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Article

Depletion of -Tocopherol and Astaxanthin in Atlantic Salmon (Salmo salar) Affects Autoxidative Defense and Fatty Acid Metabolism¹

Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, U.K.

²To whom correspondence should be addressed.

	ABSTRACT

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Duplicate groups of Atlantic salmon post-smolts were fed four purified diets supplemented with both vitamin E and the carotenoid astaxanthin (Ax) (+E, +Ax), or supplemented with either vitamin E or Ax (-E, +Ax and +E, -Ax) or deficient in both vitamin E and Ax (-E, -Ax) for 22 wk. There were no effects of diet on growth rate, but an extensive lipoid liver degenerative lesion was observed in 15% of fish fed diets deficient in vitamin E. Tissue vitamin E concentrations varied in accordance with dietary vitamin E in liver, muscle, heart, plasma, brain and eye; levels were reduced to ~3% in liver but only to 40% in eye of fish fed diets deficient in vitamin E compared with those fed diets supplemented with vitamin E. An interactive sparing of Ax supplementation on tissue vitamin E concentration was observed, but only in brain. Dietary deficiency of both vitamin E and Ax significantly increased the recovery of desaturated and elongated products of both [1-¹⁴C] 18:3(n-3) and [1-¹⁴C] 20:5(n-3) in isolated hepatocytes, suggesting that conversion of fatty acids to their long-chain highly unsaturated products can be stimulated by a deficiency of lipid-soluble antioxidants. The antioxidant synergism of vitamin E and Ax was supported by their ability to reduce malondialdehyde formation in an in vitro stimulation of microsomal lipid peroxidation and to reduce plasma levels of 8-isoprostane. The results of this study suggest that both vitamin E and the carotenoid Ax have antioxidant functions in Atlantic salmon.

KEY WORDS: • Atlantic salmon • -tocopherol • astaxanthin • autoxidation • fatty acids.

	INTRODUCTION

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Lipid oxidation, resulting from free radical–initiated reactions, is linked to the occurrence of degenerative pathological conditions in humans and other animals (Halliwell and Gutteridge 1989 ). Polyunsaturated fatty acids (PUFA),³ which are vital components of cellular membranes, are particularly susceptible to attack by reactive oxygen radicals. Uncontrolled damage to membrane PUFA and the accumulation of their oxidized breakdown products can have deleterious consequences for cell and organ function (Chan 1987 , Sies 1991 ). Numerous nutritional studies have shown a link between increased PUFA intake, resulting in increased incorporation of PUFA into cellular lipids, and increased incidence of lipid peroxidation (Cho and Choi 1994 , Sugihara et al. 1994 ). Reactive oxygen species are generated by normal physiologic processes and are, in fact, essential to normal cellular function. For this reason, cells possess a multilevel antioxidant defense system, which includes a number of radical- and peroxide-scavenging enzymes and radical-sequestering molecules, including tocopherols, ascorbate, glutathione, urate and carotenoids (Yu 1994 ). Among these smaller molecules, vitamin E and, in particular -tocopherol, is regarded as the primary lipid-soluble antioxidant that operates synergistically with vitamin C to protect lipids against peroxidative damage (Buettner 1993 , Kamal-Eldin and Appelqvist 1996 ). In addition, several recent studies have suggested that carotenoids, including ß-carotene, astaxanthin (Ax) and canthaxanthin are potent antioxidants in in vitro membrane models and that they operate synergistically with vitamin E (Nishigaki et al. 1994 , Palozza and Krinsky 1992a and 1992b ).

Fish tissues are naturally enriched in (n-3) PUFA, especially the highly unsaturated fatty acids (HUFA) eicosapentaenoic acid (EPA) [20:5(n-3)] and docosahexaenoic acid (DHA) [22:6(n-3)] (Henderson and Tocher 1987 , Sargent et al. 1989 ). As with mammals, (described above), the requirement for vitamin E in fish is closely correlated with levels of dietary and tissue PUFA (Roem et al. 1990 , Stephan et al. 1995 ) and as a result, levels of vitamin E in fish tissues appear higher than in equivalent tissues in mammals (Hamre and Lie 1995 ). Astaxanthin, derived from phytoplanktonic origins, is deposited in the muscle, ovaries and skin of wild salmonids, whereas carotenoids are present in cultured salmonids due to the addition of astaxanthin and canthaxanthin to their feed (Torrissen 1989 ). Although carotenoids have been associated with improved egg quality and larval survival in salmonids (Torrissen 1984 ) and exogenous carotenoids have been identified as singlet oxygen quenchers in vitro (Shimidzu et al. 1996 ), information on the biological activity of endogenous carotenoids is scarce.

