Tissue Distribution, Uptake, and Requirement for a-Tocopherol of Rainbow Trout (Salmo gairdneri) Fed Diets with a Minimal

Content of Unsaturated Fatty Acids

COLIN B. COWEY, JOHN W. ADRON, MICHAEL J. WALTON, JOHN MURRAY, ARTHUR YOUNGSON AND DAVID KNOX

N.E.R.C. Institute of Marine Biochemistry, St. Fittick's Road, Aberdeen, ABI 3RA, U. K. and Torn/ Research Station, Abbey Road, Aberdeen AB9 8DG, U. K. ABSTRACT The metabolism of and requirement for a-tocopherol in rainbow trout fed diets containing 1% linolenic acid as sole source of unsaturated fat and graded levels of tocopherol (0.06-10 mg/100 g) were examined. Fish grew 5-fold over a 16-week period. In liver, tocopherol was concentrated in mitochondria with little in cytosol. Orally administered [3H]-tocopherol was rapidly taken up by plasma and liver but uptake into erythrocytes and white muscle was much slower; in most tissues radioactivity reached a plateau after about 3 days but in red muscle radioactivity increased over a 10-day period. Activities of enzymes that prevent free radical initiated tissue damage did not change in tocopherol deficiency. Tocopherol-deficient trout had no gross or subcellular pathologies even though liver and muscle were severely depleted of the vitamin. Ascorbic acid-stimulated lipid peroxidation in liver organd-esindicated a tocopherol requirement of 2-3 mg/100 g diet; the molar ratios of polyunsaturated fatty acids to tocopherol in livers of trout fed diets lacking or supplemented with tocopherol (10 mg/100 g) were 980 and 170, respectively. J. Nutr. Ill: 1556-1567, 1981. INDEXING KEY WORDS tocopherol tissue distribution absorp tion trout A dietary requirement for vitamin E has been demonstrated in a number of fish including chinook salmon [Oncorhynchustshawytscha](l)carp [Cyprinus carpio] (2) channel catfish [Ictalurus punctatus] (3) and Atlantic salmon [Salmo salar] (4). Although comparatively few data are yet available, it is evident that inter-relationships between tocopherol and other nutrients, similar to those observed in birds and mammals, occur in fish. There are synergistic effects between tocopherol and selenium (4). The requirement for tocopherol may be affected by the level of polyunsaturated fatty acids in the diet (5) and by the presence of substances resulting from
their aUtOxidation (3). 1556

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It also appears from the available data on fish that some species are more sensitive to a tocopherol deficiency than are
others. Carp given partially defined diets without added tocopherol developed muscular dystrophy (5) whereas chinook salmon given similar diets showed little tissue pathology apart from hematological changes (1). Besides tocopherol, a number of enzyme systems also function to prevent or limit free radical initiated tissue damage, Adaptive changes in these systems may also be effective in the prevention of pathologies in certain species of fish under conditions of a low tocopherol
Received for publication 27 March 1981.

TOCOPHEROL

METABOLISM

AND REQUIREMENT

IN TROUT TABLE 1 of basal diet' (diet 1)

1557

intake. The systems include glutathione (GSH) peroxidase (EC 1.11.1.9] (6, 7), glutathione S-transferase [EC 2.5.1.18] (8, 9), and Superoxide dismutase [EC 1.15.1.1] (10). In the present study we examined various aspects of the tocopherol re quirement of rainbow trout (Salmo gairdneri). These include the distribution of tocopherol and the time course of its uptake in fish fed diets deficient in or supplemented with a-tocopherol, meas urements of the activities of enzymes that limit free radical damage in tissues and an assessment of the tocopherol require ment of rainbow trout from data on ascorbic acid-stimulated malonaldehyde formation.
MATERIALS AND METHODS

Composition

ComponentCasein,

freeStarch, vitamin pre-cookedPalmitic acidLinolenic acid2Vitamin pre-mix3Mineral mixture4Concentrationg/OOg55.023.213.01.02.85.0 1Vitamin E added to remaining diets to give con centrations of 0.5,1.0, 2.0,3.0, 5.0 and 10.0 mg/100 g diet in diets 2-7, respectively. 2 Sigma (London) Chemical Co. Ltd., Poole, U. K. 3Vitamin mix provided in mg/100 g diet: thiamin hydrochloride, 5; riboflavin, 20; pyridoxine hydrochloride, 5; nicotinic acid, 75; calcium pantothenate, 50; myoinositol, 200; biotin, 0.5; folie acid, 1.5; ascorbic acid, 100; choline bitartrate, 900; menaphthone, 4; cyanocobalamin, 0.009; retinyl palmitate, 4,000 lU; cholecalciferol, 10,000 lU; ascorbyl palmitate, 40. 4 Mineral mixture provided in mg/100 g diet: CaiHjPO^HjO, 3,450; CaCO3, 260; FeSO4 7HZO, 150; MgCo3, 460; KC1, 250; NaCl, 400; Al, (SO4)316H2O, 1; ZnSOJH2O, 20; CuSO4-5H2O, 5; MnSO4 4H2O, 20; KI, 1; CoSO,-7H2O, 5; Na2SeO3, 0.218.

