Free fatty acid determination kit
The components of the kit for detecting free fatty acids in serum by adjusting the enzymatic method, including the use of TOPS as the chromogen substrate, BND reacting with Coenzyme A, Triton X-100 as the surfactant and gentamicin as the preservative, solved the problems of low response and poor stability of the existing kits, and achieved higher detection accuracy and stability.
Patent Information
- Application Number
- CN202411929672.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-03-11
AI Technical Summary
The existing kits for detecting free fatty acids in serum have detection performance problems such as low reaction degree and poor stability. This is mainly because the thiol group of Coenzyme A causes the degradation of colored substances, and the impurities react with the chromogen, which inhibits enzyme activity, and the large number of enzymes lead to precipitation.
By adjusting the chromogen, surfactant, preservative and stabilizer components of the kit, specifically including using N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) as the chromogen substrate, adding 5-bromo-5-nitro-1,3-dioxane (BND) to react with Coenzyme A, using Triton X-100 as the surfactant, and using gentamicin as the preservative.
It improves the reactivity and accuracy of the kit, enhances the sensitivity and stability of the reagent, reduces costs, and avoids the inhibition and precipitation of enzyme activity.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of in vitro diagnosis and medical detection, and in particular to a free fatty acid determination kit. Background Art
[0002] Free fatty acids (NEFA), also known as non-esterified fatty acids, are fatty acids in serum that have not undergone esterification with glycerol, cholesterol, etc. They are mainly present in lipids in the human body. Under normal circumstances, their content in serum is extremely low, accounting for only 5%-10% of the total fatty acid content. Free fatty acids can not only reflect human lipid metabolism and blood lipid levels, evaluate blood sugar and assist in the diagnosis of diabetes, but also reflect a variety of other pathological and physiological conditions in the human body, such as obesity, non-alcoholic fatty liver, metabolic syndrome, and cardiovascular and cerebrovascular diseases.
[0003] The methods for determining free fatty acids in serum can be divided into: titration, colorimetry, chemiluminescence, colorimetry, high performance liquid chromatography and enzyme method. The first few methods have a narrow scope of application due to the shortcomings of cumbersome operation, poor accuracy and expensive instruments. The enzyme method is not only fast in determination and relatively accurate in results, but also easy to detect in batches, so this method is widely used. Its reaction principle is that free fatty acids in the presence of ATP and coenzyme A are acted on by acetyl-CoA synthetase to generate acetyl-CoA; acetyl-CoA is oxidized by acetyl-CoA oxidase to generate trans-enoyl-CoA and hydrogen peroxide; and the generated hydrogen peroxide reacts with the chromogenic substrate and 4-aminoantipyrine under the action of peroxidase to generate red quinone imine. The amount of this substance generated is proportional to the free fatty acid content in the sample. The concentration of free fatty acids in the sample can be determined by measuring its absorbance.
[0004] At present, the commercially available enzyme assay kits for free fatty acids in serum also have problems with detection performance such as low reactivity and poor stability. The main problems are: 1. The concentration of coenzyme A in the kit is high, and coenzyme A has a thiol group and is reductive, which causes the final generated colored substance to degrade, thereby reducing the reactivity of the entire reagent and affecting the sensitivity and accuracy of the reagent. The general method is to add N-ethylmaleimide (NEM) to eliminate excess coenzyme A, but this raw material is unstable; 2. The commonly used chromogen is N-ethyl-N-(2-hydroxy-3-benzenesulfonic acid)-3-methylaniline sodium salt (TOOS), which is easy to react with the remaining coenzyme A to reduce the reactivity; 3. The commonly used preservative methylisothiazolinone (MIT) has a certain inhibitory effect on enzyme activity; 4. Because there are many types of enzymes in reagent 2 and the concentration is high, precipitation is easy to occur, which reduces stability. Summary of the invention
[0005] In view of the problems in the detection performance of the existing enzyme method for detecting free fatty acids in serum, the present invention provides a free fatty acid determination kit, which improves the detection performance of the kit by adjusting the chromogen, surfactant, preservative and stabilizer components of the kit.
[0006] A free fatty acid detection kit comprises reagent 1 and reagent 2, wherein the main components of reagent 1 are: tris(hydroxymethylaminomethane) buffer (pH7.5), coenzyme A, adenosine triphosphate (ATP), chromogen substrate, acetyl-CoA synthetase (ACS), magnesium chloride, stabilizer, and preservative; the main components of reagent 2 are: tris(hydroxymethylaminomethane) buffer (pH7.0), 4-aminoantipyrine (4-AAP), acetyl-CoA oxidase (ACOD), peroxidase (POD), 5-bromo-5-nitro-1, 3-dioxane (BND), surfactant, and preservative.