Many years ago, researchers observed changes in tissue PUFA composition when a state of antioxidant deficiency was established (Bernhard et al. 1963 , Witting and Horwitt 1964 and 1967 , Witting et al. 1967 ). In a more recent study with rats deficient in both vitamin E and selenium, Buttriss and Diplock (1988) observed an increase in the long-chain HUFA, DHA and arachidonic acid, in mitochondrial and microsomal membranes. They theorized that this increase was due to an overproduction of these HUFA, arising from increased activity of the elongation and desaturation processes responsible for their synthesis. Infante (1986) postulated that vitamin E had a regulatory role in the microsomal electron transport chain of the desaturase complex. A later study by Despret et al. (1992) observed an increase in rat brain 6-desaturase arising from increased vitamin E levels, although the opposite effect was observed in liver. In a recent study with African catfish (Clarias gariepinus), increased hepatic DHA levels were found in fish fed diets depleted in vitamin E and containing oxidized oils (Baker and Davies 1996 ). This increase was attributed to a stimulatory effect of low vitamin E and increased peroxide tone on elongation and desaturation mechanisms.

In this study, replicate groups of Atlantic salmon post-smolts (Salmo salar) were fed diets that were supplemented with vitamin E and Ax or deficient in one or both nutrients. The effect of these diets on tissue concentrations of vitamin E and Ax, the susceptibility of muscle microsomes to oxidative challenge, the concentration of plasma 8-isoprostane and the activities of fatty acid desaturation and elongation enzymes were measured.

	MATERIALS AND METHODS

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Fish, husbandry and experimental diets.

Atlantic salmon S¹/₂ smolts (n = 424; initial mean weight, ~53 g) were obtained from Marine Harvest McConnell (Fort William, Highland, Scotland) and transferred to the F.R.S. Fish Cultivation Unit, Aultbea, Wester Ross, Scotland). The smolts were randomly distributed into eight 1-m diameter tanks of 500 L capacity supplied with non-recirculated sea water at 10 L/min. The tanks were subjected to a photoperiod regime of 12 h light:12 h dark and the temperature over the experimental period (October 1995–March 1996) was 5.2–12.5°C. The diets were supplied by automatic feeders, which were activated for a few seconds every 15 min during daylight hours and adjusted to provide 20 g/kg biomass each day. Fish were weighed individually at the start and finish of the experiment, bulk weighed every 28 d and the ration adjusted accordingly. An initial sample was taken at the start of the trial and after 12 wk; final sampling was performed after 22 wk of the feeding experiment. The experiment was conducted in accordance with the British Home Office guidelines regarding research on experimental animals.

Semipurified diets, with casein as protein source, were formulated to meet the nutritional requirements of salmon (NRC 1993 ), except for vitamin E, and contained 47% protein and 16% lipid (Table 1). Four diets were formulated from the basal diet by adding vitamin E as dl--tocopherol acetate (ICN Biomedicals, Thame, England) and/or Ax as Carophyll pink (Roche, Heanor, England) (Table 1) . The lipid component of the diet was supplied by tocopherol-stripped fish oil (Fosol, white fish body oil, Seven Seas, Hull, England). The oil was stripped by mixing 2 L of Fosol with 2 L of hexane (HPLC grade) and 150 g activated charcoal in a 5-L container. The oil/hexane/charcoal suspension was mixed on a magnetic stirrer for 4 h at room temperature in a fume cupboard. The charcoal was then filtered off using a Buchner funnel. The hexane lost by evaporation was replaced; another 150 g of activated charcoal was added and the above procedure repeated. The charcoal was removed as described above and the hexane was removed by rotary evaporation. The resulting oil contained <4 mg/kg of -tocopherol.