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Fish and diets. Rainbow trout (D. M. Brien, Almondbank, Perth, U. K.) of ap proximately 10 g mean weight were ran domly distributed among 15 tanks at the rate of 30 fish per tank. Tank dimensions and aquarium conditions (water flow rate, light regime and so on) were as described previously (11). Trout were fed a restricted ration amounting to 2 g dry diet/100 g live weight fish/day for 6 days each week; this quantity of diet was readily eaten by the fish, without wastage, at three to four feeds per day. Fish were weighed every 2 weeks over an experimental period of 16 weeks. The basal diet (diet 1, table 1) was mixed and moist pellets prepared as described previously (11); after freeze drying the pellets were stored in sealed plastic bags at -15 until fed. Only limited amounts of dietsufficient to last for 6 weeks were prepared at any one time. Analysis of different batches of this diet showed it to have a tocopherol content that varied between 60 and 66 /ng/100 g. Diets 2-6 containing graded levels of tocopherol (table 2) were formed adding appropriate amounts of DL-a-tocopherol acetate (Rovimix E 50SD, Roche Prod ucts Ltd., Dunstable, U. K.) to the basal mixture. After pelleting and storage for 6 weeks, the measured tocopherol levels were within 10% of the intended level.

The fish in three of the experimental tanks were given diet 1; diets 2-7 were each fed to duplicate tanks offish. Analysis of diets for tocopherol. Two grams of diet together with 2 g pyrogallol were refluxed 30 minutes in 100 ml 95% (v/v) ethanol containing 10 g NaOH. The flask was cooled, 200 ml water added and the mixture extracted x3 with 50-ml portions pentane; remaining traces of NaOH were removed from the com bined pentane extracts by shaking with an equal volume of water. The pentane extract was evaporated to dryness under N2 and the residue taken up in a small volume (0.2-0.5 ml) of mobile phase (1.5% isopropanol in hexane). Tocopherol in the extract was separated by high per formance liquid chromatography (HPLC) using a 250 x 4.6 mm stainless steel column containing micron silica (S5W, Phase Separations Ltd., Queensferry, Clwyd, U. K.) essentially as described by Carpenter (12). Effluent from the column was monitored at 295 nm and quantitation achieved by comparison with D-a-

1558

COWEY ET AL. TABLE 2

Weight gain and mortalities of rainbow trout given diets containing different concentrations of tocopherol for 16 weeks
Diet Tocopherol concentration mg/fcg 0.22'10.4 I234567051020305010010.3 0.0710.5 0.2910.5 0.5710.2 0.6010.2 0.0510.6 0.1251.9 1Means SEM. 2.4054.3 2.5755.8 2.1252.4 5.6751.1 4.3153.2 1.7354.3 1.430.940.970.940.991.000.981.010002011 Mean initial weight Mean final weight g Feed/g gain Total mortalities

tocopherol (Eastman Kodak Rochester, of basal diet in powder form. The ethanol NY) standards (0.2-1.0 fig). was again removed with a stream of N2 Analysis of tissues for tocopherol. and the powder made into a slurry by the Trout were killed by a sharp blow on the addition of 3.75 ml water. A portion of head and a blood sample was taken from this slurry (0.2 ml) was force fed to trout by the caudal vein and immediately cen- stomach tube. Each fish (50-60 g live trifuged to separate cells and plasma. weight) was anesthetized in water con Tissues were rapidly removed from the taining 0.1 g MS222 (ethyl m-aminobenfish, weighed, extracted and saponified zoic acid methane sulphonate, Sigma as described by Bieri (13). Tocopherol Chemical Co., Poole, U. K.) per liter. measurements were made on the final After removal from the water, the slurried extracts as described above. Subcellular radioactive diet was rapidly introduced to the stomach through a teflon catheter fractions of liver were prepared by homog leading, via an 18-gauge needle from a enizing the organ in 4 volumes ice-cold 0.3 M sucrose containing 20 mM N-2- syringe. The fish was then returned to a hydroxyethylpiperazine-N '-2-ethane sul- recovery tank before being killed at the phonic acid (HEPES), 1 mM EDTA and desired interval over the 24-hour time 2% (w/v) pyrogallol. Homogenates were course. Three individuals were used at successively centrifuged at 600 x g for 10 each interval after feeding. minutes (cell debris and nuclei), 15,000 An alternative method of administra x g for 20 minutes (mitochondria), and tion was used in a second, longer time 105,000 x g for 1 hour (microsomes and course (10 days), our intention being to cytosolic supernatant). The pelleted introduce the tocopherol as a pulse. The radioactive tocopherol dried under N2 fractions were resuspended in homog enizing medium and recentrifuged prior as previously, was dissolved in 1 ml of to extraction and analysis by HPLC. lipid extracted some days earlier from Measurements were made on tissues from trout viscera. Fifty microliters of these 20 /Ci tocopherol) six fish from each treatment, except for lipids (containing fish analyzed at 12 weeks when only two were given by stomach tube to each trout. individuals were sampled. Measurement of radioactivity. Plasma Administration of radioactive tocoph samples (200 /I) were added directly to erol to trout. D-a-[5-Methyl-3H]tocoph10 ml scintillation fluid (Instagel, Packard erol was commercially obtained (Radio- Instruments, Downers Grove, IL) and chemical Centre, Amersham, U. K.). Sol counted directly on a Tricarb liquid vent (toluene/ethanol) was removed under scintillation spectrometer (Packard In struments, Downers Grove, IL). Erythroa stream of N2 and 250 /Ci tocoph erol was taken up quantitatively in 2.0 ml cytes, other tissues and subcellular frac in ethanol and mixed thoroughly with 1.25 g tions of liver were homogenized

