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动物或植物细胞发生氧化应激（oxidative stress）时，会发生脂质氧化，丙二醛（Malondialdehyde, MDA）是一种生物体脂质氧化的天然产物，一些脂肪酸氧化后逐渐分解为一系列包括MDA在内的复杂化合物，此时通过检测MDA的水平即可检测脂质氧化的水平，因此MDA的测定被广泛用作脂质氧化的指标。生物体内的一些其它生化反应也会产生MDA，例如thromboxane synthase也可以催化产生，但只要在测定时设置适当对照即可观察到脂质氧化水平的变化。
检测原理：丙二醛在较高温度及酸性环境中可与TBA发生反应，形成红色的MDA-TBA加合物，MDA-TBA加合物在535nm处有最大吸收，据此可以通过比色法进行检测，另外MDA-TBA加合物也可以在535nm被激发产生最大发射波长553nm，据此也可以进行荧光检测。
适用样本：血浆、血清、尿液、动植物组织或细胞裂解液
自备仪器和试剂
操作步骤
1、样品的准备
1.1 血清、血浆、尿液、脑脊液样品
从待测样本中分离出的血清或血浆不应有溶血，直接检测，如超过线性范围，用生理盐水或PBS稀释后检测。
1.2 组织、细胞等样品
组织或细胞可以使用PBS或RIPA裂解液等进行匀浆或裂解，匀浆或裂解组织时组织重量占匀浆液或裂解液的比例应为10%；对于细胞，每10⁶个细胞使用0.1ml裂解液或匀浆液，匀浆或裂解后4℃ 8000~12000g离心10min，取上清用于后续测定。匀浆或裂解等样品制备步骤宜在冰浴或4℃条件下进行操作。
注：样品准备完毕后可以用BCA蛋白浓度测定试剂盒（B665595/R1491648）测定蛋白浓度，以便于后续计算单位蛋白重量组织或细胞内MDA含量。
| 试剂类别 | 化学成分 | 是否干扰 |
| 缓冲液 | HEPES (100mM) | 否 |
| 缓冲液 | Borate (50mM) | 否 |
| 缓冲液 | Phosphate (100mM) | 否 |
| 缓冲液 | Tris (25mM) | 否 |
| 去垢剂 | CHAPS (≤1%) | 否 |
| 去垢剂 | Triton X-100 (≤1%) | 否 |
| 去垢剂 | Tween 20 (≤1%) | 否 |
| 抑制剂/螯合剂 | PMSF (≤200μM) | 否 |
| 抑制剂/螯合剂 | EDTA (≤1mM) | 否 |
| 抑制剂/螯合剂 | EGTA (≤1mM) | 否 |
| 抑制剂/螯合剂 | Antipain (≤100μg/ml) | 否 |
| 抑制剂/螯合剂 | Chymostatin (≤10μg/ml) | 否 |
| 抑制剂/螯合剂 | Leupeptin (≤10μg/ml) | 否 |
| 抑制剂/螯合剂 | Trypsin (≤10μg/ml) | 否 |
| 其他 | Glycerol (≤10%) | 否 |
| 其他 | Sucrose (250mM) | 是 |
2、TBA工作液的配制
称取适量TBA，用TBA稀释液配制成浓度为0.68％的TBA工作液；例如取68mg TBA用10ml TBA稀释液配制，最终浓度即为0.68%的TBA工作液，TBA工作液需完全溶解后再使用，可以加热到60℃促溶，并可通过反复剧烈Vortex促溶；配制好的TBA工作液4℃避光保存，至少1个月内有效。
3、系列标准品的稀释
取适量MDA标准品 (1mmol/L)，用恰当溶液稀释至1、2、5、10、20μM（如果进行简易快速检测，标准品直接稀释10μM）。
注意：待测样品为血清、血浆时，标准品宜用生理盐水稀释；待测样品由匀浆液、裂解液、PBS获得时，标准品宜用相同溶液稀释，其原则是保证空白管、标准管、测定管具有可比性；配制好的MDA标准品4℃避光保存，至少3个月内有效。
4、MDA检测工作液的配制
临检测前，根据待测定的样品数（含对照），参考下表新鲜配制适量的MDA检测工作液。
检测次数 | 1次 | 10次 | 20次 |
TBA工作液 | 0.75ml | 7.5ml | 15ml |
抗氧化剂 | 0.031ml | 0.31ml | 0.62ml |
MDA检测液 | 0.075ml | 0.75ml | 1.5ml |
MDA分离液 | 2.25ml | 22.5ml | 45ml |
总体积 | 3.106ml | 31.06ml | 62.12ml |
5、MDA加样
