Malondialdehyde HPLC Assay Kit

Additional Information

Assay Background

In a healthy body, oxidative and reductive processes are in a balance. Free radicals (reactive oxygen species) are eliminated by antioxidants. In case of a lack of antioxidants free radicals react with cell structures.   A reaction of free radicals with unsaturated fatty acids leads to lipid peroxidation products. A reaction of free radicals with poly-unsaturated fatty acids generates malondialdehyde and/or 4-hydroxynonenal. These secondary lipid peroxidation products might react with other molecules in the cell.  Modifications of the DNA-based adenine or guanine result in incorrect transcripts.  A reaction of free radicals with proteins leads to an alteration or loss of function. The creation of neoantigens is possible. Neoantigens are recognized by the immune system, thus resulting in autoimmune diseases. The participation of lipid peroxidation products have been discussed in several diseases such as atherosclerosis, tumor genesis, rheumatism and reperfusion injury after transplantation.

The Eagle Biosciences Malondialdehyde (MDA) HPLC Assay kit makes it possible to determine the lipid peroxidation product in an easy, fast and precise method. The Malondialdehyde (MDA) HPLC Assay kit includes all reagents ready to use for preparation and separation of the samples with exception of the columns (IC1900rp) and the controls (IC1900ko).  Both can be supplied by Eagle Biosciences.  Beside the complete test kits it is possible to order all components separately. Please request our single component price list.

Assay Principle

For the determination of malondialdehyde a derivatisation step, in which protein bound malondialdehyde is hydrolyzed and converted into a fluorescent probe (60 min at 95 °C) is performed. The fluorescent probe is then cooled (2-8°C), centrifuged, mixed with a reaction solution and injected into the HPLC system. The isocratic separation via HPLC at 30°C, using a “reversed phase” column, lasts 4 minutes for one sample. The chromatograms are recorded by a fluorescence detector. The quantification is performed with the delivered calibrator; the concentration is calculated via integration of the peak heights.

Assay Procedure

1. Pipette into 1.5 ml reaction tubes:  20 µl sample, CAL, CTRL or blank (deionized water) + 1 ml DERIVAT
2. Mix well (15 seconds on a vortex mixer),
3. Incubate for 60 minutes at 95°C on a shaker or in a water bath;
4. Keep the incubation time exactly because only for these conditions the given MDA-concentrations for calibrator and controls are valid.
5. Cool the samples to 2-8°C and centrifuge at 10.000g for 5 minutes
6. Mix 500 µl of the supernatant + 500 µl REAL
7. Inject 20 µl of the supernatant for chromatography into the HPLC-system.  The supernatant is stable in the dark for 4 days at 2-8°C and 12 hours at 20-25 °C.

Limitations of the Method

• We recommend not to measure strong hemolytic and lipemic samples, because they might show pathological concentrations.
• Whole blood is not suited for the Malondialdehyde (MDA) HPLC Assay kit.