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TOCOPHEROL METABOLISM AND REQUIREMENT IN TROUT

1559

ethanol containing pyrogallol, saponified and extracted as described by Bieri (13). Two hundred-microliter portions of each hexane extract were finally counted. Erythrocyte fragility. Measurements were made as described by Draper and Csallany (14) except that incubations were carried out in a shaking waterbath (60 cycles/minute) at 15 for 24 hours; every 8 hours tubes were removed from the bath and cells thoroughly resuspended by careful shaking. Blood was taken by syringe from the caudal vein. Microscopy. For light microscopy tis sues (white muscle, liver, intestine) were fixed in formalin buffered with 0.05 M phosphate pH 7.4 and embedded in paraffin wax. Sections (5 /A)were stained with hematoxylin and eosin. For electron microscopy, tissues were fixed 4 hours in 0.1 M phosphate buffer containing 1% NaCl, post fixed for 1 hour in 1% aqueous osmium tetroxide, dehydrated and then embedded in Emix resin. Sections (cut on an LKB ultratone 3, LKB Instruments, Croydon, U. K.) were stained in uranyl acetate followed by lead citrate and examined in a JEOL 100 CX electron microscope (Jeol Ltd., London, U. K.). Blood smears were air dried and stained with Leishmann's stain. Lipid and fatty acid analyses in liver. Lipids were extracted from whole livers as described by Folch et al. (15) and total lipids measured gravimetrically. Lipid classes were separated by thin-layer chromatography (TLC) on silica gel G plates (0.25 mm thick) using hexane:diethyl ethenformic acid (140:60:1, by volume) as developing solvent. Methyl esters of phospholipids were prepared by H2SO4catalyzed methylation (16) and the fatty acid methyl esters were separated and quantitated by gas liquid chromatography (Fractovap 2150, Erba Science Ltd., Swindon, U. K.). A 25 m capillary column coated with liquid phase silar5CP was used, N2 being the carrier gas. Temperature programming was from 140 to 210 at 3 per minute. Individual fatty acid methyl esters were identified by comparison with known standards and by reference to the data of Ackman and Eaton (17).

Enzyme assays. GSH peroxidase (18), GSH reductase [EC 1.6.4.2] (19) and glutathione S-transferase activities were determined in cytosol from liver and intestine. Cytosol was prepared as de scribed above except that pyrogallol was omitted from the extraction medium; it was dialyzed for 4 hours at 4 against 5 mMphosphate, pH 7.5, prior to assay of GSH peroxidase and GSH reductase and non-specific oxidation of NADPH in the assay was thereby prevented. When H2O2 was used as substrate (at con centrations of 0.25 and 0.5 mM), nonen/ymatic oxidation of GSH occurred comparatively quickly and accurate meas urement of GSH peroxidase activity with the coupled assay was not possible; an interrupted assay (20) was therefore used (final concentration H2O2being 0.5 mM, GSH concentration being measured at 5minute intervals over an incubation time of 15 minutes). With eumene hydroperoxide as substrate, non-enzymatic oxidation of GSH was slow permitting accurate measurement of GSH peroxidase with the coupled assay (18). There was a linear relationship between enzyme ac tivity and protein concentration over a 4-fold range. Glutathione S-transferase activity was assayed as described by Habig et al. (21) with l,2-dichloro-4-nitrobenzene and 1chloro-2,4-dinitrobenzene as substrates. For Superoxide dismutase, tissues were homogenized in 9 volumes 0.05 M phos phate buffer, pH 7.2, containing 1% Tri ton X-100, the homogenate centrifuged 5 minutes at 12,000 g and the super natant used for assay. Activity in liver was measured by the method of Heikkila et al. (22); KCN at a concentration of 5 mM was included in the assay for measurement of cyanide insensitive superoxide dismutase. This method could not be applied to cardiac muscle because autoxidation of 6-hydroxydopamine was enhanced in the presence of cyanide; a method (23) based on the oxygen-de pendent oxidation of epinephrine to adrenochrome by xanthine oxidase plus xanthine was therefore applied to this tissue. Total and oxidized glutathione (GSSG)

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1560

COWEY ET AL. TABLE 3 Concentrations of a-tocopherol in tissues of rainbow trout given diets either lacking or supplemented with a-tocopherol for 12 weeks and 16 weeks 12 weeks Diet 1 Diet 7 2.57" 8.43 0.32" 32.92 2.27"7.04 16 weeks

Diet 1

Diet?

Liver, /ig/g tissue Muscle, fig/g tissue Adipose tissue, figlg tissue Erythrocytes, fig/ml5.64

0.731-2-" 2.44 0.06" 10.51 1.70a73.04

0.42a 2.59" 0.68 0.12C 8.40 1.28b 9.43 2.42a 46.12 8.49" 1.34 0.28-71.20 14.17 1.08" are

' Means SEM for six fish per treatment, significantly different (F < 0.01).