在离心管或其它适当容器内加入0.08ml适当溶液作为空白对照（注意：待测样品为血清、血浆时，标准品宜用生理盐水稀释；待测样品由匀浆液、裂解液、PBS获得时，标准品宜用相同溶液稀释，其原则是保证空白管、标准管、测定管具有可比性），加入0.08ml上述不同浓度系列标准品用于制作标准曲线（如果进行简易快速检测，直接加入浓度为10μM的标准品），加入0.08ml样品用于测定；随后加入3ml MDA检测工作液。可参考下表设置检测反应体系，依次加入试剂：
加入物质(ml) | 空白管 | 标准管 | 测定管 |
匀浆液、裂解液、PBS、生理盐水等 | 0.08 | － | － |
标准品 | － | 0.08 | － |
待测样品 | － | － | 0.08 |
MDA检测工作液 | 3 | 3 | 3 |
混匀，加盖，95℃水浴煮沸40min，加热时务必注意避免液体暴沸溅出；如果使用加热块（Heat block）进行加热注意用重物压紧离心管盖；如果使用沸水浴，则需使用可把盖子锁死的离心管或螺旋盖离心管，或用Parafilm封住离心管口，用针头刺一小孔；最方便和准确的加热方法是使用带有热盖并可以加热金属浴。
6、MDA测定
水浴或流水冷却至室温，3000r/min离心15min或4000r/min离心10min，取上清，其颜色为黄色至棕红色，蒸馏水调零，比色杯光径应为1cm，分光光度计测定535nm处空白管、标准管、测定管的吸光度，如果不方便也可以测定530~540nm之间的吸光度，分别记为A空白、A标准、A测定。
7、结果计算
如果进行简易快速检测，直接以10μM标准品进行计算，获得MDA的摩尔浓度；如果需要精确计算，以MDA标准品浓度为横坐标，以对应的吸光度为纵坐标，制作标准曲线，根据标准曲线计算出MDA提取液的浓度（C1，μmol/L）；对于固体状组织，可以通过单位重量的蛋白含量或组织重量等来表示最初样品中的MDA含量（C2，μmol/g），例如μmol/g 蛋白或组织。
简易快速血清、血浆、尿液等液体样品中MDA含量计算公式：
MDA浓度 (μmol/L)=C1=(A测定-A空白)/(A标准-A空白)×10
简易快速细胞、组织样品中MDA含量计算公式：
MDA含量 (μmol/g)=C2=(A测定-A空白)/(A标准-A空白)×10/蛋白质浓度(mg/ml)
=C1/蛋白质浓度(mg/ml)
MDA含量(μmol/g)=C2=(A测定-A空白)/(A标准-A空白)×10/[w/(V总×10⁻³)]
=C1/[w/(V总×10⁻³)]
参数说明：
A测定：测定管的吸光度
A标准：标准管的吸光度
A空白：空白管的吸光度
w：组织样品质量(g)
V总：样本提取液总体积(ml)
附录
参考标准曲线范围：
测定MDA标准在10μM时，通过酶标仪测定其吸光度多在1.3~2.3之间（未调零）；测定MDA标准在1、2、4、6、8、10、12、14μM时吸光度，据此作出其标准曲线：
注意：由于检测仪器和操作手法等条件的不同，参考值范围会有波动，该值仅供参考，对于要求精确计算MDA含量的，可以进行多点测定；根据测定经验显示，标准品浓度在2μmol/L以下，标准品浓度在16μmol/L以上，标准曲线会有偏差。
注意事项
1、上述低温试剂避免反复冻融，以免失效或效率下降。
2、参考取样量：血清、血浆、尿液取0.1ml；低密度脂蛋白悬液取0.1~0.2ml；食用油取0.03ml；肝脏、心肌、肌肉等，取5%或10%匀浆0.1~0.2ml。
3、测定样品吸光度值较低时，可将水浴延长至80min，但应同时延长，以免造成批间差异。
4、待测样本如不能及时测定，应置于-20℃保存，4天内稳定。
5、避免使用EDTA、枸橼酸、氟化钠、草酸等抗凝剂。
6、为了您的安全和健康，请穿实验服并戴一次性手套操作。
7、试剂开封后请尽快使用，以防影响后续实验效果。
When animals or plant cells undergo oxidative stress, lipid oxidation occurs. Malondialdehyde (MDA) is a natural product of lipid oxidation in organisms. After some fatty acids oxidize, they gradually decompose into a series of complex compounds including MDA. At this point, the level of lipid oxidation can be detected by measuring MDA levels. Therefore, the