CN105137099A - Method for detecting total cholesterol by utilization of DeuteroheminAlaHisThrValGluLys - Google Patents

Abstract

The invention provides a method for detecting total cholesterol by utilization of DeuteroheminAlaHisThrValGluLys, and belongs to the biological and medical technology field. The method is as follows: change delta A of absorbance values before and after a reaction is detected at a 520nm wavelength of a fully automatic biochemical analyser, through comparison with a cholesterol standard substance detected in the same steps, the amount of total cholesterol is calculated; the amount of the cholesterol m mol/L=delta A sample/delta A sample * a standard substance concentration. The beneficial effects are that the developed detection method combined with a modern biological technology principle has characteristics of high sensitivity and good stability, is economical and practical, and can be widely used in fields of medical health, food production enterprises, farm product markets, quality supervision departments and the like for monitoring a total cholesterol amount in a liquid sample.

Description

A method for measuring serum total cholesterol with hypoheme hexapeptide

technical field

The invention belongs to the technical field of biology and medicine, and relates to an improvement of a detection method of biology and medicine.

Background technique:

Cholesterol, also known as cholesterol, is a derivative of cyclopentane polyhydrophenanthrene with a chemical formula of C ₂₇ H ₄₆ O.

Cholesterol is widely present in animals, especially in brain and nerve tissue, and is also high in kidney, spleen, skin, liver and bile. Cholesterol is an indispensable and important substance in animal tissue cells. It not only participates in the formation of cell membranes, but also is a raw material for the synthesis of bile acids, vitamin D and steroid hormones.

Cholesterol exists in lipoproteins in the blood, and its existing forms include high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and very low-density lipoprotein cholesterol. The reference range of the total cholesterol concentration in the blood is 2.9-5.2mmol/L. When the total cholesterol concentration in the blood is higher than the reference range, it is easy to cause heart disease and cerebrovascular diseases. Therefore, the determination of total cholesterol is a routine test item in disease diagnosis.

There are currently more than 200 methods for the determination of total cholesterol, which can be divided into five categories: chemical reagent colorimetry, enzyme analysis, fluorescence, gas phase and high performance liquid chromatography. Due to the high conditions of gas phase and high performance liquid chromatography, chemical reagent method and enzyme analysis method are most commonly used in clinical laboratories. The chemical reagent method is rarely used now due to the disadvantages of complicated operation, strong corrosive reagent (concentrated sulfuric acid) and difficulty in automation. At present, the enzymatic determination of total cholesterol in serum is carried out according to the national standard WS/T120-1999, its principle:

Total cholesterol (TC) in serum (plasma) includes two parts: free cholesterol (FC) and cholesteryl ester (CE). CE in serum can be hydrolyzed into FC and free fatty acid (FFA) by cholesterol esterase (CEH). FC generates △4-cholesterenone and hydrogen peroxide (H ₂ O ₂ ) under the action of cholesterol oxidase (CHOD), and H ₂ O ₂ reacts with 4-aminoantipyrine (4AAP) and 3,5- In the presence of sodium dichloro-2-hydroxybenzenesulfonate (DHBS), it is catalyzed by oxidase (POD), and the red compound of subquinone amine with the maximum absorption wavelength at 520nm is generated in the reaction, and its absorbance value is proportional to the TC content in the sample. This method has the characteristics of simple operation, non-corrosive reagents and fully automatic operation, and is currently the most widely used determination method in clinical laboratories.

Among the reagents for the enzymatic determination of total cholesterol, peroxidase (POD) is the key substance for the generation of color sources.

POD is a class of oxidoreductases extracted from plants, animals, and microorganisms. In the process of life activities, it mainly catalyzes the decomposition of oxides or peroxides in organisms and oxidative decomposition of other toxins. Since POD is a protein, it can lead to a decrease in enzyme activity during storage, transportation, application, etc., thereby affecting the accuracy of total cholesterol determination.

