The effect of storage on the lipids and proteins in Atlantic mackerel stored for up to 24 months at À20 and À30°C was studied. Traditional methods including the peroxide value, thiobarbituric acid-reactive substances (TBARS) and a reverse phase HPLC method were used to determine the primary and secondary lipid oxidation products. All tests showed an increase in lipid oxidation products with storage time and at a higher storage temperature of À20°C compared with samples stored at À30°C. Antioxidants had a signi®cant effect (P < 0.01) on the inhibition of lipid oxidation, as shown by the reduction in peroxide value and hydroxides, and malondialdehyde formation. Similarly, deterioration of protein structure and functionality in mackerel stored for 3, 6, 12 and 24 months was greater at À20 than À30°C. ATPase activity in the myosin extract of Atlantic mackerel showed a signi®cant decrease (P < 0.01) with progressive frozen storage. Protein solubility in high salt concentration (0.6 M NaCl) decreased (P < 0.01) during storage at both À20 and À30°C but was greater at À20°C. Interestingly, antioxidants BHT, vitamin C and vitamin E protected the proteins against complete loss of ATPase activity and protein solubility to a signi®cant level (P < 0.01) for up to 1 year at À20°C compared with samples stored without antioxidants. This study con®rms the deleterious effect of lipid oxidation products on protein structure and function in frozen fatty ®sh.

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Lipoxygenase was prepared from Atlantic mackerel muscle using differential centrifugation, ammonium sulphate precipitation and gel permeation (phenyl Sephadex G-50) column chromatography. The crude lipoxygenase enzyme preparation was characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), which showed two prominent bands with molecular weights of 119 and 125 kDa. Fractions collected from the chromatography column were tested for enzyme activity by reacting with arachidonic acid and determining the production of hydroxyeicosatetraenoic acid (12-HETE) using reverse phase HPLC and GC±MS. The 12-HETE peak was absent from the fresh arachidonic acid control sample and from arachidonic acid treated with heatinactivated lipoxygenase. Esculetin, a known inhibitor of lipoxygenase, inhibited the production of 12-HETE from the reaction of lipoxygenase with arachidonic acid, thus con®rming that the enzyme was lipoxygenase. The HETE peak was partially reduced in the presence of antioxidants, namely synthetic butylated hydroxytoluene (BHT) and natural antioxidants vitamins C and E. The presence of lipoxygenase in Atlantic mackerel muscle indicates the possibility that the lipid oxidation mechanism is initiated enzymatically in chilled and frozen stored ®llets of mackerel and that this oxidative deterioration could be inhibited by antioxidants (BHT, vitamins C and E) which are used widely in the food industry.

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The formation of stable hydroxy derivatives from hydroperoxides produced during the oxidation of linoleic acid methyl ester and fish oil were studied by reverse-phase highperformance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and 13 C nuclear magnetic resonance (NMR) spectroscopy. The oxidation products identified were mixtures of four isomeric hydroxy derivatives: 13-hydroxy-9-cis,11-trans-octadecadienoic,13-hydroxy-9-trans,11-trans-octadecadienoic, 9-hydroxy-10-trans,12-cis-octadecadienoic, and 9-hydroxy-10-trans,12-trans-octadecadienoic acids. The presence of hydroxy compounds was confirmed by 13 C NMR, which gave rise to a hydroxy carbon peak at 87 ppm, and by GC-MS, which showed three peaks corresponding to isomeric mixtures of trimethylsilyl ethers of the oxidized linoleic acid methyl ester. The mass spectra scans of the three peaks showed that they represent isomers of molecular weight 382 and are consistent with the molecular formula C 22 H 42 O 3 Si. In oil extracted from stored frozen mackerel, 13-hydroxy-9-cis,11-transoctadecadienoic acid was more prominent compared to the model lipid systems. HPLC offered a sensitive means of detection of hydroxy compounds produced both in the initiation and latter stages of oxidation. The effect of antioxidants added to the fish mince prior to storage can also be monitored by HPLC. Thus, the monitoring of lipid oxidation hydroxy derivatives by HPLC is of practical value in the efficient processing and quality control of fish, fish oils, and other fatty foodstuffs in order to enhance the acceptability, nutritional, and safety aspects.

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