Sampling procedure.

An initial sample of six fish was taken at the start of the experiment to determine baseline levels of vitamin E in liver, white muscle and plasma. A similar sample of four fish per dietary treatment was taken after 10 wk of the trial. After 22 wk, each analytical measurement was performed on at least three fish, selected at random, from each tank. Fish used to measure the production of malondialdehyde (MDA) in NADPH-stimulated liver microsomes were transported from the experimental site to an aquarium facility on the campus of the University of Stirling. Fish were killed by a blow to the head; blood samples were collected from the caudal vein into syringes containing Na EDTA as anticoagulant. Plasma was prepared by centrifugation at 12,000 x g for 2 min. Samples of liver, heart, spleen, white muscle, brain and eye were dissected and placed immediately in liquid nitrogen. Samples were stored at -40°C before analysis.

Vitamin E measurement.

Vitamin E was measured in the diets by the method of McMurray et al. (1980) and in tissue samples by the method described in detail by Bell et al. (1998) . In both cases, vitamin E was saponified and extracted in hexane, dried under nitrogen and the dry residue dissolved in a small volume of methanol before analysis by HPLC. Separation and quantification of -tocopherol was performed using a 5-µm Hypersil ODS column (4.6 mm x 25 cm, Capital HPLC, Broxburn, Scotland). The chromatographic system was equipped with a Waters Model 501 pump, and -tocopherol was detected at 293 nm using a Waters 490E multiwavelength UV/vis detector [Millipore (U.K.), Watford, England]. An isocratic solvent system was used containing 98% methanol (v/v) at a flow rate of 1 mL/min. Sample concentrations were calculated using an external standard of d--tocopherol.

Astaxanthin measurement.

Total carotenoid was extracted from salmon muscle largely by the method of Barua et al. (1993) . Tissue samples were homogenized in 5 mL of absolute ethanol and 5 mL of ethyl acetate using an Ultra-Turrax tissue disrupter (Fisher Scientific UK, Loughborough, UK). The homogenate was centrifuged (1000 x g, 5 min) and the supernatant removed to a stoppered glass tube. The pellet was rehomogenized in 5 mL of ethyl acetate and recentrifuged, and the supernatant was combined with the first supernatant. Finally, the pellet was rehomogenized in 10 mL of hexane and recentrifuged, and the supernatant combined with the pooled supernatant. The pooled supernatant was dried under a stream of nitrogen and vacuum-desiccated for 2 h before redissolving the residue in 2 mL of hexane containing 0.2g/L BHT. Total carotenoid was measured spectrophotometrically at 470 nm using the E_1% (wt/v) of 2100. Measurement of Ax was carried out using the HPLC column, pump and detector described above. An isocratic solvent system was used containing ethyl acetate/methanol/water (20:72:8, v/v/v) at a flow rate of 1 mL/min. Ax was detected at 470 nm and quantified using an external standard of Ax obtained from Roche (Heanor, England). Ax in diets was extracted and measured similarly after an enzymatic digestion of the ground extruded pellets with Maxatase (International Biosynthetics, Rijswijk, Netherlands). Portions of ground diet (1 g) were mixed with 10 mL water and 10 mg of Maxatase in a 50-mL stoppered glass tube followed by incubation in a water bath at 50°C for 30 min.

Muscle microsome preparation and in vitro oxidative challenge.

