferred in the present invention.

It has further been found that various microorganisms contain particularly active cholesterin esterases and, therefore, within the scope of the present process of the invention can be used directly without separating and purifying the cholesterin esterases. This, in addition to the particularly simple availability, also has the advantage that, in the absence of any separation and enrichment of the cholesterol esterase, the danger of making a quantitative determination of all bound cholesterol by avoiding separating the present mixture of cholesterol esterases that are specific to different choesters in nesters. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

In addition, the purification of the majority of lipid-2 membrane-bound cholesterol esterases is cumbersome, and therefore 3 leads to a preparation which, because of its cost, is less suitable 4 for a routine diagnosis than a microorganism preparation usable without any enzyme purification.

6 7 Particularly advantageous results are obtained by the method of the invention 8 using a cholesterol esterase of Candida 9

rugosa (also referred to as Cylindracea) AICC 14830 and WS respectively

10 90031 and by Aspergillus spec. WS 90030. Within the scope of the present invention, these two microorganisms 11, 12 can be used directly 13 as such or in an open form, e.g. as acetone dry powder, however, of course, the use of an enriched cho = 14 lesterin esterase preparation of these microorganisms is also possible, of which 15 a particular advantage consists in the fact that some enrichment here can be obtained very simply 16. Said Candida rugosa is a commercially produced microorganism, which is produced on a large scale. The usual form of trade is an acetone dry stabilized lactose powder which has proved particularly advantageous for the invention.

4 151396

As already mentioned, a particularly important advantage of the process according to the invention therein is that a complete enzymatic determination of the total cholesterol content is possible. Hereby, it is important that with the preferred cholesterin ester 5 preparations of microorganisms, it is possible for a quick and quantitative release of the cholesterin by its esters. Especially with the above preferred microorganisms, it is possible, by direct addition thereof, in a very small amount and while maintaining the pH and temperature conditions as desired by the subsequent enzymatic cholesterol determination, to obtain within a few minutes a quantitative release of the cholesterol. In doing so, it was found that the usual carbohydrate-based stabilizers used for such microorganisms do not, within the scope of the fully enzymatic process 15, interfere with cholesterol determination.

As mentioned, a separated and enriched cholesterol esterase can also be used for the process of the invention. Suitable enrichment can be achieved by starting from an acetone drying powder of a microorganism and subjecting it to dialysis, a treatment with weakly basic anion exchanger and an ammonium sulfate fractionation. In this way, a 20 to 30 times enrichment of cholesterol in the nesterase easily succeeds. As a weakly basic anion exchanger, it is found that a modified carbohydrate-based modified diethylaminoethanol group is particularly suitable. In the case of ammonium sulfate fraction ions, the fraction between 1.3 and 2.4 moles of ammonium sulfate is preferably recovered. The enzyme fraction thus obtained is then preferably chromatographed on said exchange material.

Particularly good results are obtained within the scope of the invention with microorganisms grown on a cholesterol ester-containing nutrient medium. Thus, the cholesteric nester or a cholester in the nester blend can be used as the sole carbon source during cultivation, or a different carbon source can be used simultaneously. Particular preference is given to the use of microoranisms prepared by a multi-step culture method in which, in the first step, microorganisms were cultured on a suitable carbon-providing substance as glycerol, and in the second step on a cholesterol ester. A suitable cultivation method is e.g. described in the published German Specifications Nos. 2,224,133 and 2,307,518.

5

The cholesterinesterase used according to the invention by Candida rugosa AICC 14830 exhibits very good stability in the slightly acidic range between pH 3 and 6.5. The pH optimum of the enzyme is at 7.5. The enzyme is peculiar in that a particularly good course of the catalytic reaction occurs at relatively high salt content in the reaction medium. Therefore, it is preferably employed in a 0.5-0.8 M, preferably 0.4-0.6 M buffer solution. The pH may range from 4.5 to 7.5, preferably in the above range from pH 5 to 6.5. The effect of cholesterol esterase is preferably enhanced by the addition of surfactants. An addition of hydroxypolyethoxydodean is particularly preferred.

As already mentioned, it is particularly preferred to carry out the method according to the invention completely enzymatically, i.e. the subsequent cholesterol determination also occurs enzymatically, preferably using cholesterol oxidase. However, cholesterol dehydrase or cholesterin dehydrogenase can also be used.