2 Values in the same line with different superscripts

concentrations in liver and whole blood were assayed as the sum of reduced and oxidized forms expressed in GSH equiv alents (GSH + 2 GSSG) or as GSSG alone using the catalytic assay with 5,5'dithiobis (nitrobenzoate) and glutathione reductase (24). Glutathione estimations and enzyme measurements were per formed on tissues from six individuals from each of dietary treatments 1 and 7. Ascorbic acid-stimulated lipid peroxidation in microsomes and mitochondria. Microsomes and mitochondria were pre pared as described above except that sucrose was replaced by sorbitol in homog enizing medium which did not contain pyrogallol. Portions (0.5 ml) of washed microsomal or mitochondrial suspensions (0.5 mg protein/ml) were added to 2 ml of a medium containing (final concentra tions) 0.033 M phosphate, pH 7.5, 0.1 M KC1,0.133 mM FeCl3 and 1.0 mM ascorbic acid. Incubations were carried out for 1 hour under air at 15. The reaction was stopped by addition of 1 ml of 20% (w/v) trichloroacetic acid (TCA); zero time con trols were included with each series of incubations, trichloroacetic acid being added before incubation. Quantities of malonaldehyde formed were measured with thiobarbituric acid as described by Noguchi et al. (25). Statistical procedures. Statistical anal ysis of results (mean SEM, significance of difference between treatments) was carried out as described by Fisher (26).
RESULTS

Weight gains, mortalities and food con version ratios of trout given the seven ex

perimental diets are shown in table 2. There was a 5-fold increase in weight in all treatments over the experimental period but no differences occurred be tween treatments. Only four fish died throughout the experiment and these had no discernible pathologies; mortalities were not associated with a low or basal dietary intake of tocopherol. Food con version rate was also unaffected by the tocopherol level in the diet, values were close to unity in all treatments. There were no gross pathologies such as exudative diathesis, dermal depigmentation or muscular dystrophy in fish fed diet 1 lacking supplementary tocopherol. Con ventional histology and electron micros copy applied to liver, intestine and mus cle of trout fed diets 1 and 7 gave similar, normal pictures. Hepatosomatic index (weight of liver in grams/body weight in grams) of fish given diet 1 was 1.5 0.07% that of fish given diet 7, 1.53 0.01. Erythrocyte fragility in fish fed diet 1 (46.87 9.95% haemolysis) was, however, significantly greater than that of fish given diet 7 (5.53 1.06%hemolysis; P < 0.01) and the former group had a sig nificantly lower hematocrit (21 3.4% compared with 41 2.1%; P < 0.01). Concentrations of tocopherol in mus cle, liver and erythrocytes of trout given the basal diet (table 3) were about 10-fold less than those in trout fed diet 7 com taining 100 mg a-tocopherol/kg diet. Dif ferences in adipose tissue were less marked. Between weeks 12 and 16 there was apparently a decrease in tocopherol content of the white muscle in trout given diet 1. By contrast tocopherol

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TOCOPHEROL METABOLISM AND REQUIREMENT IN TROUT

1561

TABLE 4 Concentrations of a-tocopherol in subcellular fractions of liver from rainbow trout given diets lacking or supplemented with a-tocopherol for 16 weeks
Diet 1 Diet?

(-glfraction-g liver % total 0.20'-2-a1.50 CytosolNuclear 0.56a3.76 fractionMitochondriaMicrosomes0.56 0.42a1.22 0.49a7.921.353.417.32.57

1Means SEM for six fish per treatment, are significantly different (P < 0.01).

fig/fraction/g liver % total 1.17a16.03 3.00"32.21 3.51"20.39 3.04"3.622.545.228.6

2 Values in the same line with different superscripts

content of the liver and adipose tissue did not decrease over this period despite the fact that the mean weight of the fish increased on average by 15.9 g (43%). There was a marked reduction in the tocopherol content of all four subcellular fractions from fish given the diet 1 (table 4), however the proportion of tocopherol in the mitochondria of these tocopheroldepleted fish increased relative to that of other organelles. The time course of assimilation of radio active tocopherol is shown in figure 1. Under the experimental conditions used, tocopherol was rapidly taken up into both blood stream and liver. In fact appreciable amounts of radioactivity from [3H]-tocopherol were detected in the livers of deficient fish within 1 hour of adminis tering the dose. Rapid assimilation of tocopherol has also been observed in nor mal chicks (27). Uptake of radioactivity into adipose tissue (not shown in fig. 1) was also rapid but fluctuated somewhat erratically over the time course. Incor poration of isotope into erythrocytes and muscle was much slower than into liver. There were no significant differences in the time course between tocopheroldepleted fish and those given supplemen tary tocopherol (100 mg/kg diet). Radioactive tocopherol was rapidly taken up by liver mitochondria in depleted fish (fig. 2) and highest levels of radioactivity were found in this fraction over the time course. Isotope concen tration pattern in other subcellular frac tions ranged in the same order as the pattern of mass concentration shown in

table 4. A somewhat different time course was evident in control fish (fig. 2)high levels of radioactivity were present in cytosol after 4 hours with a more gradual transfer to subcellular organelles and much of the activity remaining in the nuclear fraction. In rats given an intraperitoneal dose of [14C]tocopherol, the greater part (approximately 90%) of the radioactivity in liver was located in mito chondria and microsomes (28). Radioactivity was still increasing in both plasma and tissues 24 hours after administration of the [3H]-tocopherol (fig. 1) presumably because isotope was still being taken up from the food bolus in the gastrointestinal tract. In order to examine the fate of a pulse of isotopie tocopherol, we carried out a second, much longer (10 days) time course. In this ex periment the isotope was administered in oil rather than in a complete diet. The concentration of radioactivity in the plasma reached a peak after about 3 days and slowly declined thereafter. In most tissues radioactivity reached a plateau level after about 3 days and re mained relatively constant thereafter, in red muscle however, isotope concentra tion was still increasing after 10 days. The total amounts of isotope accumu lated in whole organs or tissues of fish of 10 g in this experiment are shown in table 5. Values for the relative size of each organ or the relative quantity of each tis sue were obtained by dissection of fish and from literature values (29, 30). Those used (as percent whole fish) were white muscle 60, red muscle 5, plasma 1.42,

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1562

COWEY ET AL.

