Lipid composition of tissue and plasma in two mediterranean fishes, the gilt-head sea bream (Chrysophyrys auratus) and the European seabass (Dicentrarchus labrax) Grant McClelland, Georges Zwingelstein, Jean-Michel Weber, and Gerard Brichon

Abstract: The fatty acid (FA) composition of phospholipid (PL), triacylglycerol (TAG), and total lipid (TL) fractions was determined for liver, adipose tissue, white muscle, and plasma of European seabass (Dicentrarchmrs babrax) and gilt-head sea bream (Chrysophyrys auratus) fed on the same diet. FA composition of plasma nonesterified fatty acids (NEFA) and TAG as well as tissue TAG and TL correlates with that of the diet. FA composition of PL in tissues and plasma was different from that of the diet and different in the two species. Contribution to tissue wet weight of the different lipid fractions showed that both species store lipid primarily outside of muscle (only 3% in both species) as TAG in adipose tissue (about 80% lipid-wet weight-'). Seabass store 33% wet weight of lipid in liver, while sea bream store only 5% wet weight. The plasma concentration of NEFA and TAG is 2223 and f 2 260 nmo1.m~-' for seabass and 2790 and 9670 nrnol-m~- ' for sea bream. The data show that these two fishes with similar lifestyle store lipid in the same fashion and that dietary FA composition affects the FA composition of TL and TAG fractions but not PL in tissue and plasma.

RCsumC : Nous avons rnesurC la composition en acides gras (FA) des phospholipides (PL), des triacylglycerides (TAG) et des lipides totaux (TL) dans le foie, le tissu adipeux, le muscle blanc et le plasma chez le bar commun (Dicerztrarchus labrax) et la dorade royale (Ch~pssophy~pss aurabus), ces deux poissons recevant la meme alimentation. La composition en AG des acides gras libres (NEFA) et de TAG du plasma ainsi que de TAG et de TL des tissus correspond B la proportion cabservee dans le rCgime alimentaire. Par contre, la composition en FA de PL dans les tissus et le plasma Ctait diffkrente de celle du rkgime alimentaire, et diffkrait chez les deux espkces. La contribution des diffkrentes fractions lipidiques au poids humide des tissus rkvkle que les esp5ces stockent les lipides essentiellement B 19extCrieur du muscle (3 % seulement chez les deux espkces) sous forme de TAG dans le tissu adipeux (environ $0 % des lipides, en psids humide). Le bar stoeke 33 % en pcaids humide des lipides dans le foie, contre 5 96 seulement chez la dorade. La concentration plasmique de NEFA et de TAG est de 2 223 and 12 260 nmol-rnl-' pour le bar et 2 790 and 9 678 nmol-rn~- ' pour la dsrade. Les donnCes montrent que ces derax poissons ara mode de vie similaire stockent les lipides de la meme fa~ow, et que la composition dra rCgime alimentaire en FA agit sur la composition en FA de TL et de TAG mais non sur celle de BL dans les tissus et le plasma.

[Traduit par la RCdaction]

Introduction represent the largest energy source for fishes (Moyes and West 1994). Phospholipids (PL), the major component of

Lipids serve many roles in vertebrates. Among the most cell membranes, help maintain membrane fluidity and func- prominent are (1 ) an energy source and (2) the main corn- tion despite changes in water salinity andlor temperature ponent of cellular membranes. They are stored primarily (Bell et al. 1986). The major lipid storage sites in fish are as triacylglyceroIs (TAG), whole-body stores of which liver, adipose tissue, and skeletal muscle. The principal

I Received December 13, 1993. Accepted June 15, 1994. 512193

G, ~ c ~ l e l l a n d ' and J.-M. Weber. University of Ottawa, Ottawa, ON KIN 6N5, Canada. G. Zwingelsbeirm and @. Brickon. Institut Michel Pasha, Laboratoire maritime de physiolsgie, UniversitC de Lyon I , 83500 La Seyne Sur Mer, France.

1 ' Current address: Department of Zoology, University of British Columbia, Vancouver. BC V6T 124, Canada.

Can J. Fish. Aqaaat. Sci. 52: 161-170 (1995). Printed in Canada / Imprime au Canada

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Can. J. Fish. Aquat. Sci. Vol. 52, 1995

lipid storage organ varies greatly among fish species. Some species store most of their TAG in muscle, while others use either the liver or adipose tissue as their principal site for lipid (Sheridan 1988). It has been suggested that dif- ferences in storage site may be attributed to life-style. Sedentary fish tend to store lipid in Biver and/or adipose tissue, relying heavily on the circulation for transport, while more active fish use muscle for fat storage where it is more readily available to power ltscsrncatisn (Larsson and Farage 1977; Weber and Zwingelstein 1994).