[0007] Furthermore, the chromogenic substrate is N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS).
[0008] Furthermore, the surfactant is one or more of Triton X-100, Tween 20, and lauryl alcohol polyoxyethylene ether (Brij-35).
[0009] Furthermore, the stabilizer is one or more of sucrose, trehalose, and bovine serum albumin (BSA).
[0010] Furthermore, the preservative is one or more of methylisothiazolinone (MIT), Proclin series, gentamicin, and sodium azide.
[0011] Furthermore, in the reagent 1: the concentration of tris hydroxymethylaminomethane buffer (pH7.5) is 50-500 mmol / L, the concentration of coenzyme A is 1-10 mmol / L, the concentration of adenosine triphosphate (ATP) is 5-20 mmol / L, the concentration of TOPS is 0.6-1.2 g / L, the concentration of acetyl-CoA synthetase (ACS) is 0.4-2.0 KU / L, the concentration of magnesium chloride is 5 mmol / L, the concentration of stabilizer is 0.5-5 g / L, and the concentration of preservative is 0.2-0.4‰; in the reagent 2: the concentration of tris hydroxymethylaminomethane buffer (pH7.0) is 50-500 mmol / L, the concentration of 4-aminoantipyrine (4-AAP) is 5-10 mmol / L, the concentration of acetyl-CoA oxidase (ACOD) is 10-20 KU / L, and the concentration of peroxidase (POD) is 12-36 KU / L, BND concentration is 0.1-1.0‰, surfactant concentration is 0.5-5 g / L, and preservative concentration is 0.2-0.4‰.
[0012] The present invention has the following beneficial effects: 1. Add 5-bromo-5-nitro-1, 3-dioxane (BND) to reagent 2. BND can react chemically with the sulfhydryl group of coenzyme A, thereby reducing the interference of coenzyme A with hydrogen peroxide, and ultimately will not lead to a decrease in the amount of red quinone imine generated, thereby improving the reactivity and accuracy of the entire reagent; 2. Coenzyme A has a thiol group and is reductive, which can cause the degradation of the colored substance generated by the reaction. The appropriate concentration of coenzyme A, on the one hand, can provide a sufficient amount of substrate for the reaction and reduce the cost of the kit; on the other hand, it will not cause the degradation of the red colored substance and reduce the reactivity; 3. The remaining coenzyme A in the reagent will react with the chromogen as an impurity, thereby reducing the reactivity of the entire reagent. The chromogen substrate N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) added in the present invention is not easily interfered by coenzyme A and has a higher sensitivity; 4. Compared with methylisothiazolinone (MIT), the preservative gentamicin in reagent 1 of the present invention does not interfere with the sulfhydryl group of coenzyme A, has no inhibitory effect on enzyme activity, and has better stability.
[0013] 5. Since reagent 2 contains many types of enzymes and a high concentration, it is easy to produce precipitation, which leads to reduced stability. The present invention improves the problem of poor stability of reagent 2 due to precipitation by adding Triton X-100 to reagent 2. DETAILED DESCRIPTION
[0014] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.
[0015] Example 1 The present invention provides a free fatty acid detection kit, comprising reagent 1 and reagent 2, the main components of which are as follows:
[0016] This example is different from Example 1 in that 5-bromo-5-nitro-1, 3-dioxane (BND) is not added.
[0017] Comparative Example 2 The difference between this embodiment and embodiment 1 is that, while embodiment 1 uses N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) as the chromogenic substrate, this embodiment uses TOOS, a commonly used chromogenic substrate for free fatty acids on the market, and does not add 5-bromo-5-nitro-1, 3-dioxane (BND).
[0018] 1. Operation method The embodiments of the present invention all use a fully automatic biochemical analyzer Hitachi 7180 for detection, with a detection wavelength of 546 nm as the main wavelength and 600 nm as the secondary wavelength, to detect the accuracy, analytical sensitivity and stability of the reagents. The specific operation is as follows: 2. Accuracy Example 1, Comparative Example 1 and Comparative Example 2 were used to measure serum samples with low and high concentrations of 0.62 mmol / L and 1.12 mmol / L, respectively. The test was repeated 3 times. The average value of the test results was recorded as (M), the reference material labeled value was recorded as (T), and the relative deviation was recorded as (B). B = (M- T ) / T ×100%, the accuracy of the three reagents was tested respectively, and the results are shown in Table 1.
