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Determination of Phospholipid Oxidation by UV/VIS Spectroscopy

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

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.
    1. Rinse the sample cell with the solvent three times to remove any residual contaminants from the cell.
    2. Fill 3/4 of the sample cell with the reference.
    3. Use a Kimwipe to wipe off all fingerprints and contaminants on the exterior of the sample cell.
    4. Insert the sample cell into the spectrophotometer and run a blank spectrum.
  4. Run the DPPC Sample.
    1. Pour the blank solvent out of the sample cell.
    2. Fill 3/4 of the sample cell with the DPPC sample solution.
    3. Remove fingerprints from the exterior of the sample cell.
    4. Insert the sample cell into the spectrophotometer and run a DPPC spectrum.
  5. Run the Sample.
    1. Pour the DPPC sample out of the sample cell.
    2. Rinse the sample cell with the solvent three times to remove any residual DPPC from the cell.
    3. Fill 3/4 of the sample cell with the phospholipid sample.
    4. Remove fingerprints from the exterior of the sample cell. Insert the sample cell into the spectrophotometer and run a sample spectrum.
    5. 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

  • R.S. Kim & F.S. LaBella (1987). Comparison of analytical methods for monitoring autoxidation profiles of authentic lipids. J. Lipid Res. 28:1110-1117.