A method for improving the accuracy of determining the content of malondialdehyde in a biological sample

Abstract

The application discloses a method for improving the determination accuracy of the content of malondialdehyde in a biological sample, which comprises adding a buffer solution, a protease solution and a thiobarbituric acid (TBA) into a malondialdehyde standard solution, and determining the absorbance of the malondialdehyde standard solution; subsequently, a standard curve is constructed, and the actual concentration of the malondialdehyde in the biological sample is calculated through the measured absorbance of the biological sample solution. The sample processing process of the method is simple, the Buffer A buffer solution and the protease used can release the MDA combined with the protein, can not only avoid the adsorption interference of the protein precipitation on the red complex of the MDA and the TBA and the extraction limitation of the supernatant, but also can detect the protein combined state MDA, can significantly improve the detection limit of the sample, is good in selectivity, improves the accuracy of the detection result, and can be used for the trace detection of clinical precious samples.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples. Background Technology

[0002] When organisms are subjected to abiotic stress, they produce large amounts of superoxide free radicals, leading to peroxidation of membrane lipids and the generation of malondialdehyde (MDA). Excessive accumulation of MDA can cause cross-linking and polymerization of biomolecules such as proteins and nucleic acids, resulting in alterations in the structure and function of the cell membrane. Therefore, membrane lipid peroxidation is an important marker of cell membrane damage, and the level of peroxidation in the cytoplasmic membrane is typically characterized and measured using MDA, a product of membrane lipid peroxidation. Furthermore, under physiological conditions, the interaction between various proteins and MDA has been shown to produce various potentially toxic adducts and induce protein cross-linking. When MDA and MDA-protein adducts are produced endogenously in cells and tissues as part of stress-induced oxidative processes, they are considered potential pathogenic factors for many human diseases, such as different types of cancer, atherosclerosis, diabetes, Alzheimer's disease, and aging.

[0003] Currently, methods for detecting MDA include fluorescence methods, high-performance liquid chromatography (HPLC), gas chromatography (GC), and the thiobarbituric acid (TBA) method. Fluorescence methods are easily affected by various types of scattered light, such as Rayleigh scattering from the solvent, scattering from the container surface, scattering from colloidal particles, and Raman scattering from the solvent. This significantly impacts the fluorescence spectrum, resulting in a substantial reduction in detection sensitivity and accuracy. HPLC and GC are complex to operate, require highly skilled personnel, and involve expensive equipment, making them difficult to implement in general laboratories. Therefore, the TBA method is the most widely used for MDA detection. The thiobarbituric acid (TBA) method is simple to operate. Under conditions such as high temperature (95℃) and low pH, MDA reacts with TBA to form a relatively stable reddish-brown trimethylolamine (3,5,5-trimethyloxazol-2,4-dione) complex. Its maximum absorption wavelength is at 532 nm, therefore it can be determined using ultraviolet spectrophotometry. The TBA method offers good reproducibility, requires no expensive equipment, and is suitable for general laboratory use. However, traditional TBA methods produce significant protein precipitation upon heating biological samples (including tissue lysates, blood, serum, plasma, semen, seminal plasma, etc.). These protein precipitates adsorb the red reaction complex of MDA and TBA, leading to measurement bias and hindering the separation and extraction of the supernatant. Furthermore, traditional TBA methods can only detect free MDA, while protein-bound MDA, which better reflects cytotoxicity, is often difficult to detect. Therefore, a larger sample volume is usually required for the colorimetric reaction, lacking high sensitivity for detecting trace samples. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples, so as to eliminate the adsorption interference of protein precipitation, and at the same time detect MDA bound to proteins, so as to achieve the purpose of accurate and trace detection of MDA in biological samples.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide a method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples, comprising the following steps:

[0006] S1: Add buffer and protease solution to malondialdehyde standard solution, incubate at 50-60℃ for 10-30 min, then add TBA reagent, seal and react at 90-110℃ for 20-40 min, and finally measure the absorbance A1 of malondialdehyde standard solution at 532 nm.