Contents of the invention

The object of the present invention is to provide a method for measuring serum total cholesterol with hypoheme hexapeptide, which utilizes the principle that ferric porphyrin DhHP-6 has the function of peroxidase to replace peroxidase to prepare a total cholesterol Cholesterol detection reagent, to establish a detection method for quantitative determination of total cholesterol in liquid samples. By detecting the change of light absorbance after the reaction, the amount of total cholesterol in the liquid sample to be tested is calculated. Assay method of the present invention is:

Although various PODs in nature have common biological activities, they do not have the same or similar structure and mechanism of action. Among them, heme peroxidase (HPX) containing metalloporphyrin ring is a kind of enzymes with similar structure and same function, belonging to the superfamily of heme PX protein. Based on the structure of peroxidase with heme as the prosthetic group, a mimic with peroxidase activity was designed and synthesized, named: DeuteroheminAlaHisThrValGluLys (DhHP-6).

DhHP-6 has a molecular weight of 1228.48 and a molecular formula of C ₅₉ H _{74 FeN 13O13} . It is a small molecular compound with very stable chemical properties, no activity decline during storage, transportation, and application, and it is cheap.

a. Detection steps

Measure the change (△A) of the absorbance value before and after the reaction at a wavelength of 520nm in an automatic biochemical analyzer, and calculate the amount of total cholesterol by comparing it with the same measured cholesterol standard.

The amount of cholesterol (mmol/L) = △A _sample /△A _standard × standard concentration.

b. Reagent composition

① Phosphate buffer, ionic strength 300mmol/L, pH 7.7.

② Cholesterol esterase (Cholesterolesterase, CEH; EC3.1.1.13)

③Cholesteroloxidase (Cholesteroloxidase, CHOD; EC1.1.3.6)

④DhHP-6

⑤Triton X-100

⑥Sodium cholate

⑦4-Aminoantipyrine (4-AAP)

⑧Sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS)

Add the above items into the phosphate buffer solution respectively, and store at 2-8°C after dissolving for later use.

C. The operation process

① Add the reagents to the three test tubes (F, B, C) in a 37°C water bath, add the liquid sample, standard solution and water to the test tubes F, B, and C respectively after 1 minute, mix well, and react for 10 minutes. minute;

② Measure the absorbance values of F, B, and C at a wavelength of 520nm in a spectrophotometer (automatic biochemical analyzer), △A _sample =A _F —A _C , △A _standard =A _B —A _C . The amount of total cholesterol was calculated by comparing with the same measured cholesterol standard.

The amount of cholesterol (mmol/L) = △A _sample /△A _standard × standard concentration.

The beneficial effects of the present invention are: the detection method developed by the present invention combined with the principle of modern biological technology has the characteristics of high sensitivity, good stability, economical and practical, and can be widely used in medical and health and food production enterprises, farmers' markets, quality supervision, etc. The sector monitors the amount of total cholesterol in liquid samples.

Description of drawings

Figure 1 is a molecular structure diagram of DhHP-6.

Figure 2 shows the linearity and correlation (3h) of the two reagents measuring different concentrations of H2O2.

Figure 3. Correlation between two reagents for measuring total cholesterol in serum samples.

Figure 4 is the determination of H ₂ O ₂ by the DhHP-6 reagent in Table 1.

Fig. 5 is the determination of H ₂ O _{2 by the total cholesterol assay kit in Table 2} .

Figure 6 is a comparison of the stability of the two reagents in Table 3 for measuring H2O2 at different concentrations.

Fig. 7 is the DhHP-6 reagent in Table 4 to measure serum total cholesterol.

Detailed ways

Example 1

In Figure 2, , the x-axis is H ₂ O ₂ (mmol/L), and the y-axis is the absorbance value.

Figure 3, , the x-axis is the total cholesterol concentration (mmol/L), and the y-axis is the absorbance value.

(1) Reagent preparation:

1. DhHP-6 reagent composition: in 300mmol/L phosphate buffer solution with pH 7.7, add CEH≥800U/L, CHOD≥400U/L, DhHP-61.0mmol/L, TritonX-1003%, cholic acid Sodium 3.0mmol/L, 4-AAP0.5mmol/L, DHBS3.5mmol/L.

