|The reaction of lipid peroxidation products, such as
malonaldehyde, with amino groups of proteins, free amino acids,
aminophospholipids, or nucleic acid bases, produces fluorescent
lipid-peroxidation products known as conjugated Shiff bases with the
general structure RN=CHCH=CHNHR’ (N,N’-disubstituted 1-amino-
3-iminopropenes) . When these products are excited at 360 nm the
fluorescence maximum is in the region 430-440 nm.
|Spectrofluorimetric analysis of fluorescent lipid peroxidation
products is covered in detail elsewhere [2,3]. The method described
here is a simple procedure for detection of lipid-soluble and watersoluble
fluorescent products formed during lipid peroxidation of
|Lipid-Soluble Fluorescent Products
|1. Place membrane sample (e.g. oxidized tissue homogenate,
plasma, mitochondria, microsomal suspension, or lipoproteins;
0.5 mL) and ethanol-ether (3:1, v/v, 1.5 mL) in screw-capped
tubes (1.3×10 cm, Pyrex).
|2. Mix on a vortex mixer for 1 min.
|3. Centrifuge for 10 min at 3,000 rpm (4°C).
|4. Wash the sediment at the bottom of tube twice with ethanolether
(3:1, v/v, 2 mL) repeating assay steps 2 and 3.
|5. Pipette the solution (1.5 mL) into a 1-mL quartz cuvette.
|6. Measure fluorescence (emission) and excitation spectra
by spectrofluorimetry. (The fluorescent lipid-peroxidation
products have usually an excitation maximum in the region of
355ー365 nm and an emission maximum at 430-440 nm).
|Water-Soluble Fluorescent Products
|1. Dissolve sediments from assay of lipid-soluble fluorescent
products (above) in 2.0 mL 15% SDS-PBS (phosphate-buffered
saline, pH 7.4) on a vortex mixer for 5 min.
|2. Centrifuge for 10 min at 3,000 rpm (room temperature).
|3. Pipette the solution (1.5 mL) into a 1-mL quartz cuvette.
|4. Measure fluorescence (emission) and excitation spectra by
|The ftuorescence intensity of quinine sulfate (1 mg mL-1) in H2SO4
solution (0.05M) is the standard for the relative ftuorescence intensity
of the sample . Quinine sulfate in the solution has a ftuorescence
maximum at 457 nm when excited at 360nm.
|Fluorescent products which bind to proteins are insoluble in
common organic solvents and their structures (ftuorophores) can be
disrupted, in part, during extraction of lipids with 2:1 (v/v) chloroformmethanol.
They are, however, stable in 3:1 (v/v) ethanol-ether, as
described elsewhere . It should be noted that the lipid extracts from
biological membranes and tissue homogenates contain retinol as one of
interfering ftuorescent compounds. Retinol, a lipid-soluble compound,
has a ftuorescence maximum at 478nm when excited at 335 nm  and
its ftuorescence can be removed from the lipid extracts by exposing it to
high-intensity ultraviolet light for 30s .
- Tappel AL (1980) Free radicals in Biology.(4thedn), WA Pryor. Academic Press, New York: 1-47.
- Dillard CJ, Tappel AL (1984) Fluorescent damage products of lipid peroxidation. Methods Enzymol 105: 337-341.
- Shimasaki H (1994) Assay of fluorescent lipid peroxidation products. Methods Enzymol 233: 338-346.
- Shimasaki H, Ueta N, Privett OS (1982) Covalent binding of peroxidized linoleic acid to protein and amino acids as modeIs for lipofuscin formation. Lipids 17: 878-883.
- Shimasaki H, Ueta N, Privett OS (1980) Isolation and analysis of age-related fluorescent substances in rat testes. Lipids 15: 236-241.