[0007] S2: A standard curve y = ax + b is constructed by linear fitting with the concentration c1 of the malondialdehyde standard solution as the abscissa and the absorbance A1 as the ordinate, where a and b are constants;

[0008] S3: Preparation of biological sample solutions;

[0009] S4: Replace the malondialdehyde standard solution in step S1 with an equal volume of biological sample solution, add buffer and protease solution, and incubate and react under the same conditions. Then measure the absorbance A2 of the biological sample solution at 532 nm. Finally, calculate the concentration c2 of malondialdehyde in the biological sample solution based on the standard curve obtained in step S2.

[0010] S5: The actual concentration c of malondialdehyde in the biological sample was calculated. 实际值 The calculation formula is as follows:

[0011]

[0012] In the formula, A 空白值 The absorbance of deionized water at 532 nm is given.

[0013] Based on the above technical solution, the present invention can be further improved as follows:

[0014] Furthermore, the buffer solution is Buffer A buffer.

[0015] Furthermore, Buffer A consists of 0.5-1 wt% Triton X-100, 0.8-1.2 wt% sodium deoxycholate, and 0.05-0.15 wt% SDS, with the balance being water.

[0016] Furthermore, the protease solution is a proteinase K, pepsin, or papain solution.

[0017] Furthermore, the concentration of the protease solution was 0.015-0.02 g / mL.

[0018] Furthermore, the preparation steps for TBA reagent are as follows: dissolve TBA, trichloroacetic acid, and EDTA in water at a mass ratio of 0.1-2:7.5-9.5:0.1-0.2 to obtain the reagent.

[0019] Furthermore, the concentration of TBA reagent is 0.01-0.05 mol / L.

[0020] Furthermore, the volume ratio of malondialdehyde standard solution, buffer solution, protease solution and TBA reagent is 10-15:10-15:1-5:1-10.

[0021] Furthermore, biological samples include tissue lysates, blood, serum, plasma, semen, or seminal plasma.

[0022] Furthermore, the mass fraction of the biological sample solution is 10-20%.

[0023] The beneficial effects of the present invention are as follows: The sample processing of the present invention is simple. The Buffer A buffer and protease used can release MDA bound to proteins. This not only avoids the adsorption interference of protein precipitation on the red complex of MDA and TBA and the limitation of supernatant extraction, but also detects protein-bound MDA. It can significantly improve the detection limit of the sample, has good selectivity, improves the accuracy of the detection results, and can also be used for the trace detection of precious clinical samples. Attached Figure Description

[0024] Figure 1 This is a standard curve graph of Example 1;

[0025] Figure 2 This is a comparison diagram of the reaction precipitation of Comparative Example 1, Example 1, and Comparative Example 2;

[0026] Figure 3 This demonstrates the linear relationship between sample volume and measured concentration in Examples 1, 1, and 2.

[0027] Figure 4 The method described in Example 1 was used to detect the MDA content in diabetic patients before and after the onset of the disease.

[0028] Figure 5 The method used in Comparative Example 1 was used to detect the MDA content of diabetic patients before and after the onset of the disease. Detailed Implementation

[0029] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0030] Example 1:

[0031] A method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples includes the following steps:

[0032] S1: Mix 1 wt% Triton X-100, 1 wt% sodium deoxycholate, 0.1 wt% SDS and 97.9 wt% water to obtain Buffer A; dissolve TBA, trichloroacetic acid and EDTA in water at a mass ratio of 1:8:0.15 to obtain TBA reagent; then simultaneously add 12 mL of Buffer A and 3 mL of proteinase K solution with a concentration of 0.015 g / mL to 12 mL of malondialdehyde standard solutions with concentrations of 2, 5, 10, 20 and 50 μmol / L, incubate at 55 °C for 20 min, then add 5 mL of TBA reagent, seal and react at 100 °C for 30 min, and finally measure the absorbance A1 of malondialdehyde standard solutions of different concentrations at 532 nm.

[0033] S2: A standard curve was constructed by linearly fitting the concentration c1 of the malondialdehyde standard solution to the x-axis and the absorbance A1 to the y-axis. The linear relationship was y = 0.0033x + 0.0788, R0. 2 =0.9997;

[0034] S3: Mix plasma and deionized water to obtain a 15 wt% biological sample solution;

[0035] S4: Replace the malondialdehyde standard solution in step S1 with an equal volume of biological sample solution, add Buffer A and proteinase K solution, and incubate under the same conditions. Then measure the absorbance A2 of the biological sample solution at 532 nm. Finally, based on the standard curve obtained in step S2 (y = 0.0033x + 0.0788, R...),... 2 =0.9997), the concentration c2 of malondialdehyde in the biological sample solution was calculated;

[0036] S5: The actual concentration c of malondialdehyde in the biological sample was calculated. 实际值The calculation formula is as follows:

[0037]

[0038] In the formula, A 空白值 The absorbance of deionized water at 532 nm is given by a = 0.0033 and b = 0.0788.