Microsomes were prepared by homogenizing ~2 g of white muscle in 9 volumes of 5 mmol/L HEPES/0.25 mol/L sorbitol + 1 mmol/L Na EDTA, adjusted to pH 7.4 with KOH (homogenization buffer), and then centrifuged at 14,000 x g for 20 min at 4°C to sediment nuclear material and mitochondria. The supernatant was centrifuged at 100,000 x g for 1 h and the resulting microsomal pellet resuspended in 10 mL of 0.15 mol/L KCl and recentrifuged at 100,000 x g for 1 h. The microsome pellet was then resuspended in 2 mL of 50 mmol/L Tris base + 0.14 mol/L NaCl, adjusted to pH 7.4 with HCl (incubation buffer). The time course of MDA production in NADPH-stimulated muscle microsomes was measured as follows. Reactions were performed in 25-mL "Reacti-flasks" [Pierce & Warriner (UK), Chester, England] in a shaking water bath at 18°C. Each flask contained 1.5 mL incubation buffer, 2.0 mL ADP/FeCl₃ solution (1:1, v/v; 10 mmol/L ADP:60 µmol/L FeCl₃ · 6H₂O) and 0.5 mL 3 mmol/L NADPH; the reaction was started by the addition of 1.0 mL of microsome suspension. Aliquots (0.5 mL) were removed after 0, 10, 30, 60 and 90 min. and added to stoppered test-tubes containing 1.0 mL trichloroacetic acid (300 g/L), 1.0 mL thiobarbituric acid (TBA) solution (7.5 g/L) and 10 µl BHT (20 g/L in ethanol). Tubes were kept on ice until the last sample was collected and then placed in a boiling water bath for 15 min. The samples were transferred to 2-mL plastic centrifuge tubes and centrifuged at 12,000 x g for 2 min. The supernatant was read on a spectrophotometer at 532 nm. The extinction coefficient used for the TBA-MDA adduct was 1.56 x 10⁵ (mol/L)^-1 · cm^-1.

Protein determination.

Protein concentration in hepatocyte and microsome suspensions was determined according to the method of Lowry et al. (1951) after incubation with 0.25 mL of 2.5 g/L SDS/1mol/L NaOH for 45 min at 60°C.

Preparation of isolated hepatocytes.

The gall bladder and main blood vessels were carefully dissected from the liver. The liver was perfused via the hepatic vein with solution A [calcium- and magnesium-free Hank’s balanced salt solution (HBSS) + 10 mmol/L HEPES + 1mmol/L EDTA] to clear blood from the tissue. The liver was chopped finely with scissors and incubated with 20 mL of solution A containing 1 g/L collagenase in a 25 mL "Reacti-flask" in a shaking water bath at 20°C for 45 min. The digested liver was filtered through 100-µm nylon gauze and the cells collected by centrifugation at 1000 x g for 5 min. The cell pellet was washed with 20 mL of solution A containing fatty acid–free bovine serum albumin (10 g/L, FAF-BSA) and recentrifuged. The hepatocytes were resuspended in 10 mL of Medium 199 containing 10 mmol/L HEPES, 2 mmol/L glutamine, 1 x 10⁵ U/L penicillin and 0.1 g/L streptomycin. Cell suspension (100 µL) was mixed with 400 µL of trypan blue, hepatocytes were counted and their viability assessed using a hemocytometer. Cell suspension (100 µL) was retained for protein determination as described above.

Assay of hepatocyte fatty acyl desaturation/elongation activities.

Five milliliters of each hepatocyte suspension was dispensed into two 25-cm² tissue culture flasks. Hepatocytes were incubated with 0.25 µCi of either [1-¹⁴C] 18:3(n-3) or [1-¹⁴C] 20:5(n-3), added as complexes with FAF-BSA. Briefly, 25 µCi of fatty acid (0.5 µmol) in ethanol was placed in a reaction vial, solvent-evaporated under a stream of nitrogen and 100 µL of 0.1mol/L KOH added. The mixture was stirred for 10 min at room temperature before 5 mL of 50 g/L FAF-BSA in HBSS containing 10 mmol/L HEPES buffer was added and the mixture stirred for 45 min at 20°C. After addition of isotope, the flasks were incubated at 20°C for 3 h. After incubation, the cell layer was dislodged by gentle rocking, the cell suspension transferred to glass conical test tubes and the flasks washed with 1 mL of ice-cold HBSS containing 10 g/L FAF-BSA. The cell suspensions were centrifuged at 300 x g for 2 min, the supernatant discarded and the cell pellets washed with 5 mL of ice-cold HBSS/FAF-BSA and recentrifuged. The supernatant was again discarded and the tubes placed upside down on paper towels to blot for 15 s before extraction of total lipid using ice-cold chloroform/methanol (2:1, v/v) containing 0.1 g/L BHT essentially as described by Folch et al. (1957) and as described in detail previously (Tocher et al. 1988 ).

