Determination of Phospholipid Oxidation by UV/VIS Spectroscopy - Avanti^® Polar Lipids

Definition

The percent oxidation of a phospholipid can be determined by using Beer’s Law to calculate the peroxidized phospholipid’s concentration from its absorbance at 234 nm.

Scope

Applicable to all unsaturated phospholipids.

Apparatus

• UV/VIS Spectrophotometer
• HELLMA UV Quartz Sample Cell (QS 1.000)
• Kimwipes
• Disposable Glass Tubes

Reagents

• Ethanol (200 proof, dehydrated USP)
• Deionized water
• 16:0 PC (DPPC)

Procedure

1. Prepare the DPPC Sample. Prepare a minimum of 3 mL of a 1mg/mL solution of DPPC dissolved in ethanol/DI water (9:1).
2. Prepare the Sample. Prepare a minimum of 3 mL of a 1mg/mL solution of the phospholipid sample dissolved in ethanol/DI water (9:1).
3. Run the Blank. The blank (background) is the solvent used to dissolve the phospholipid samples.
   • Rinse the sample cell with the solvent three times to remove any residual contaminants from the cell.
   • Fill 3/4 of the sample cell with the reference.
   • Use a Kimwipe to wipe off all fingerprints and contaminants on the exterior of the sample cell.
   • Insert the sample cell into the spectrophotometer and run a blank spectrum.
4. Run the DPPC Sample.
   • Pour the blank solvent out of the sample cell.
   • Fill 3/4 of the sample cell with the DPPC sample solution.
   • Remove fingerprints from the exterior of the sample cell.
   • Insert the sample cell into the spectrophotometer and run a DPPC spectrum.
5. Run the Sample.
   • Pour the DPPC sample out of the sample cell.
   • Rinse the sample cell with the solvent three times to remove any residual DPPC from the cell.
   • Fill 3/4 of the sample cell with the phospholipid sample.
   • Remove fingerprints from the exterior of the sample cell. Insert the sample cell into the spectrophotometer and run a sample spectrum.
   • Subtract the DPPC spectrum from the sample spectrum and record the phospholipid sample’s absorbance reading (OD) at 234 nm.

Calculations

Concentration of peroxidized lipid (D) = A/(B*C)
A = Absorbance reading (OD) at 234 nm
B = Extinction coefficient in (OD*mL)/(mmol*cm) (27,000 for peroxidized lipid)
C = Path length in cm Percent oxidation = D/E * 100
D = Concentration of peroxidized lipid in mg/mL
E = Concentration of lipid sample = 1.0 mg/mL

References

1. Kim R, LaBella F. 1987. Comparison of analytical methods for monitoring autoxidation profiles of authentic lipids. J. Lipid Res. 28:1110-1117.