[0039] Example 2:

[0040] A method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples includes the following steps:

[0041] S1: Mix 0.5wt% Triton X-100, 1.2wt% sodium deoxycholate, 0.05wt% SDS and 98.25wt% water to obtain Buffer A; dissolve TBA, trichloroacetic acid and EDTA in water at a mass ratio of 2:9.5:0.1 to obtain TBA reagent; then simultaneously add 15mL of Buffer A and 5mL of 0.02g / mL pepsin solution to 10mL of malondialdehyde standard solutions with concentrations of 2, 5, 10, 20 and 50μmol / L, respectively, incubate at 50℃ for 30min, then add 10mL of TBA reagent, seal and react at 110℃ for 20min, and finally measure the absorbance A1 of malondialdehyde standard solutions of different concentrations at 532nm.

[0042] S2: A standard curve y = ax + b is constructed by linear fitting with the concentration c1 of the malondialdehyde standard solution as the abscissa and the absorbance A1 as the ordinate, where a and b are constants;

[0043] S3: Mix plasma and deionized water to obtain a 10 wt% biological sample solution;

[0044] S4: Replace the malondialdehyde standard solution in step S1 with an equal volume of biological sample solution, add Buffer A and pepsin solution, and incubate and react under the same conditions. Then measure the absorbance A2 of the biological sample solution at 532 nm. Finally, calculate the concentration c2 of malondialdehyde in the biological sample solution based on the standard curve obtained in step S2.

[0045] S5: The actual concentration c of malondialdehyde in the biological sample was calculated. 实际值 The calculation formula is as follows:

[0046]

[0047] In the formula, A 空白值 The absorbance of deionized water at 532 nm is given.

[0048] Example 3:

[0049] A method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples includes the following steps:

[0050] S1: Mix 1 wt% Triton X-100, 0.8 wt% sodium deoxycholate, 0.15 wt% SDS and 98.05 wt% water to obtain Buffer A; dissolve TBA, trichloroacetic acid and EDTA in water at a mass ratio of 0.1:7.5:0.2 to obtain TBA reagent; then simultaneously add 10 mL of Buffer A and 1 mL of 0.015 g / mL papain solution to 15 mL of malondialdehyde standard solutions with concentrations of 2, 5, 10, 20 and 50 μmol / L, respectively, incubate at 60 °C for 10 min, then add 1 mL of TBA reagent, seal and react at 90 °C for 40 min, and finally measure the absorbance A1 of malondialdehyde standard solutions of different concentrations at 532 nm.

[0051] S2: A standard curve y = ax + b is constructed by linear fitting with the concentration c1 of the malondialdehyde standard solution as the abscissa and the absorbance A1 as the ordinate, where a and b are constants;

[0052] S3: Mix plasma and deionized water to obtain a 20 wt% biological sample solution;

[0053] S4: Replace the malondialdehyde standard solution in step S1 with an equal volume of biological sample solution, add Buffer A and papain solution, and incubate and react under the same conditions. Then measure the absorbance A2 of the biological sample solution at 532 nm. Finally, calculate the concentration c2 of malondialdehyde in the biological sample solution based on the standard curve obtained in step S2.

[0054] S5: The actual concentration c of malondialdehyde in the biological sample was calculated. 实际值 The calculation formula is as follows:

[0055]

[0056] In the formula, A 空白值 The absorbance of deionized water at 532 nm is given.

[0057] Comparative Example 1:

[0058] A traditional method for determining malondialdehyde (MDA) content in biological samples includes the following steps:

[0059] S1: TBA, trichloroacetic acid and EDTA were dissolved in water at a mass ratio of 1:8:0.15 to obtain TBA reagent; then 12 mL of malondialdehyde standard solutions with concentrations of 2, 5, 10, 20 and 50 μmol / L were incubated at 55 °C for 20 min, then 5 mL of TBA reagent was added, sealed and reacted at 100 °C for 30 min, and finally the absorbance A1 of malondialdehyde standard solutions of different concentrations was measured at 532 nm.