10.0 9.0
8.0

7.0

= 6.0 o

i
b

5.0
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o 4.0 * 3.0 E

0 2.0

1.0

O 8 12 16 Hours after dosing 20

24

12

16

20

24

Hours after dosing

Fig. 1 Time course of D-a-[5-methyl-3H] tocopherol uptake by rainbow stomach tube; a. tocopherol-deficient trout (diet 1); b. trout given diet 7, Radioactivity in plasma ( ), in liver (A A), in red blood cells (0 0). Standard errors are shown by vertical lines; extremely small

trout following a single dose by supplemented with tocopherol. (O O) and in white muscle standard errors are not shown.

liver 1.5, gills 1.25, kidney 0.75, heart 0.2, brain 0.17 and spleen 0.15. Because of its large mass, more radioactivity accumu lated in white muscle than in other tis sues. This is line with an earlier estimate of the tocopherol distribution in the body of rainbow trout (31). There was also a steady accumulation of tocopherol in red muscle, total radioactivity exceeding that in any other tissue apart from white muscle. Concentrations of lipid in the livers of trout given tocopherol-deficient diets (3.78 0.18%) were not significantly dif ferent from those of control trout (3.54 0.09%). These values are similar to

that (4.30%) obtained by Castell et al. (32) in trout fed casein-based diets containing 1% linolenic acid and 4% oleic acid as lipid component. Linolenic acid levels were very low in phospholipids from liver and intestine of both tocopherol-deficient and control fish (table 6). Substantial conversion of linolenic acid to docosahexanoic acid had clearly occurred in both tocopherol deficient and control trout; the ratio of 20:3 (n-9)/22:6 (n-3) in liver phospholipids suggested as an in dex of essential fatty acid status in trout (32) was less than 0.1 for both dietary treatments indicating that the fish were normal in this respect. There were no sig-

TOCOPHEROL METABOLISM AND REQUIREMENT IN TROUT

1563

60
40

S 20

a m
4*

b.

s? 60 in
I g o 1 20 40

8 12 16 Hours after dosing

20

24

Fig. 2 Time course of uptake of radioactivity from D-a-[5-methyI-3H]tocopherol into subcellular fractions of rainbow trout liver following a single dose by stomach tube into (a) trout given diet 7 supplemented with tocopherol and into (b) tocopherol-deficient trout (diet 1). Radioactivity in cytosol (), nuclear fraction (O G), mitochondria (A A) and microsomes (El El).

nificant differences between treatments in fatty acid composition of phospholipids of these tissues. Glutathione peroxidase activities in liver and intestine of trout given diet 1

(tables 1, 2) were 12.0 3.0 and 5.4 0.2 nmole substrate converted/minute/mg protein; activities in these same tissues from trout given diet 7 were significantly different. These values were obtained with eumene hydroperoxide as substrate and may thus include activity due to both Se-dependent and Se-independent en zymes (33). The presence of Se-depend ent GSH peroxidase in trout liver was demonstrated by interrupted assays using H2O2as substrate (20). Activity measured with H2O2as substrate was approximately half that obtained with eumene hydroperoxide. Se-independent GSH peroxi dase activity in mammals is now known to be due, at least in part, to glutathione S-transferase activity (8). The presence of this enzyme in trout liver was demon strated recently (34) but the activity of the enzyme was not altered in tocopherol deficiency, mean values in the liver and intestine of tocopherol-deficient fish being 0.56 0.6 and 0.21 0.06 /.mole substrate converted/minute/mg protein respectively. It is probable that gluta thione S-transferase activity contributed to the GSH peroxidase activity measured with eumene hydroperoxide as substrate. Virtually all the glutathione in liver and blood was in the reduced form in both tocopherol-deficient and control trout. There were no significant differences between treatments; mean values were 2.45 0.19 imole/g and 1.30 0.01 /mole/mlfor liver and blood, respec-

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TABLE 5 Accumulation of radioactivity in tissues of rainbow trout following a pulse of [3H]-(ocop/ieroigiven by stomach tube Days 1 2 3 4 6 cpmltotal tissue/100 g fish x 10~ 258 490 235 123 324 182 2
22

10

Plasma Liver Kidney

112

112
30

Gills
White muscle Red muscle Brain Heart Spleen

12
86 26

215 315 141 77 174

96

2
3

3
17

139 228 176 197 510 274 1 25

20

30

162 382 297 186 618 261 1 25 24

91 230 136 129 762 391 2 22 26

72 310 214 163 726 456 3 27 24

1564

COWEY ET AL. TABLE 6

Proportions of fatty acids in phospholipids from liver and intestine of rainbow trout given diets either lacking or supplemented with a-tocopherol for 16 weeks Liver 1 (tocopherol dficient)Diet 7 (tocopherol supplemented)Diet % by wt 0.12'25.7 14:016:016:l(n-7)18:018:l(n-9)18:3 0.1924.9 0.1533.03 0.1533.53 0.326.67.929.80.400.932.60.600.931.117.60.551.041.110.010.090.470.010.070.061.484.341.53 0.407.03 2.41.9 0.861.98 0.685.6 0.2612.4 0.3212.5 0.3228.93 0.3410.07 0.447.43 1.580.50 1.711.77 0.291.93 0.021.43 0.171.47 0.202.1 0.132.7 0.260.56 0.124.2 0.44.67 0.021.23 0.452.4 0.131.33 0.492.4 0.1920.1 0.1528.23 0.0631.67 1.43.21.35 1.994.161.33 0.610.42 1 (tocopherol deficient)Diet Intestine 7(tocopherol supplemented)