Total lipid was transmethylated and fatty acid methyl esters prepared as described by Tocher et al. (1988) . The methyl esters were redissolved in 100 µL hexane containing 0.1 g/L BHT and applied as 2.5-cm streaks to TLC plates impregnated by spraying with 2 g silver nitrate in 20 mL acetonitrile and preactivated at 110°C for 30 min. Plates were fully developed in toluene/acetonitrile (95:5, v/v) (Wilson and Sargent 1992 ). Autoradiography was performed with Konica A2 film (Anachem Ltd., Laton, UK) for 4–7 d at room temperature. Silica corresponding to individual PUFA was scraped into scintillation mini-vials containing 2.5 mL of scintillation fluid (Ecoscint A. National Diagnostics, Atlanta, GA) and radioactivity determined in a TRI-CARB 2000CA scintillation counter (United Technologies Packard, Pangbourne, England). Results were corrected for counting efficiency and quenching of ¹⁴C under exactly these conditions.

Extraction and measurement of 8-isoprostane in plasma.

Free (nonesterified) 8-isoprostane (8-epi prostaglandin F₂) was extracted and purified by the method described in the enzyme immunoassay (EIA) kit protocol (Cayman Chemical Ann Arbor, MI). An aliquot of a known quantity of plasma (0.5–1.0 mL) was mixed with 2 mL of ethanol on a vortex mixer. The samples were allowed to stand at 4°C for 5 min and then centrifuged (12,000 x g, 2 min) to remove precipitated protein. The supernatant was decanted into a clean test tube, 8 mL of deionized water (Milli-Q) added, and the pH reduced to <4.0 by addition of 0.5 mL of 2 mol/L formic acid. The samples were purified using Sep-Pak C₁₈ mini-columns (Millipore Ltd., Watford, UK), which were activated by washing with 5 mL methanol followed by 5 mL deionized water. The acidified sample prepared as described above was passed through the Sep-Pak followed by 5 mL deionized water and 5 mL hexane. The 8-isoprostane was eluted with 5 mL of ethyl acetate containing 10 mL/L of methanol. The ethyl acetate was dried under a stream of nitrogen and the residue dissolved in 1 mL of EIA buffer. Fifty microliters was assayed for 8-isoprostane as described in the EIA kit protocol (Cayman Chemical).

Histology.

Samples of liver, heart and white muscle were fixed in 200 mL/L buffered formol saline and embedded in paraffin wax. Sections (5 µm) were cut and stained with hematoxylin and eosin for histological analysis. Pathological assessment was carried out on coded, randomized slides to eliminate bias in interpretation. Examination of slides was performed using a Wild M20 compound microscope (Wild-Heerbrugg, Chatham, UK) at an objective magnification of X20 or X40. All organs were examined for general abnormalities such as tumors, infections, inflammation, and loss or replacement of tissues.

Statistical analysis.

Significance of difference (P < 0.05) among dietary treatments was determined by two-way ANOVA. Differences between means were determined by Tukey’s test. Data identified as nonhomogeneous, using Bartlett’s test, were subjected to log or arcsin square-root transformation before ANOVA. ANOVA and linear regression analyses were performed using a Graphpad Prism (version 2.0) statistical package (Graphpad Software, San Diego, CA).

	RESULTS

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There were no significant differences among initial weights at the outset of the trial (Table 2). The final weights also were not significantly different among dietary treatments nor were there any differences among replicate tanks from the same treatment. All groups increased in weight by > 2.5-fold and although the specific growth rates were relatively low, this could be attributed to the use of purified diets containing nonfish protein. All treatments suffered a number of mortalities (up to 17%), which were due mainly to an outbreak of Vibrio anguillarum ~8 wk after the start of the dietary trial. This infectious outbreak may have affected growth rates because antibiotic was added to feed (oxytetracycline, 80 mg/kg) for a period of 2 wk, and the altered palatability can affect food consumption.