determination of MDA is widely used as an indicator of lipid oxidation. Some other biochemical reactions in organisms also produce MDA, such as catalysis by thromboxane synthase. However, as long as appropriate controls are set during the assay, changes in lipid oxidation levels can be observed.
Detection Principle
Under conditions of high temperature and in an acidic environment, MDA can react with TBA to form a red MDA-TBA adduct. The MDA-TBA adduct has a maximum absorption at 535 nm, allowing detection via colorimetry. Additionally, the MDA-TBA adduct can also be excited at 535 nm, producing a maximum emission wavelength at 553 nm, enabling fluorescence detection.
Applicable Samples: Plasma, serum, urine, animal/plant tissues, or cell lysates.
Reagents, consumables and Equipments not provided
Spectrophotometer or microplate reader (capable of measuring absorbance at 535 nm)
Centrifuge tubes or 96-well plate, Centrifuge, Water bath or incubator
Physiological saline or PB
Procedure
1. Sample Preparation
1.1 Serum, Plasma, Urine, Cerebrospinal Fluid Samples
Serum or plasma separated from the test sample should be free of hemolysis. Assay directly. If exceeding the linear range, dilute with physiological saline or PBS before assay.
1.2 Tissue, Cell, and Other Samples
Tissues or cells can be homogenized or lysed using PBS or RIPA lysis buffer, etc. The tissue weight should constitute 10% of the homogenization or lysis buffer. For cells, use 0.1 mL of lysis buffer or homogenization buffer per 10⁶ cells. After homogenization or lysis, centrifuge at 4°C, 8,000~12,000 g for 10 minutes. Collect the supernatant for subsequent assays. Steps such as homogenization or lysis should be performed on ice or at 4°C.
Note: After sample preparation, the protein concentration can be measured using a BCA Protein Assay Kit (B665595/R1491648) to facilitate subsequent calculation of MDA content per unit protein weight in tissues or cells.
