Hypoxia induces free radical damage to rat erythrocytes and spleen: analysis of the fluorescent end-products of lipid peroxidation.
Several studies have shown that hypoxia induces alterations in the lipid membranes of many cell types. The mechanism of these changes might consist in membrane lipid peroxidation. Lipid peroxidation in erythrocytes and spleen is easily detected by measurement of the concentration of fluorescent end-products. Exposure of rats to hypoxia for various time periods induced formation of lipophilic fluorescent products both in erythrocytes and spleen. A new kind of fluorophore was found in chloroform extracts from erythrocytes with excitation maximum at 270 nm and emission maximum at 310 nm. Additionally, two minor fluorophores were observed, emitting at 360 nm and in the region of 415-440 nm. Only one type of fluorophore was detected in spleen, emitting at 445 nm after excitation at 315 nm. The concentration of fluorophores was dependent on the time of hypoxic exposure both in erythrocytes and spleen. In erythrocytes there was a decrease of the predominant fluorophore after 3 hours (54%, P < 0.05) and 21 days (54%, P < 0.05) of hypoxia in relation to normoxic controls, accompanied by changes in spectral patterns of tridimensional fluorescence spectra. There was also a significant increase in the concentration of fluorophore in spleen (to 164%, P < 0.05, after 3 h, and to 240%, P < 0.05, after 21 days). The fluorophores, both in erythrocytes and spleen, were resolved into several distinct fractions with HPLC. The presented results support the hypothesis of hypoxia-induced lipid peroxidation and create a basis for further characterization of the fluorescent products.
Department of Medical Chemistry and Biochemistry, Second Medical School, Charles University, Prague, Czech Republic. jiri.wilhelm@lfmotol.cuni.cz
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J
Wilhelm
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J
Herget
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism*; D015227:Lipid Peroxidation; D008062:Lipofuscin / Q000737:chemistry; D008297:Male; D018384:Oxidative Stress; D010860:Pigments, Biological / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000378:metabolism*
D008062:Lipofuscin; D010860:Pigments, Biological