[0019] Table 1. Accuracy test results In Example 1, by adding the novel chromogenic substrates N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) and 5-bromo-5-nitro-1, 3-dioxane (BND), the relative deviation was 2.69% when measuring low-concentration samples, and the relative deviation was only 0.89% when measuring high-concentration samples, which was superior to Comparative Example 1 and Comparative Example 2. Comparative Example 2 used a commonly used chromogenic substrate, and the relative deviation of the reagent when measuring high-concentration samples was less than 10%, which met the conventional detection requirements, and the relative deviation was too large when measuring low concentrations.
[0020] 3. Analytical sensitivity The sample with a concentration of 1.04 mmol / L was measured, and the absorbance difference produced in 5 minutes was recorded and converted into the change in its absorbance at a concentration of 1 mmol / L, i.e., the analytical sensitivity. The test results are shown in Table 2.
[0021] Table 2. Analytical sensitivity test results The experimental results show that the analytical sensitivity of Example 1 is significantly better than that of Comparative Example 1, and the analytical sensitivity increases by 22.00% and 31.38% compared with Comparative Example 1 and Comparative Example 2, respectively.
[0022] 4. Stability The stability difference between Example 1 and the comparative example was observed. The specific operation was as follows: Example 1 and Comparative Example 2 respectively measured samples at low and high levels of 0.62 mmol / L and 1.12 mmol / L, and the measurement was repeated 10 times. The average value of the test results was used as the initial value (0th day). Then the samples were placed in a 37°C constant temperature drying oven for accelerated testing, and the samples were tested once each after acceleration for 1d, 3d, 5d, 7d, and 9d. Each sample was measured 10 times and the average value was taken. The data were recorded. The test results are shown in Table 3.
[0023] Table 3. Results of 37℃ accelerated stability test The test results show that the initial values of the two samples in Example 2 are lower than the labeled values. During the acceleration process, the measured values continue to decrease. After 7 days of acceleration, the relative deviations of the low and high values are -12.50% and -10.89%, respectively, both higher than 10%, which do not meet the measurement requirements. During the acceleration process, the maximum relative deviation of Example 1 is -4.84%, both less than ±10%, which meets the requirements for the stability of the reagent. It can be seen that the chromogen substrate is replaced by N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) instead of TOOS, and 5-bromo-5-nitro-1, 3-dioxane (BND) is added, which can effectively improve the stability of the reagent.
Claims
1. A free fatty acid determination kit, comprising reagent 1 and reagent 2, wherein the main components of reagent 1 are: tris(hydroxymethylaminomethane) buffer (pH 7.5), coenzyme A, adenosine triphosphate (ATP), chromogen substrate, acetyl-CoA synthetase (ACS), magnesium chloride, stabilizer, and preservative; the main components of reagent 2 are: tris(hydroxymethylaminomethane) buffer (pH 7.0), 4-aminoantipyrine (4-AAP), acetyl-CoA oxidase (ACOD), peroxidase (POD), 5-bromo-5-nitro-1, 3-dioxane (BND), surfactant, and preservative.
2. A free fatty acid determination kit as claimed in claim 1, characterized in that: The chromogen substrate in the reagent 1 is N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS).
3. A free fatty acid determination kit according to claim 1, characterized in that: The surfactant in the reagent 2 is one or more of Triton X-100, Tween 20, and lauryl alcohol polyoxyethylene ether (Brij-35).
4. A free fatty acid determination kit as claimed in claim 1, characterized in that: The stabilizer in the reagent 1 is one or more of sucrose, trehalose, and bovine serum albumin (BSA).
5. A free fatty acid determination kit as claimed in claim 1, characterized in that: The preservatives in the reagents 1 and 2 are one or more of methylisothiazolinone (MIT), Proclin series, gentamicin, and sodium azide.
6. A free fatty acid determination kit according to claim 1, characterized in that: In the reagent 1, the concentration of tris(hydroxymethyl)aminomethane buffer (pH7.5) is 50-500 mmol / L, the concentration of coenzyme A is 1-10 mmol / L, the concentration of adenosine triphosphate (ATP) is 5-20 mmol / L, the concentration of TOPS is 0.6-1.2 g / L, the concentration of acetyl-CoA synthetase (ACS) is 0.4-2.0 KU / L, the concentration of magnesium chloride is 5 mmol / L, the concentration of stabilizer is 0.5-5 g / L, and the concentration of preservative is 0.2-0.4‰; in the reagent 2, the concentration of tris(hydroxymethyl)aminomethane buffer (pH7.0) is 50-500 mmol / L, the concentration of 4-aminoantipyrine (4-AAP) is 5-10 mmol / L, the concentration of acetyl-CoA oxidase (ACOD) is 10-20 KU / L, and the concentration of peroxidase (POD) is 12-36 KU / L, BND concentration is 0.1-1.0‰, surfactant concentration is 0.5-5 g / L, and preservative concentration is 0.2-0.4‰.