The determination of cholesterol oxidase is e.g. described in the published German specification No. 2,224,132. The process described above can advantageously be combined with the method according to the invention. In principle, oxygen consumption, H 2 O 2 formation or cholesterol formation can be measured. 1 2 3 4 5 6

The determination of the oxygen consumption can e.g. gas chromatographic, 2 polarometric or by the polarization method. These determination methods are known per se. Hydrogen peroxide formed 4 can be determined titremetric, potentiometric, polarographic and 5 colorimetric as well as enzymatic. The enzymatic assay under the use of catalase or peroxydase, especially the determination by catalase in the presence of β-diketones, such as acetylacetone, lower alcohols and ammonium ion-containing buffer, or the determination by peroxydase in the presence of a chromogen such as 2.2, -amino = benzthiazoline sulfonic acid, preferred. Cholestones are determined by ketor reagents, e.g. 2,4-dinitrophenylhydrazine or photometric at 240 nm.

If the complete enzymatic determination of the total or bound amount of cholesterin occurs with cholesterol oxidase, preferably one of Nocardia erythropolis ATCC 17895, Nocardia erythropolis ATCC 4277, Nocardia formica AHCC 14811 or Proacti = nomyces erythropolis NCIB 9158 is used.

10

The present invention further relates to a reagent for the determination of cholesterin, consisting of cholesterine esterase of microbiological origin, and a system for the determination of free cholesterol. Preferably, a reagent of this kind consists of a microbiological origin cholesterine esterase, cholesterine oxidase, a H2O2 determination system or a cholestoneone determination system. This is particularly preferred for a reagent which uses as a cholesterin esterase a cholesterin esterase made from one of the 20 additional microorganisms mentioned above, especially in the form of an acetone dry powder or a protein fraction obtained with cholester in the nes asea act in the field.

The aforementioned preferred reagent combinations, in addition to the required constituents, may further contain usual solvents, stabilizers or / and surfactants. All of these additives are known to those skilled in the art and are customary in systems for detecting hydrogen peroxide and cholesterol, respectively.

With the method and reagent of the invention, an extremely rapid and complete digestion of bound cholesterol can be accomplished. Eg. For example, under the conditions of cholesterol in the determination of cholesterol oxidase according to the invention, a quantitative cleavage of bound cholesterin is achieved within 1 to 3 minutes by the 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.

7 151396

Example 1.

Using the procedure described in Example 1 of published German Specification No. 2,224,132, the serum free cholesterol content is determined to be 63 mg% (63 mg in 100 ml). To

determination of bound cholesterol became a comparative sample of 5O

serum treated for 30 minutes with alcoholic potash at 70 ° C.

After neutralization and reassessment of the cholesterol present, a total content of $ 181 mg of cholesterol was found. From this it appears that 118 mg of cholesterol / 100 ml was present in the bound form.

The procedure was repeated with untreated serum, however, at the beginning of the assay, 0.3 mg (calculated on the protein) of Candida rugosa ATCC 14830 acetone dry powder was added in the form of a conventional commercial product. After 3 minutes, the polarographic determination showed a total cholesterol content of $ 183 mg. Example 2.

To enrich the cholesterol esterase activity, a commercial product of acetone dry powder of Candida rugosa ATCC 14830 was dissolved in potassium phosphate buffer pH 6.0 and dialyzed against the same buffer. Following the removal of the stabilizing agent lactose, there was a specific cholesterol esterase activity of 0.3 U / 25 mg protein in the dialyzed solution.

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

The solution thus obtained was subjected to an ammonium sulfate fractionation. The protein fraction between 1.8 and 2.4 M ammonium sulfate precipitated was separated. It exhibits a specific cholesterol = esterase activity of 2.5 U / mg.

The resulting product was redissolved in phosphate buffer, pH 6.0, dialyzed against the same buffer until free of salt, and then pH 6.0. In the fraction with cholesterol esterase activity, a specific activity of 7 U / mg protein was found.

The enriched cholesterol esterase preparation thus obtained was used as described in Example 1 in the cholesterol determination. However, the amount used was only 0.001 mg, calculated on the protein. The result was similar to Example 1.

