SU717994A3 - Reagent for determining cholesterin in blood serum - Google Patents

Description

(54) A REACTIVE FOR DETERMINATION OF TOTAL CHOLESTEROL IN BLOOD SERUM but to be used together with other carbon sources. Especially preferred is the use of microorganisms that are obtained by a multi-stage expression process, with the cultivation in the first stage being produced on suitable carbon sources (glycerol) and in the second stage neither cholesterol esters. The use of cholesterol esterase and Candida rugosa AflCC 14830 in a weakly acidic form, pH 5-6.5 shows high stability. The optimum pH of the enzyme is 7.5. The activity of cholesterol rinesterase is enhanced by the addition of surfactants. Especially preferred additive: hydroxypolyethoxydodecane. Cholesterol is determined by fermentation when using cholester inoxidase, but cholesterol dehydrase or cholesterine dyrogenase can also be used. Enzymatic determination of total or bound cholesterol is metabolized using Sydase cholesterol, for example, Nocardia erythropoeis AMCC 17895, Nocardia erythropoEis AMCC 4277, Nocardia formica AMCC 14811 or Proactin Synthesis, or Nocardia erythropoEis AMCC 4277, Nocardia formica AMCC 14811, or Proactin Synthesis. Cholesterol cholesterol, cholesterol esterase: from microorganisms, catalysis, acetylacetone, methanol and containing ammonium ions taken separately or in a mixture. The reagent may also consist of cholesterol oxidase, a preparation of microorganisms with cholesterol eterase activity, peroxidase, a chromogen, and a buffer individually or in a mixture. 2,2-aminobenzthiazlinsulfonic acid is used as the chromogen. . The reagent may also consist of cholesterol oxidase, a preparation of cholesterol esterase, and a derivative of hydrazine, which reacts with keto groups to form idrazone and a buffer. As a derivative guide Razin use 2,4-dinitrophenylglyrazine. In addition, the reagent may additionally contain solvents, stabilizers and surfactants. Preferably, the reagents contain the following components: 1. Cholesterol oxidase 13-150 units / mg Cholesterol esterase from - microorganism .. 0.05-0.5 mg. Catalase 2 10 --5-10 units. Acetylacetone 0.05-0.2 ml Methanol 2-10 ml in 100 ml of buphene containing ammonium ions; pH 5-7. Hydroxypolyethoxidodecane 0.02-0.3 ml 2. Cholesterol oxidase. 3-40 units / mg Cholesterol esterase from a microorganism, 05-0.5 mg Peroxidase 2-10 -140 2,2-Aminobenzthiazolinesulfonic acid 50-200 mg Hydroxypolyethoxydodecane 0.05-0.5 ml in 100 ml of buffer with pH 6-8 3 .Cholesterol-; oxidase 0.1-1.0 units Cholesterol esterase from the microorganism O, O5-0., 5 mg 1 mm solution of 2,4-dinitrophenylhydrazine 1-5 ml PAV0.005-0.1 ml Per 10 ml of buffer with pH 6-8. 4. Cholesterol oxidase 2-100 units. Cholesterol esterase from microorganism 0.05-0.5 mg surfactant (hydroxy polyethoxydodecane 0, 1-2.0 ml in 50 ml of buffer with pH 5-9. Using the proposed reagent you can quickly and completely wash the bound cholesterol. For example, under cholesterol determination conditions using cholesterol oxidase, quantitative cleavage of bound cholesterol is achieved within 1-3 minutes with the addition of a dry powder from acetone Candida rugosa AMCC 14830 or AspergiE € spec. WS .90030 in an amount of 0.1-0.3 mg. Example 1. In the serum, the content of free cholesterol is determined to 63% (63 100 ml) -. To determine bound cholesterol, a comparative serum sample is treated for 30 minutes with an alcohol solution of caustic, potassium at 70 C. After neutralization and new measurement of existing cholesterol, the total cholesterol content is 181 mg%. cholesterol / 100 ml is in a bound form.The method with the untreated, serum is repeated, but at the beginning of the test, 0.3 mg (relative to protein) of a dry powder from acetone Candida rugosa AMCC 14830 in standard form is added. After 3 minutes, a polar determination gives a total cholesterol content of 183 mg%.

Example 2. To increase cholesterol ester activity, standard dry powder from acetone Candida rugosa AMCC 14830 is dissolved in potassium phosphate buffer pH 6.0 and dialyzed against the same buffer. After removal of the lactose contained as a stabilizer, a proper cholesterol esterase activity of 0.3 units / mg protein in the dialyzed solution is obtained.

The resulting solution is mixed with a dextran-based ion exchanger modified with diethylamino-ethanol groups, the ion exchanger is separated and eluted with O, 2 M phosphate buffer pH 6.0. In the eluate, the specific activity of cholesterol esterase 1.2 units / mg is obtained. .

