Chuo University, School of Science, 1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan
Citation: Shintani H (2013) IFluorescent Products. Pharmaceut Anal Acta 4:e152. doi: 10.4172/2153-2435.1000e152
Copyright: © 2013 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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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 biological membranes.
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?365 nm and an emission maximum at 430-440 nm).
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 spectrofluorimetry.
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 .