The cholesterol esterase of Candida rugosa can be further purified according to the usual methods of fine purification of enzymes. Instead of the aforementioned enrichment steps, other conventional biochemical purification steps such as e.g. precipitation or fractionation with polyethyleneimine, organic solvents or salts, chromatography over molecular materials or weak anion exchangers with functional groups other than diethylaminoethanol groups, protamine = sulfate precipitation and the like.

Example 3

To 0.5 ml of serum and cholesteric standard, respectively, were added 1.0 ml of 0.5 M potassium phosphate buffer, pH 7.5 containing 0.4 $ hydro ^ polyethoxydodecane and 2.5 U of cholesterin esterase of Example 2. This reaction mixture was incubated for 40 minutes at 37 ° C. Then 0.25 ml of this solution was added to 25 ml of cholesterin reagent containing 2 parts acetic acid, 3 parts acetic anhydride and 1 part sulfuric acid (Iiebermann-Burchardt reagent).

Using a standard as the reference size, a total cholesterol level of $ 170 mg was found for a typical sample. The comparative assay of saponification of the cholesterol ester with alcoholic potash gave $ 165 mg.

Example 4

35 0.02 ml of serum are treated with 10 ml of 0.5 M potassium phosphate buffer containing 0.4 $ hydroxypolyethoxydodecane and 0.2 U cholesterinesterase of Example 2. The reaction solution is incubated for 60 minutes at 37 ° C. Then, the extinction (E 1) is read at 240 nm in a hydrase of Brevibacterium sterolicum. After 15 minutes, the extinction (E ^) is read again. The concentration of the Δ ^ -cholesterone formed and thus the cholesterin is obtained from the difference between the first and second readings, taking into account the molar extinction coefficients of Δ ^ -cholestenone at 240 nm. The measurement of a typical sample yielded a total cholesterol level of $ 183 mg.

The comparison assay with a cholesterol oxidase (of Nocardia erythropolis) instead of the sterine dehydrase gave a total chole = sterin amount of $ 181 mg.

Example 5

Dissolve 10 g of diammonium hydrogen phosphate in 100 ml of water and adjust the pH to 7.0 with $ 85 phosphoric acid. Then 10 µl of catalase is added. With the solution thus obtained, a mixture of 0.2 ml of acetylacetone, 10 ml of methanol and 0.1 g of hydroxypolyethoxydodecane 20 is made up to 100 ml. To this solution is added 2.5 U cholesterin = esterase of Rhizopus spec. (WS 90027).

5.0 ml of the solution thus obtained is mixed with 0.02 ml of serum, 0.02 ml of a standard cholesterol solution, respectively, containing 200 mg of cholesterol. Portions of the serum-containing as well as the cholesterol-containing sample are each treated with 0.1 U of cholesterol oxidase and incubated for 60 minutes at 37 ° C. Then, the dye formed at 405 nm is measured photometrically, taking into account the blank value of the sample.

The cholesterol content of the serum-containing sample was, using a standard reference amount of 154 mg $ total cholesterol amount. The control assay with cholesterinesterase of 35 Candida rugosa ATCC 14830 instead of Cholesterinesterase of Rhizopus spec. (WS 90027) gave the same value.

Claims (9)

10 151396 A method for determining the total cholesterol or bound cholesterol content by releasing the bound cholesterol content and subsequently determining the released cholesterol content according to known methods, characterized in that the bound cholesterin is released with a cholesterol esterase of 10 microorganisms. Process according to claim 1, characterized in that a cholesterine esterase of Candida rugosa ATCC 14830, Rhi-zopus spec. WS 90027 or Aspergillus Spec. WA 90030. 15 A method according to any one of claims 1-2, characterized in that the assay is carried out completely enzymatically by combination with an enzymatic assay for free cholesterol. Process according to claim 3, characterized in that cholesterol esterase and an enzyme for the determination of free cholesterol are simultaneously added. The method according to claim 3 or 4, characterized in that the cholesterol released is determined by cholesterol oxidase. Process according to any one of claims 1-5, characterized in that a cholesterol esterase is used by a microorganism grown on a cholesterol ester-containing nutrient medium. 30 Reagent for carrying out the process according to claim 1, characterized in that it consists of cholesterol esterase of microbiological origin and a system for the determination of free cholesterol. 3 5 Reagent according to claim 7, characterized in that it consists of a cholesterol esterase of microbiological origin, cholesterol oxidase, a system for determining or a system for determining cholesterol. Reagent according to any one of claims 7 and 8, characterized