Fatty acidDiet

(n-3)20:1
(n-9)20:220:3 (n-9)20:5 (n-3)22:5 (n-3)22:6 (n-3)Other1.5

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1 Means SEM.

tively. Thus there was neither a fall in reducing potential nor in capacity for detoxication by GSH-dependent enzymes in tocopherol-deficient tissues. Superoxide dismutase neutralizes un controlled formation of Superoxide radi cals by conversion to H2O2 which in turn may be decomposed by GSH peroxidase or catalase. No significant changes in activity of either the cupro-zinc or manganese enzymes in liver or heart of tocopherol-deficient fish were found. Ascorbic acid stimulated lipid peroxide formation in mitochondria and microsomes from trout given graded dietary levels of tocopherol are shown in figure 3. Large amounts of malonaldehyde were formed when organd-es from tocopheroldeficient fish were incubated; peroxidation decreased significantly (P < 0.01) as dietary tocopherol increased until the limit of response (level at which malonal dehyde formation was effectively pre vented) was reached. By extrapolation of the linear regression of both parts of the curve, the dietary requirement for tocoph erol was determined as 2-3 mg/100 g diet from curves from both microsomes and mitochondria.

DISCUSSION The diets used in these experiments were formulated to measure tocopherol uptake and requirement in the absence of complicating factors such as a high polyunsaturated fatty acid intake, the pres ence of peroxides or other autoxidation products or a selenium deficiency. Under these conditions tocopherol deficiency did not lead to any lowering of growth rate of Atlantic salmon (4) and channel catfish (3). In other species, carp (2) and chinook salmon fed diets containing herring oil triglycrides (1), tocopherol deficiency did lead to a reduction in weight gain. Pathologies that have been associated with tocopherol deficiency per se include, in Atlantic salmon, depigmentation, exu dative diathesis, a stress (fainting) re sponse and hematological disorders (4); in carp the appearance of muscular dystrophy was used as an indication of tocopherol deficiency (5). By contrast no gross pathologies (other than an increase in erythrocyte fragility and a decrease in hematocrit) were detected by us in rain bow trout depleted of tocopherol; light

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and electron microscopy demonstrated that in these fish, tissues were normal at 40 both the cellular and subcellular level. Micnosomes Concentrations of tocopherol present in tissues offish exhibiting pathologies were I 30 in Atlantic salmon 5.0 iu/100 gin liver and I 0.15 iu/100 g in carcass [equivalent to 45 a and 1.4 /u,g/g,respectively] (4) and in carp 20 2.7 /ug/g in hepatopancreas and 2.8 /g/g in muscle. Comparing these values with those obtained in the present study (table ^ 10 3), we reasonably inferred that trout given diet 1 were depleted of tocopherol to an extent that resulted in pathologies in these other species. The failure to induce 40 pathologies in rainbow trout similar to Mitochondria those occurring in a closely related salmonid may be due to the different c 30 fatty acid intakes in the two experiments _o a and to the fact that fish differed in size by an order of magnitude [those of Poston S 20 et al. (4)were between 0.9 and 5.2 g, those o in the present experiment 10-50 g] Tocopherol concentration in the white c 10 muscle of trout decreased between weeks 12 and 16. That in liver did not change (the quantities of adipose tissue present in the trout were so small that lack of 0 2 4 6 8 10 change in tocopherol concentration was Dietary tocopherol mg / 100g unremarkable). The decrease in tocoph erol concentration in the white muscle is Fig. 3 Effect of concentration of a-tocopherol in greater that can be accounted for by the the diet on in vitro ascorbic acid-stimulated peroxidiluent effect of an increase in weight. dation in mitochondria and microsomes from the It seems unlikely that tocopherol is livers of rainbow trout. utilized more rapidly in white muscle than in liver or other tissues and there previous findings in mice that the distri remains the possibility that tocopherol is bution of intravenously administered translocated from muscle to other tissues [14C]tocopherol is not significantly dif under conditions of tocopherol depletion. ferent in dystrophic mice and normal It was considered possible that during litter mates.1 The pattern of uptake of [3H]tocopherol tocopherol deficiency certain tissues might become chronically depleted more into red muscle differed from that into rapidly than others; these tissues might most other tissues. The level of radio then take up tocopherol most avidly when activity was still increasing 10 days after a limited supply became available. Eryth- the pulse (fig.3, table 5)presumably by rocytes are rapidly affected by tocopherol uptake from plasma and other tissues deficiency and an increase in erythrocyte whereas that in other tissues had by then fragility is used to test for this condition. reached a plateau. The reasons for this Such an increase was observed in the are not obvious but red muscle is an current experiments. However the rate of aerobic tissue and is active catabolically uptake of radioactive tocopherol by eryth- being used continuously in normal swimrocytes was similar in both deficient and 1Simon, E. J., Gross, C. S. & Milhorat, A. T. (1957) Intracellular control fish and was relatively slow in distribution of radioactivity following intravenous administration of both (fig. 1). This result accords with ["CJlabelled vitamin E. Fed. Proc. 16, 249 (abs. 1067).