View larger version (171K): [in this window] [in a new window]   Figure 1. Light micrograph of a liver section showing a mild early focal lesion (arrowed) adjacent to a blood vessel (bv) in hepatic tissue of a fish fed the diet deficient in both vitamin E and astaxanthin (Ax). Similar lesions were seen in all dietary treatments, representing 17% of all fish sampled. Scale bar = 100 µm.

View larger version (163K): [in this window] [in a new window]   Figure 2. Light micrographs of a liver section at two levels of magnification. (A) The occurrence of extensive areas of degenerate vacuolated hepatocytes (d) among normal cords of hepatocytes from a fish fed a diet deficient in both vitamin E and astaxanthin is shown. These extensive lesions were only found in fish fed the diet deficient in both vitamin E and astaxanthin and were observed in 15% of the fish assayed from this treatment group. Blood vessel (bv). Scale bar = 100 µm. (B) Light micrograph of the same liver section at higher power. Details are as for panel A except (C) shows an area of early cellular change. Scale bar = 10 µm.

Table 3

Table 4

View larger version (20K): [in this window] [in a new window]   Figure 3. Production of malondialdehyde (MDA), a secondary product of lipid peroxidation vs. time in NADPH/Fe3+-stimulated muscle microsomes prepared from fish fed diets supplemented with both vitamin E and astaxanthin (Ax) (+E, +Ax) or supplemented with either vitamin E or Ax (-E, +Ax and +E, -Ax) or deficient in both vitamin E and Ax (-E, -Ax) for 22 wk. Values are means ± SD, 6 replicates. Although total MDA production after 1.5 h was not significantly different (P = 0.086) among dietary treatments, the slopes of the regression lines were significantly different with values of 0.70 ± 0.14,a 1.25 ± 0.14,b 0.46 ± 0.06a and 1.90 ± 0.17c for the following diets, respectively: + E, +Ax; +E, -Ax; -E, +Ax; -E, -Ax.

View larger version (18K): [in this window] [in a new window]   Figure 4. Concentration of plasma 8-isoprostane in salmon fed diets supplemented with both vitamin E and astaxanthin (Ax) (+E, +Ax) or supplemented with either vitamin E or Ax (-E, +Ax and +E, -Ax) or deficient in both vitamin E and Ax (-E, -Ax) for 22 wk. Results are mean ± SD, n = 6 fish per dietary treatment. The concentrations were not significantly different among dietary treatments. (P = 0.089)

	DISCUSSION

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In all tissues examined, vitamin E concentrations were reduced significantly in fish fed diets deficient in vitamin E compared with those supplemented with 100 mg/kg of -tocopherol. Values for the vitamin E content of liver and muscle in this experiment with Atlantic salmon given diets deficient in vitamin E were similar to the values found in two previous studies using rainbow trout (Cowey et al. 1981 , Hung et al. 1981 ). However, the vitamin E concentrations in liver were greater in this study compared with an experiment investigating deficiency of vitamin E and/or selenium in rainbow trout (Bell et al. 1985 ) but were comparable to values found in a study in which rainbow trout were fed diets containing fresh or moderately oxidized oil in the presence or absence of supplementary vitamin E (Cowey et al. 1984 ). A recent study on Atlantic salmon parr induced vitamin E levels less than half those found in this study with postsmolts, although no effects on growth rate, mortality or tissue histopathologies were reported (Parazo et al. 1998 ). In the study of Bell et al. (1985) , exudative diathesis was seen in fish fed diets deficient in both vitamin E and selenium, although no histopathological lesions were observed. In the study by Cowey et al. (1984) , a severe myopathy was observed in fish fed diets lacking supplementary vitamin E although no evidence of liver lipoid degeneration was reported. The appearance of myopathy in the study of Cowey et al. (1984) was apparently due to the low water temperatures at which the trial was conducted (6–12°C) compared with previous studies conducted at a constant 15°C (Cowey et al. 1981 and 1983 , Hung et al. 1981 ). However, although the temperature range was similar to that found by Cowey et al. (1984) , no myopathy of the skeletal muscle was observed in this study.