[0060] S2: A standard curve y = ax + b is constructed by linear fitting with the concentration c1 of the malondialdehyde standard solution as the abscissa and the absorbance A1 as the ordinate, where a and b are constants;

[0061] S3: Mix plasma and deionized water to obtain a 15 wt% biological sample solution;

[0062] S4: Replace the malondialdehyde standard solution in step S1 with an equal volume of biological sample solution, incubate and react under the same conditions, then measure the absorbance A2 of the biological sample solution at 532 nm, and finally calculate the concentration c2 of malondialdehyde in the biological sample solution based on the standard curve obtained in step S2.

[0063] S5: The actual concentration c of malondialdehyde in the biological sample was calculated. 实际值 The calculation formula is as follows:

[0064]

[0065] In the formula, A 空白值 The absorbance of deionized water at 532 nm is given.

[0066] Comparative Example 2:

[0067] A traditional method for determining malondialdehyde (MDA) content in biological samples includes the following steps:

[0068] S1: TBA, trichloroacetic acid and EDTA were dissolved in water at a mass ratio of 2:9.5:0.1 to obtain TBA reagent; then 10 mL of malondialdehyde standard solutions with concentrations of 2, 5, 10, 20 and 50 μmol / L were incubated at 50 °C for 30 min, then 10 mL of TBA reagent was added, sealed and reacted at 110 °C for 20 min, and finally the absorbance A1 of malondialdehyde standard solutions of different concentrations was measured at 532 nm.

[0069] S2: A standard curve y = ax + b is constructed by linear fitting with the concentration c1 of the malondialdehyde standard solution as the abscissa and the absorbance A1 as the ordinate, where a and b are constants;

[0070] S3: Mix plasma and deionized water to obtain a 10 wt% biological sample solution;

[0071] S4: Replace the malondialdehyde standard solution in step S1 with an equal volume of biological sample solution, incubate and react under the same conditions, then measure the absorbance A2 of the biological sample solution at 532 nm, and finally calculate the concentration c2 of malondialdehyde in the biological sample solution based on the standard curve obtained in step S2.

[0072] S5: The actual concentration c of malondialdehyde in the biological sample was calculated. 实际值 The calculation formula is as follows:

[0073]

[0074] In the formula, A 空白值 The absorbance of deionized water at 532 nm is given.

[0075] Experimental example:

[0076] 1. Prepare five groups of biological sample solutions with different concentrations: Sample 1: 10 μL plasma + 90 μL ddH2O; Sample 2: 20 μL plasma + 80 μL ddH2O; Sample 3: 30 μL plasma + 70 μL ddH2O; Sample 4: 40 μL plasma + 60 μL ddH2O; Sample 5: 50 μL plasma + 50 μL ddH2O. The experiments were conducted according to the methods of Example 1, Comparative Examples 1 and 2, respectively. The results are shown in Table 1.

[0077] Table 1 Comparison of the method of the present invention with traditional malondialdehyde detection methods.

[0078]

[0079]

[0080] Figure 2 The figures show a comparison of the reaction precipitation of Comparative Example 1, Example 1, and Comparative Example 2. The left test tube shows the actual precipitate of Comparative Example 1, the middle tube shows the actual precipitate of Example 1, and the right tube shows the actual precipitate of Comparative Example 2. As can be seen from the figures, protein precipitation occurred in both Comparative Examples 1 and 2, with the red precipitate in Comparative Example 2 being particularly prominent. The supernatant was difficult to extract, leading to a significant deviation in the malondialdehyde (MDA) content detection results. In contrast, no precipitation occurred in Example 1. This indicates that the Buffer A and proteinase K used in the method of this invention can release MDA bound to proteins, not only avoiding the interference of protein precipitation on the adsorption of the red MDA-TBA complex, but also allowing the detection of protein-bound MDA. This demonstrates good selectivity and improves the accuracy of the detection results. Figure 3This represents the linear relationship between sample volume and measured concentration in Examples 1, 1, and 2.