2. Cholesterol standard solution 5.2mmol/L (Changchun Huili Biotechnology Co., Ltd.).

3. 3% hydrogen peroxide (Shandong Dezhou Xinkang Disinfection Products Co., Ltd.)

4. Total cholesterol determination kit (Changchun Huili Biotechnology Co., Ltd., Jishi Food and Drug Administration (Quasi) Word 2011 No. 2400064, YZB/Ji 0208-2010).

(2) Experimental method (manual):

1. Experimental steps using H ₂ O ₂ as substrate:

(1) DhHP-6 reagent has the function of peroxidase, which can cause the decomposition of H ₂ O ₂ to generate O ^- . Therefore, different concentrations of H ₂ O ₂ can be used for experimental observation.

(2) Dilute 3.0% H ₂ O ₂ to concentrations of 22.0, 11.0, 5.50, 2.75, 1.375 mmol/L for later use.

(3) Take distilled water, DhHP-6 reagent (self-prepared), total cholesterol assay kit (Huili Biological), and H ₂ O ₂ solutions with different concentrations. Operate according to Tables 1 and 2, and measure the absorbance at 520 nm at 37°C for 1/6 hour (10 minutes), 1.0 hour, 3.0 hour and 24 hours, respectively. The linearity, stability and correlation of the two reagents for measuring different concentrations of H2O2 are shown in Table 3 and Figure 2.

The experimental results using H ₂ O ₂ as the substrate showed that the DhHP-6 reagent had better linearity and stability in the determination of different concentrations of H2O2 and had a good correlation with commercial kits.

2. Total cholesterol detection method:

(1) Serum samples with total cholesterol of 8.62mmol/L were taken and diluted with normal saline to concentrations of 4.31, 2.16, 1.08, and 0.54mmol/L for later use.

(2) Serum samples with different concentrations were taken, and tested with DhHP-6 reagent (self-prepared) and total cholesterol reagent on Hitachi 7180 automatic biochemical analyzer. Reagent 500 μl, serum sample 5.0 μl, react at 37° C. for 10 minutes, and measure the absorbance value at 520 nm. See Table 4 and Figure 3 for the correlation of total cholesterol in serum samples with different concentrations measured by the two reagents.

The results of measuring serum total cholesterol on an automatic biochemical analyzer showed that the DhHP-6 reagent had good linearity and had a good correlation with the commercial kit.

Claims (1)

1. A method for measuring serum total cholesterol with hypoheme hexapeptide, the method is: a. Detection steps Measure the change of absorbance value before and after the reaction at 520nm wavelength of the automatic biochemical analyzer, that is, △A, and calculate the amount of total cholesterol by comparing with the same measured cholesterol standard; The amount of cholesterol is mmol/L=△A _sample /△A _standard ×standard concentration; b. Reagent composition ① Phosphate buffer, ionic strength 300mmol/L, pH 7.7; ② Cholesterol esterase is Cholesterolesterase, CEH; EC3.1.1.13; ③Cholesteroloxidase, namely Cholesteroloxidase, CHOD; EC1.1.3.6; ④ DhHP-6; ⑤ Triton X-100; ⑥Sodium cholate ⑦4-Aminoantipyrine is 4-AAP; ⑧Sodium 3,5-dichloro-2-hydroxybenzenesulfonate is DHBS; Add the above items to the phosphate buffer solution respectively, and store at 2-8°C for later use after dissolving; C. The operation process ①Add the reagents to the three test tubes F, B, and C in a water bath at 37°C. After 1 minute, add the liquid sample, standard solution and water to the F, B, and C test tubes respectively, mix well, and react for 10 minutes ; ②Measure the absorbance values of F, B and C at the wavelength of 520nm in the spectrophotometer, △A _sample =A _F —A _C , △A _standard =A _B —A _C ; by comparing with the same measured cholesterol standard, calculate the amount of total cholesterol; The amount of cholesterol is mmol/L=△A _sample /△A _standard ×standard concentration.