1.3 Compatibility of Common Chemical Components in Samples with this Kit (Reference Table):
| Reagent Category | Chemical Component | Interference? |
| Buffer | HEPES (100mM) | No |
| Buffer | Borate (50mM) | No |
| Buffer | Phosphate (100mM) | No |
| Buffer | Tris (25mM) | No |
| Detergent | CHAPS (≤1%) | No |
| Detergent | Triton X-100 (≤1%) | No |
| Detergent | Tween 20 (≤1%) | No |
| Inhibitor/Chelator | PMSF (≤200μM) | No |
| Inhibitor/Chelator | EDTA (≤1mM) | No |
| Inhibitor/Chelator | EGTA (≤1mM) | No |
| Inhibitor/Chelator | Antipain (≤100μg/ml) | No |
| Inhibitor/Chelator | Chymostatin (≤10μg/ml) | No |
| Inhibitor/Chelator | Leupeptin (≤10μg/ml) | No |
| Inhibitor/Chelator | Trypsin (≤10μg/ml) | No |
| Other | Glycerol (≤10%) | No |
| Other | Sucrose (250mM) | Yes |
2. Preparation of TBA Working Solution
Weigh an appropriate amount of TBA and prepare a 0.68% TBA working solution using the TBA Diluent. For example, take 68 mg TBA and dilute with 10 mL TBA Diluent to obtain a final concentration of 0.68% TBA working solution. The TBA working solution must be completely dissolved before use. Heating to 60°C can aid dissolution, and repeated vigorous vortexing can also help. The prepared TBA working solution should be stored at 4°C protected from light and is valid for at least one month.
3. Dilution of Series Standards
Take an appropriate amount of MDA Standard (1 mmol/L) and dilute it with a suitable solution to concentrations of 1, 2, 5, 10, and 20 µM (if performing a simple rapid assay, dilute the standard directly to 10 µM).
Note: When the test sample is serum or plasma, the standard should preferably be diluted with physiological saline. When the test sample is obtained from homogenate, lysate, or PBS, the standard should preferably be diluted with the same solution. The principle is to ensure comparability among the blank, standard, and test tubes. Prepared MDA standards can be stored at 4°C protected from light and are valid for at least 3 months.
4. Preparation of MDA Assay Working Solution
Immediately before detection, prepare a sufficient amount of fresh MDA Assay Working Solution according to the number of samples to be tested (including controls), as a reference:
Number of Assays | 1 time | 10 times | 20 times |
TBA Working Solution | 0.75ml | 7.5ml | 15ml |
Antioxidant | 0.031ml | 0.31ml | 0.62ml |
MDA Assay Solution | 0.075ml | 0.75ml | 1.5ml |
MDA Separation Solution | 2.25ml | 22.5ml | 45ml |
Total Volume | 3.106ml | 31.06ml | 62.12ml |
5. MDA Sample Loading
Add 0.08 mL of an appropriate solution to a centrifuge tube or other suitable container as a blank control (Note: When the test sample is serum or plasma, the standard should preferably be diluted with physiological saline. When the test sample is obtained from homogenate, lysate, or PBS, the standard should preferably be diluted with the same solution. The principle is to ensure comparability among the blank, standard, and test tubes.). Add 0.08 mL of the above series of different concentration standards to generate a standard curve (if performing a simple rapid assay, directly add the 10 µM standard). Add 0.08 mL of sample for measurement. Then add 3 mL of MDA Assay Working Solution. The assay reaction system can be set up as follows, adding reagents sequentially:
Additive (mL) | Blank Tube | Standard Tube | Test Tube |
Homogenate, lysate, PBS, physiological saline, etc. | 0.08 | － | － |
Standard | － | 0.08 | － |
Test Sample | － | － | 0.08 |
MDA Assay Working Solution | 3 | 3 | 3 |
Mix well, cap, and boil in a 95°C water bath for 40 minutes. Take care to avoid violent boiling and splashing during heating. If using a heat block for heating, ensure the tube caps are tightly pressed with a weight. If using a boiling water bath, use centrifuge tubes with lockable caps or screw-cap tubes, or seal the tube mouth with Parafilm and pierce a small hole with a needle. The most convenient and accurate heating method is to use a metal bath with a heated lid.