The resulting solution is subjected to fractionation with ammonium sulphate. The protein fraction that precipitated between 1.8 and 2.4 M ammonium sulfate was separated. It shows the specific activity of cholesterol esterase 2.5 units / mg

The product obtained is again dissolved in phosphate buffer with a pH of 6.0, dialyzed against the same buffer until desalting, and then chromatographed over columns that are filled with the same anion exchange resin as indicated above. Again, elution is carried out with 0.2 M phosphate buffer pH 6.0. In the fraction, the specific activity of cholesterol esterase reaches 7 units .. / mg protein

The resulting enriched cholesterol esterase preparation is used to determine cholesterol, as described in Example 1. The amount used is only 0.001 mg with respect to protein. The results correspond to the result of example 1.

Cholesterol esterase from Candida rugosa can be cleaned in the usual way by thin layer chromatography. Instead of enrichment stages, biochemical purification steps can be applied, for example, precipitation or fractionation using polyethyleneimine, organic solvents or salts, chromatography through molecular sieve materials or weaker anion exchangers with other functional groups than diethylaminoethanol groups, wasp): Deny with protamine sulfate. .

Drimer 3. K0.5ml of serum or standard cholesterol is added with 1.0 ml of 0.5 M potassium phosphate buffer based on pH 7.5, the KpTdry contains 0.4% hydroxypolyethoxydodecane and 2.5 U / mg cholesterol basics, according to example 2.

This reaction mixture is incubated for 40 minutes at 37 ° C. Then.

0.25 ml of this solution is added to 3 ml of cholesterol reagent, which contains two parts of acetic acid, three parts of acetic anhydride and one part of sulfuric acid (Lieberman-Burchardt reagent).

When using the standard as a relative value for a typical sample, 170 mg% total was found. cholesterol. A comparative definition in the saponification of cholesterol esters with an alcohol solution of potassium hydroxide gives 16 5 mg%.

Example 4, 0.02 ml of serum is mixed with 10 ml of 0.5 M potassium phosphate-based buffer, which contains 0.4% hydroxy polyethoxydodecane and 0.2 U / mg cholesterol esterase, according to Example 2.

The reaction solution is incubated for 60 minutes at. Then calculate

0, the extinction of E at 240 nm in a suitable spectrophotometer and the reaction is started with 0.1 U / mg sterine dehydrase from Brevibacterium steirolicum. After 15 minutes, the extinction Eg- is determined again. The concentration of d-cholestenonone formed is formed, and with it cholesterol is obtained from the difference between the first and second definitions.

taking into account the molar extinction coefficient dl d-cholestenone

at 240 nm. Measuring a typical sample gives 183 mg% total cholesterol. Comparative determination using cholesterol oxidase (from Nocardia erythropotis) instead of sterine dehydrase gives 181 mg% of total cholesterol.

Example 5. The south of diammonium hydrogen phosphate is dissolved in 100 ml of water and the pH is adjusted to 7.0 with 85% phosphoric acid. Then 10 U / mg catalase is added. Using the solution obtained, a mixture of 0.2 ml of acetylacetone, 10 ml of methanol and 0.1 g of hydroxypolyethoxydol,

dean was adjusted to 100 ml. To this solution, 2.5 units / mg of cholesterol esterase from Rhizopus spec. (WS 90027), 5 ml of the resulting solution is mixed with 0.02 ml of serum or 0.02 ml of a standard solution

cholesterol containing 200 mg% cholesterol. Aliquots of the sample containing the serum and also the standard of cholesterol are mixed with O, 1 U / mg of cholesterol oxidase and incubated for 60 minutes at 37 ° C. The resulting dye was then measured photometrically at 405 nm, taking into account the values of the control samples.

Claims (1)

The cholesterol content of the serum-containing sample is, when using the standard, as a relative value, 154 mg% of total cholesterol. Control determination using cholesterol esterase from Candida rugosa AMCC 14830 instead of cholesterol esterase from Rhi opus spec. (WS 90027) gives the same value. The proposed reagent improves the accuracy of determining total cholesterol in blood serum. Claims of the invention: A reagent for determining total serum serine serum, o t h h S (and y with the fact that, in order to increase the determination accuracy, it contains cholesterol esterase, cholesterol oxidase, catalase, acetyl acetate, methanol, detergent and ammonia in the following ratio, koment, wt.%: Cholesterol 0, 05. 10 esterase Cholesterol 10-b. oxidase 10 -10 Catalase 10-2- Acetylacetone 5 Methanol 2,0-10,0 2,0. 10-2-1,01 Detergent Amg Iaachny Else Sources of information, taken into account during examination 1, Predtechenskiy V. Clinical laboratory guide atorna isovaniya M. M., 1964, pp. 223-224.