Effect of oxidized lipids on protein structure and cultured human cells.

Many foods, particularly fish, change in texture and become tough on frozen storage. In addition to ice crystal formation that causes protein denaturation, lipid oxidation products including aldehydes may induce protein cross-linking. In this study, Atlantic Mackerel was stored for up to 26 weeks at -80 and -10 °C. The formation of aldehydes was investigated by thiobarbituric (TEARS) test, HPLC and LC-MS. As expected, there was an increase in the production of aldehydes during storage at -10 °C compared with the control fillets at -80 °C. However, in addition to malonaldehyde (MDA) and hexanal (HEX), we report the formation of gluteraldehyde (GLA) and hydroxynonenal (4-HNE) in frozen fish for the first time. Frozen storage also decreased protein solubility, induced changes in protein conformation and increased formaldehyde and DMA formation. Changes in texture during storage, assessed by small deformation rheology showed increased elastic modulus (G') values. Aldehyde formation in fish and consequently the G' values were reduced in samples that were treated with antioxidant (green tea). A model study using BSA with various aldehydes showed a decrease in lysine availability, increased G' values and conformational changes including increased beta-sheet and tyrosine doublet ratio and decreased tryptophan and disulphide groups. Increased MW of BSA-aldehyde adducts as well as fragmentations were observed by LC-MS. Methyl linoleate (ML) and extracted mackerel oil were oxidised under ultraviolet (UV) radiation for 24, 48 and 72 hours and lipid peroxidation was assessed by measuring peroxide value (PV) and TEARS. To assess the cytotoxicity, oxidized lipids (0, 20, 40, 80 and 100 mug/ml) (ML or fish oil) were added to human colon cancer (Caco-2) cells (density 2 x 104 cells/well) and incubated for 24 h. Using the MTT assay, a decrease in cell viability was observed in all samples treated with UV oxidized ML or fish oil, at concentrations above 40 mug/ml. Caco-2 cells treated with 24 and 72 h oxidized mackerel oil were less damaged compared to cells treated with oxidized ML. Improved cell viability and decrease in lipid peroxidation were shown in cells pretreated with 50 muM EGCG. The mechanism of cell death either by necrosis or apoptosis was investigated. Oxidized 100 mug/ml ML or fish oil-treated caco-2 cell line showed characteristics of apoptosis. Morphological changes resulted from cellular membrane rupture, the formation of apoptotic bodies, DNA fragmentation and caspase-3 activation, detected by Western blotting; these features were visualized by bioimaging techniques such as light microscopy, fluorescence microscopy, Raman microspecfroscopy and atomic force microscopy