6. MDA Assay
Cool in water or under running water to room temperature. Centrifuge at 3,000 rpm for 15 minutes or 4,000 rpm for 10 minutes. Take the supernatant, which will be yellow to reddish-brown in color. Zero the instrument with distilled water. Using a cuvette with a 1 cm light path, measure the absorbance of the blank, standard, and test tubes at 535 nm with a spectrophotometer. If not convenient, absorbance between 530~540 nm can also be measured. Record the values as A<sub>Blank</sub>, A<sub>Standard</sub>, A<sub>Test</sub>.
7. Calculation of Results
If performing a simple rapid assay, calculate directly using the 10 µM standard to obtain the molar concentration of MDA. If precise calculation is required, use the MDA standard concentrations as the x-axis and the corresponding absorbance values as the y-axis to plot a standard curve. Calculate the concentration of MDA in the extract (C1, µmol/L) based on the standard curve. For solid tissues, the initial MDA content in the sample (C2, µmol/g) can be expressed per unit weight of protein or tissue weight, e.g., µmol/g protein or tissue.
Formula for Simple Rapid Calculation of MDA Content in Liquid Samples (Serum, Plasma, Urine, etc.):
MDA Concentration (µmol/L) = C1 = (ATest - ABlank) / (AStandard - ABlank) × 10
Formula for Simple Rapid Calculation of MDA Content in Cell or Tissue Samples:
MDA Content (µmol/g) = C2 = (ATest - ABlank) / (AStandard - ABlank) × 10 / Protein Concentration (mg/mL)= C1 / Protein Concentration (mg/mL)
or
MDA Content (µmol/g) = C2 = (ATest - ABlank) / (AStandard - ABlank) × 10 / [w / (VTotal × 10⁻³)]= C1 / [w / (VTotal × 10⁻³)]
Parameter Description:
ATest: Absorbance of the test tube
AStandard: Absorbance of the standard tube
ABlank: Absorbance of the blank tube
w: Mass of tissue sample (g)
V<sub>Total</sub>: Total volume of sample extraction solution (mL)
Reference Standard Curve Range
When measuring the MDA standard at 10 µM, its absorbance measured by a microplate reader is mostly between 1.3~2.3 (without zeroing). Measure the absorbance of MDA standards at 1, 2, 4, 6, 8, 10, 12, and 14 µM to generate a standard curve.
Note: Due to differences in detection instruments and operational techniques, the reference range may vary. This value is for reference only. For accurate calculation of MDA content, multi-point determination can be performed. Based on assay experience, the standard curve may deviate for standard concentrations below 2 µmol/L and above 16 µmol/L.
Notes
Avoid repeated freeze-thaw cycles for the aforementioned low-temperature reagents to prevent loss of efficacy or reduced efficiency.
Recommended sample amounts: Serum, plasma, urine: take 0.1 mL; Low-density lipoprotein suspension: take 0.1~0.2 mL; Edible oil: take 0.03 mL; Liver, myocardium, muscle, etc.: take 0.1~0.2 mL of 5% or 10% homogenate.
If the measured sample absorbance is low, the water bath time can be extended to 80 minutes, but this should be done consistently for all samples to avoid batch-to-batch variation.
If test samples cannot be assayed promptly, store at -20°C. They are stable for 4 days.
Avoid using anticoagulants such as EDTA, citrate, sodium fluoride, oxalate, etc.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.
| M1508248 | Component | 50T | 100T | Storage |
| M1508248A | TBA | 0.4g | 0.8g | RT. Store in the dark. |
| M1508248B | TBA稀释液 | 50 mL | 100 mL | RT. |
| M1508248C | 抗氧化剂 | 2.5 mL | 5 mL | -20℃. Store in the dark. |
| M1508248D | MDA标准品 (1mmol/L) | 0.2 mL | 0.4 mL | -20℃. Store in the dark. |
| M1508248E | MDA检测液 | 5 mL | 10 mL | RT. |
| M1508248F | MDA分离液 | 75 mL×2 | 150 mL×2 | RT. |
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