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ming. It has high concentrations of myoglobin and cytochrome C and is a tissue with a high O2 consumption and a high rate of fatty acid oxidation. These con ditions may be conductive to the forma tion of free radicals, the level of which could be controlled by tocopherol. As tocopherol functions as a free radical reaction chain terminator, the thesis has been developed that the adequacy of tocopherol intake should be reflected at the cellular level by the polyunsaturated fatty acid (PUFA): tocopherol ratio (35), the ratios in membranous structures, microsomes or mitochondria being par ticularly apposite (36). Evarts and Bieri (35) examined this ratio in tissues of the rat to assess tocopherol adequacy of vege table oils; in rats given corn oil the value for the ratio /molesPUFA > 18;2//amoles a-tocopherol was 1,200 in liver. Biomembranes in cold water fish are characterized by (n-3) fatty acids having a high degree of unsaturation presumably to maintain membrane fluidity at low temperatures. Lower molar ratios of PUFA:tocopherol than those found in rats by Evarts and Bieri (35) would appear necessary to ensure adequate protection against peroxidation. Values found in the livers of fish in the present experiment fit this expectation being 980 and 170 for trout given diets deficient in or supplemented with a-tocopherol. PUFA intake of these fish was much lower than that of the rats studied by Evarts and Bieri (35) and this undoubtedly contributed to the low ratio found. A value of 200 for this ratio in rainbow trout liver should certainly indi cate an adequate vitamin E intake; a value of the order of 1,000 may indicate an adequate intake. Synergistic effects between Se and vitamin E in preventing exudative di athesis in chicks have been related to the role of Se as a component of GSH peroxidase (25). A similar situation has been shown to exist in salmon (Salmo salar) with muscular dystrophy of the greatest severity occurring in fish given a diet deficient in both tocopherol and Se (4); plasma GSH peroxidase activity, measured with H2O2 as substrate, re sponded to dietary Se intake but was not affected by tocopherol intake. Because

hepatic GSH peroxidase activity in mice increased as tocopherol intake decreased (37) as did that in adipose tissue and muscle (but not liver) of rats (7), two components of the "glutathione peroxi dase system" (GSH peroxidase and GSH reductase) were assayed in liver and intestine of rainbow trout but in neither tissue was either enzyme affected by tocopherol deficiency. It has been known for some time that the extent of lipid peroxidation in tissue homogenates in vitro is inversely related to the vitamin E status of the animal (38). Consequently peroxidation of microsomal and mitochondrial lipids has been used as a criterion in establishing the tocopherol requirement of animals. The values so obtained generally agree well with those obtained by other parameters including growth (39). In the present ex periments, membrane lipid peroxidation was the only criterion on which tocoph erol requirement of trout could be based as neither reductions in growth rate nor pathologies occurred. The value ob tained, 2-3 mg a-tocopherol/100 g diet, agrees well with values of 0.5-3 mg/100 g for chinook salmon (Oncorhynchus tshawytscha) (1) previously derived from growth experiments.
LITERATURE CITED

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1. Woodall, A. N., Ashley, L. M., Halver, J. E., Olcott, H. S. & Van Der Veen, J. (1964) Nu trition of salmonid fishes. XII. The a-tocoph erol requirements of chinook salmon. J. Nutr. 84, 125-135. 2. Watanabe, T., Takashima, F., Ogino, C. & Hibiya, T. (1970) Effects of a a-tocopherol deficiency on carp. Bull. Jpn. Soc. Sci. Fish. 36, 623-630. 3. Murai, T. & Andrews, J. W. (1974) Inter actions of dietary a-tocopherol, oxidized men haden oil and ethyoxyquin on channel catfish (Ictalurus punctatus) J. Nutr. 104, 1416-1431. 4. Poston, H. A., Coombs, G. F. & Leibovitz, L. (1976) Vitamin E and selenium interrela tions in the diet of Atlantic salmon (Salmosalar): gross, histological and biochemical deficiency signs. J. Nutr. 106, 892-904. 5. Watanabe, T., Takeuchi, T., Matsui, M., Ogino, C. & Kawabata, T. (1977) Effect of a o-tocopherol deficiency on carp. VII. The relation ship between dietary levels of linoleate and a-tocopherol requirement. Bull. Jpn. Soc. Sci. Fish. 43, 935-946. 6. Little, C. & O'Brien, P. J. (1968) An intra-