The largest reduction in tissue vitamin E concentration as a result of feeding a diet deficient in vitamin E for 22 wk was in liver, where the concentration fell to 3% of the value in livers of fish fed a diet supplemented with vitamin E. In plasma, muscle and heart, the vitamin E present in fish fed the deficient diet was 13, 17 and 18%, respectively, of the values found in fish fed the vitamin E–supplemented diet. However, in brain and eye, vitamin E was reduced to only 35 and 40%, respectively, of the value in fish fed the supplemented diet as a result of feeding the vitamin E–deficient diet for 22 wk. Similar results for the retention of - and -tocopherol were also seen in salmon parr brain in the study by Parazo et al. (1998); although they did not measure eye vitamin E, they found an equally high retention of vitamin E in testicular tissue when feeding diets deficient in vitamin E. Brain, retina and testes are particularly enriched in the long-chain (n-3) HUFA, not only in mammals (Tinoco 1982 ) but particularly in fish (Bell and Dick 1991 , Bell et al. 1996 , Tocher and Harvie 1988 ). Clearly, when diets are deficient in vitamin E, there is a preferential and highly effective retention of this powerful antioxidant in tissues that are enriched in and have a high functional requirement for the long-chain (n-3) HUFA, especially 22:6(n-3).

When salmon muscle microsomes were stimulated by incubation with Fe³⁺/ATP and NADPH, the lag time before onset of MDA production was increased by the presence of vitamin E, Ax and by both vitamin E and Ax. This suggests that Ax can function as a coantioxidant with vitamin E in a stimulated muscle microsome preparation. Ax and the structurally related canthaxanthin have been observed to suppress lipid peroxidation both in vitro and in vivo (Krinsky 1993 ); in a study with rats, Ax was found to be a more potent inhibitor of peroxidation than -tocopherol (Nishigaki et al. 1994 ). In a study by Shimidzu et al. (1996) , eight carotenoids, including Ax and canthaxanthin, extracted from a variety of marine organisms, were tested for their ability to quench singlet oxygen production after thermodissociation of 1,4 dimethylnaphthalene endoperoxide. The quenching activity of each carotenoid was found to be 40–600 times greater than that of -tocopherol, which suggests that they may play a fundamental role in protection against active oxygen species in fish and other organisms. In a recent study in which survival of salmon larvae was compared with egg and larval Ax concentration, a correlation was found suggesting that low Ax resulted in very poor larval survival (Pickova et al. 1998 ).

Recently, 8-iso-prostaglandin F₂ or 8-isoprostane, which is formed via non-enzymatic lipid peroxidation, has been suggested as a potentially important indicator of oxidative stress in vivo (Roberts and Morrow 1997 ). In this study, mean values for plasma 8-isoprostane were elevated more than four-fold in salmon fed diets deficient in Ax or both Ax and vitamin E, although large variation among individual fish meant that the increases were not significant compared with the other two treatments. However, this result does tend to support the theory described above that Ax can provide important antioxidant protection, both in vivo and in vitro, and may have a potency similar to or, in some cellular systems, greater than that provided by -tocopherol (Krinsky 1993 , Nishigaki et al. 1994 , Shimidzu et al. 1996 ). The measurement of 8-isoprostane may provide a particularly sensitive indicator of oxidative damage, both in vivo and in vitro, in addition to the rather nonspecific assessment provided by the current range of TBA-reacting substances assays (Roberts and Morrow, 1997 ).