[0081] 2. Feasibility Verification

[0082] Three groups of 30 μL plasma sample solutions were prepared for spiking and recovery tests: Sample 1: 30 μL plasma + 70 μL ddH2O; Sample 2: 30 μL plasma + 65 μL ddH2O + 5 μL standard; Sample 3: 30 μL plasma + 69.5 μL ddH2O + 0.5 μL standard (standard was 1 mM malondialdehyde solution). The experiments were conducted according to the methods of Example 1, Comparative Examples 1 and 2, respectively. The results are shown in Table 2.

[0083] Table 2 Comparison of the recovery rates of the method of the present invention and the traditional malondialdehyde detection method.

[0084]

[0085]

[0086] Results Analysis: The control recovery rate range for the spiking recovery experiment in biological sample analysis is 80-120%. The closer the result is to 100%, the higher the accuracy of the analytical method. As shown in Table 2, the spiking recovery rate of Comparative Example 1 was 19.72-30.07%, and that of Comparative Example 2 was 9.06-15.46%, indicating that in traditional malondialdehyde (MDA) detection methods, proteins have a strong adsorption effect on added MDA, causing significant deviations in the detection results. In contrast, the spiking recovery rate of Example 1 was 83.03-102.24%, indicating that the detection method of this invention has high accuracy and meets the standards for spiking recovery experiments.

[0087] 3. By tracking the health of the population, the differences in plasma MDA levels before and after the onset of diabetes were compared, and the advantages and disadvantages of the method of this invention compared with traditional methods were compared. Plasma samples (34×2) from 34 diabetic patients before and after the onset of diabetes were tested for malondialdehyde (MDA) content according to the detection methods of Example 1 and Comparative Example 1. The results are as follows: Figure 4 and 5 As shown. Statistical analysis revealed a significant difference between the two groups of plasma samples measured in Example 1, with a P-value of 0.045. Figure 4 The p-value for the two groups of plasma samples measured in Comparative Example 1 was 0.28. Figure 5 The difference was not statistically significant, indicating that the detection method of Example 1 is more sensitive than that of Comparative Example 1.

Claims

1. A method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples, characterized in that, Includes the following steps: S1: Add Buffer A and protease solution to the malondialdehyde standard solution, incubate at 50-60℃ for 10-30 min, then add TBA reagent, seal and react at 90-110℃ for 20-40 min, finally measure the absorbance A1 of the malondialdehyde standard solution at 532 nm; the Buffer A consists of 0.5-1 wt% Triton X-100, 0.8-1.2 wt% sodium deoxycholate and 0.05-0.15 wt% SDS, with the balance being water; the protease solution is proteinase K, pepsin or papain solution; S2: A standard curve y=ax+b is constructed by linear fitting with the concentration c1 of the malondialdehyde standard solution as the abscissa and the absorbance A1 as the ordinate, where a and b are constants; S3: Preparation of biological sample solutions; S4: Replace the malondialdehyde standard solution in step S1 with an equal volume of biological sample solution, add buffer and protease solution, and incubate and react under the same conditions. Then measure the absorbance A2 of the biological sample solution at 532 nm. Finally, calculate the concentration c2 of malondialdehyde in the biological sample solution based on the standard curve obtained in step S2. S5: The actual concentration c of malondialdehyde in the biological sample was calculated using the following formula: ， In the formula, A 空白值 The absorbance of deionized water at 532 nm is given.

2. The method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples according to claim 1, characterized in that: The concentration of the protease solution is 0.015-0.02 g / mL.

3. The method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples according to claim 1, characterized in that, The preparation steps of the TBA reagent are as follows: dissolve TBA, trichloroacetic acid and EDTA in water at a mass ratio of 0.1-2:7.5-9.5:0.1-0.2 to obtain the reagent.

4. The method for improving the accuracy of malondialdehyde content determination in biological samples according to claim 3, characterized in that: The concentration of the TBA reagent is 0.01-0.05 mol / L.

5. The method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples according to claim 1, characterized in that: The volume ratio of the malondialdehyde standard solution, buffer solution, protease solution and TBA reagent is 10-15:10-15:1-5:1-10.

6. The method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples according to claim 1, characterized in that: The biological samples are tissue lysates, blood, serum, plasma, semen, or seminal plasma.

7. The method for improving the accuracy of malondialdehyde (MDA) content determination in biological samples according to claim 6, characterized in that: The biological sample solution has a mass fraction of 10-20%.

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