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cellular GSH-peroxidase with a lipid peroxide substrate. Biochem. Biophys. Res. Commun.31, 145-150. 7. Chow, C. K., Reddy, K. & Tappe!, A. L. ( 1973) Effect of dietary vitamin E on the activities of the glutathione peroxidase system in rat tissues. J. Nutr. 103, 618-624. 8. Lawrence, R. A., Parkhill, L. K. & Burk, R. F. (1978) Hepatic cytosolic non selenium-de pendent glutathione peroxidase activity: its nature and the effect of selenium deficiency. J. Nutr. 108, 981-987. 9. Prohaska, J. R. & Ganther, H. E. (1977) Glutathione peroxidase activity of glutathione S-transferase purified from rat liver. Biochem. Biophys. Res. Commun. 76, 437445. 10. De Rosa, G., Keen, C. L., Leach, R. M. & Hurley, L. S. (1980) Regulation of Superoxide dismutase activity by dietary manganese. J. Nutr. 110, 795-804. 11. Cowey, C. B., Higuera, M. & Adron, J. W. (1977) The effect of dietary composition and of insulin on gluconeogenesis in rainbow trout. Br. J. Nutr. 38, 385-389. 12. Carpenter, A. P. (1979) Determination of tocopherols in vegetable oils. J. Am. Oil Chem. Soc. 56, 668-671. 13. Bieri, J. G. (1969) Chromatography of to copherols. In: Lipid Chromatographie Analysis, vol. 2 (Marinetti, G. V., ed.), p. 459, Marcel Dekker Inc., New York. 14. Draper, H. H. & Csallany, A. S. (1969) A simplified hemolysis test for vitamin E defi ciency. J. Nutr. 98, 390-394. 15. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509. 16. Christie, W. W. (1973) Lipid Analysis, pp. 87-92, Pergamon Press, Oxford. 17. Ackman, R. G. & Eaton, C. A. (1978) Some contemporary applications of open-tubular gasliquid chromatography in analyses of methyl esters of longer-chain fatty acids. Fette Seifen Anstrichm 80, 21-37. 18. Lawrence, R. A. & Burk, R. F. (1976) Glu tathione peroxidase activity in selenium-defi cient rat liver. Biochem. Biophys. Res. Com mun. 71, 952-958. 19. Racker, E. (1955) Glutathione reduc-ase (liver and yeast). In: Methods in Enzymology, vol. 2 (Colowick, S. P. & Kaplan, N. O., eds.), pp. 722-725, Academic Press, New York. 20. Hafeman, D. G., Sunde, R. A. & Hoekstra, W. G. (1974) Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J. Nutr. 104, 580-587. 21. Habig, W., Pabst, M. J. & Jakoby, W. B. (1974) Glutathione S-transferases. The first enzymatic step in mecapturic acid formation. J. Biol. Chem. 249, 7130-7139. 22. Heikkila, R. E., Cabbat, F. S. & Cohen, G. (1976) In vivo inhibition of Superoxide dismutase in mice by diethyldithiocarbamate. J. Biol. Chem. 251, 2182-2185. 23. Panchenko, L. F., Brusov, O. S., Gerasimov, A. M. & Loktaeva, T. D. (1975) Intramito-

chondrial localization and release of rat liver Superoxide dismutase. FEES Lett. 55, 84-87. 24. Hazelton, G. A. & Lang, C. A. (1980) Glu tathione contents of tissues in the aging mouse. Biochem. J. 188, 25-30. 25. Noguchi, T., Cantor, A. H. & Scott, M. L. (1973) Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103, 1502-1511. 26. Fisher, R. A. (1950) Statistical Methods for Research Workers, p. 354, Oliver & Boyd, Edinburgh. 27. Thompson, J. N. & Scott, M. L. (1970) Im paired lipid and vitamin E absorption related to atrophy of the pancreas in selenium-deficient chicks. J. Nutr. 100, 797-809. 28. Csallany, A. S.& Draper, H.H. (1960) Deter mination of N,N'-diphenyl-p-phenylenediamine in animal tissues. Proc. Soc. Exp. Biol. Med. 104, 739-742. 29. Dentn, J. E. & Yousef, M. K. (1976) Body composition and organ weights of rainbow trout, Salmo gairdneri.]. Fish. Biol. 8,489-499. 30. Holmes, S. N. & Donaldson, E. M. (1969) The body compartments and the distribution of electrolytes. In: Fish Physiology, vol. 1 (Hoar, W. S. & Randall, D. J., eds.), pp. 1-89, Academic Press, New York. 31. Bugii, K. & Kinumaki, T. (1968) Distribu tion of vitamin E in a few species offish. Bull. Jpn. Soc. Sci. Fish. 34, 420-428. 32. Castell, J. D., Lee, D. J. & Sinnhuber, R. A. (1972) Essential fatty acids in the diet of rain bow trout (Salmo gairdneri): lipid metabolism and fatty acid composition. J. Nutr. 102, 93-100. 33. Lawrence, R. A. & Burk, R. F. (1978) Species, tissue and subcellular distribution of non Sedependent glutathione peroxidase activity. J. Nutr. 108, 211-215. 34. Nimmo, I. A., Clapp, J. B. & Strange, R. C. (1979) A comparison of the glutathione Stransferases of trout and rat liver. Comp. Bio chem. Physiol. 63B, 423-427. 35. Evarts, R. P. & Bieri, J. G. (1974) Ratios of polyunsaturated fatty acids to a-tocopherol in tissues of rats fed corn or soybean oils. Lipids 9, 860-864. 36. Kornbrust, D. J. & Mavis, R. D. (1980) Rela tive susceptibility of microsomes from lung, heart, liver, kidney, brain and testes to lipid peroxidation: correlation with vitamin E con tent. Lipids 15, 315-322. 37. Trostler, N., Brady, P. S., Romsos, D. R. & Leveille, G. A. (1979) Influence of dietary vitamin E on malondialdehyde levels in liver and adipose tissue and on glutathione peroxi dase and reductase activities in liver and erythrocytes of lean and obese (Ob/Ob) mice. J. Nutr. 109, 345-352. 38. Bieri, J. G. & Anderson, A. A. (1960) Peroxi dation of lipids in tissue homogenates as re lated to vitamin E. Arch. Biochem. Biophys. 90, 105-110. 39. Coombs, G. F. & Scott, M. L. (1974) Dietary requirements for vitamin E and selenium meas ured at the cellular level in the chick. J. Nutr. 104, 1292-1296.

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