Infante (1986) proposed that not all of the biological activities of vitamin E could be explained by a simple antioxidant role, and it was suggested that vitamin E could have a regulatory role in the desaturation of (n-3) and (n-6) PUFA by modulation of the microsomal electron transport chain, which is a component of the desaturase complex. Over 30 years ago, a number of investigators found changes in tissue fatty acid composition when a state of antioxidant deficiency was established (Bernhard et al. 1963 , Witting and Horwitt 1964 and 1967 , Witting et al. 1967 ). In a more recent study by Buttriss and Diplock (1988) , a marked increase in the 20:4(n-6) and 22:6(n-3) content of liver microsomes was observed in rats fed diets deficient in both vitamin E and selenium, whereas a further study found 22:6(n-3) alone was elevated in brain and liver microsomes from vitamin E–deficient rats (Clement and Bourre 1993 ). In a recent study with African catfish (Clarias gariepinus), a similar increase in 20:4(n-6) and 22:6(n-3) was observed in liver total lipid of fish fed diets containing oxidized fish oil or oxidized fish oil and low vitamin E (Baker and Davies 1996 ). In both studies, it was suggested that the increased PUFA levels were due to a compensatory overproduction of these fatty acids resulting from their loss due to peroxidation. In this study, we observed a significant increase in 6- and 5-desaturation products of [1-¹⁴C] 18:3(n-3) in hepatocytes isolated from fish fed diets deficient in both vitamin E and Ax, although no changes were observed in the liver polar lipid fatty acid compositions (results not shown). Production of DHA from [1-¹⁴C] 20:5(n-3) was significantly increased in fish fed diets deficient in Ax, suggesting that the absence of Ax has a greater stimulatory effect on conversion of 20:5(n-3) to 22:6(n-3) than does the absence of vitamin E. This study has demonstrated these effects in isolated intact hepatocytes, whereas most previous studies have utilized subcellular fractions, which are less representative of in vivo conditions. In rats fed diets deficient in vitamin E, 6-desaturase activity was increased in liver microsome suspensions but decreased in brain microsome suspensions (Despret et al. 1992 ). However, the activity of 9-desaturase was found to be reduced in rat liver microsomes when vitamin E was fed either to excess or was deficient (Okayasu et al. 1977 ), whereas recent studies of rats fed oxidized cholesterol or linoleic acid showed an increased activity of 6-desaturase (Hochgraf et al. 1997 , Osada et al. 1998 ). All of these studies, including the present one, suggest that an alteration in oxidation potential or "peroxide tone" may be responsible for an increased cellular synthesis of long-chain PUFA. An increase in peroxide tone, whether achieved by restricted dietary intake of one or more antioxidant components and/or by inclusion of dietary pro-oxidants, in the form of oxidized triglyceride oils or other lipid classes, results in modulation of the enzymes involved in fatty acyl desaturation and elongation. However, although the exact mechanism of this modulation remains unclear, increased oxidative stress results in increased phospholipase A₂ activity, which removes peroxidized acyl groups followed by reacylation of lysophospholipids by acyl-CoA:lysophospholipid acyl transferase (McLean et al. 1993 ). The requirement for replacement PUFA in repairing the damaged phospholipids and/or a direct effect of peroxides on the desaturase complex remain the most likely mechanisms to explain the observed increase in fatty acyl desaturation and elongation activities.

This study has demonstrated that, although Atlantic salmon post-smolts may be relatively resistant to many of the gross pathological lesions associated with "classical" vitamin E deficiency models in other animals, they still demonstrate a number of important biochemical changes resulting from dietary vitamin E depletion. In particular, this study has provided evidence for the antioxidant activity of the carotenoid astaxanthin in vivo as well as describing the effects of a moderate oxidative insult on fatty acid metabolism in isolated hepatocytes.

	ACKNOWLEDGMENTS

 
We would like to thank Philip MacGlaughlin and the staff of the Fisheries Research Services Marine Research Unit for their expert assistance with fish husbandry.

	FOOTNOTES

 
¹ Supported by a research award from the MAFF LINK Aquaculture initiative (Project SAL04) including support from industrial co-sponsors, the Scottish Salmon Growers Association Ltd. and the Crown Estates Commissioners. The study was also supported by the "Aquagene" initiative funded jointly by the Natural Environment Research Council and the Scottish Higher Education Funding Council.

³ Abbreviations used: Ax, astaxanthin; BSA, bovine serum albumin; DHA, docosahexaenoic acid; EIA, enzyme immunoassay; EPA, eicosapentaenoic acid; FAF, fatty acid–free; HBSS, Hank’s balanced salt solution; HUFA, highly unsaturated fatty acid; MDA, malondialdehyde; PUFA, polyunsaturated fatty acid; TBA, thiobarbituric acid.

Manuscript received October 15, 1999. Initial review completed November 15, 1999. Revision accepted February 7, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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