Plasma is used as the means of transport from either the gut or storage organ to catabolic or storage tissues and in so doing can also serve as a storage site itself. The major circulatory lipid class in fish are nonesterified fatty acids (NEFA), which are used for rapid delivery, whereas TAG and PL are used for slow delivery. Of the circulating NEFA, 20-30% are unsaturated, having carbon chain lengths of 20-22; their major function is to serve as pre- cursors for PL and eicosanoid compounds. Monoenes and saturated NEFA of shorter chain lengths (18 C and less) are thought to be important for energy production, and those present in the plasma in highest concentrations should be those preferred by catabolic tissues. There is some evi- dence of preferential oxidation of these fatty acids (FA) at the level of red muscle mitochondria (Kiessling and Kiessling 1993). TAG, Pk, and cholesterol must also play an important role in energy metabolism because they can contain 10-40 times the energy stored in circulating NEFA (Weber and Zwingelstein 1994).

Dietary lipids, specifically in respect to their FA com- position, are thought to affect the NEFA composition of fish plasma (Henderson and Tocher 1987) and may con- tribute directly to blood NEFA, although this is not well established (Greene and Selivonchick 1987). This suggests that, Iike nonruminant mammals, fish digest lipids with very little preabsorptive modification of individual FA and that, like mammals, their adipose tissue (TAG) FA com- position (Argenzio 1984) and consequentially their plasma NEFA composition (JCquier 1992) should reflect their dietary FA composition.

The purpose of this study was to see if two fish species with similar life-styles and also fed the same diet had (I) similar lipid storage sites and (2) similar FA compo- sitions in their different lipid classes.

Materials and methods

Experimental animals Gilt-head sea bream (Chrysophyys aurabus) and European seabass (Dicentrarchus labrax) were originally obtained

(Centre de production aquacole HFWEMEW, Palavas les Flots) and were kept in indoor tanks with con- tinuously flowing seawater at 22°C. Males from both species were used; they were at least 2 years old and weighed 200-300 g. Experiments were carried out in July and August 1 992.

Diet Both species were fed commercial sea fish feed (Aqualim, Nersac, France; 49% cmde protein, 10% cmde fat, 3% crude cellulose, 13% crude ash). Feeding continued up until the

time of the experiments. The total lipid content sf the feed was found to be 10.6%. Lipids were extracted from feed for analysis using chloroform-methanol (Folck et al. 1957) and total FA were liberated by saponification for 3 hours at 88°C. Nonsaponifiable contaminants were removed with hexane extraction and the saponified FA were acidified with 6 N HCl to convert them back to free fatty acids (FFA). FFA were extracted with hexane and the solvent was evaporated under nitrogen. Methylation of FFA for analysis by gas-liquid chromatography (GkG) was per- formed with acetyl chloride (0.5 M) in anhydrous methanol and benzene.

Plasma

Extraction gdf kipids and methylation of FFA Blood samples were obtained either via the caudal vein or heart puncture with a 19-gauge needle. Blood was cen- trifuged and plasma samples were frozen immediately. Lipids were extracted and plasma WEFA were methylated as previously described (Tserng et al. 1981) with modifi- cation to improve extraction (see below).

In a Teflon screw-capped glass culture tube, 200 pL of plasma was mixed with 100 FE of internal standard hepta- decanoic acid (1 2 pg. 100 pL methanol-'). 2,2-Dimetho- xypropane (DMP) was added (2 mL) as a water scavenger, deproteinizing agent, and methylating agent. (Unlike other techniques (see Hallaq et al. 1993), BMP converts FFA to their methyl esters but is not reactive enough at low temperatures to transmethylate esterified FA from TAG car PL, which remain unmodified as shown by thin-layer chro- matography (TLC) (unpublished data).) Hydrochloric acid was then added (40 FL) and the resulting reaction was allowed to proceed at room temperature for a minimum of 40 minutes to allow all FFA to be methylated. (This time was determined to be sufficient for complete methy- lation of NEFA by using standard mixtures of NEFA and comparing GLC analysis with those of classic methods using separation by TLC and methylation with methanol-HCl (unpublished data).) The reaction was then stopped by the addition of 20 pL of pyridine and the mix- ture was evaporated to 100-200 FL using a heated water bath and a steady of nitrogen. Distilled water (1 mL) was added and all lipids were extracted twice with 2.5 mL of 2,2,4-trimethylpentane (isooctane). The isooctane extracts were evaporated to dryness and then redissolved in 50 ~ L L of isooctane, 2.5 pL of which was used for gas ckroma- tography of methyl esters.

FA esmposifion of plasma PL and triglycerides TL were extracted from plasma and methylated as described previously (Anderton et al. 1982). Triglycerides and PL were also separated on silica TLC plates (Riedel-de Haen silica gel 60 special) using a solvent mixture of heptane - isopropyl ether - acetic acid in the proportions 60:30:3. Lipids from approximately 100-200 pL of plasma were deposited on the TEC plate and after solvent migration the plates were dried and sections containing PL and trigly- cerides that were revealed under UV using a primuline reagent (Skipski 1973) were scraped off and placed in sep- arate tubes. 'To each tube, 0.5 mL of a benzene-WC1 mixture

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(60:30) was added followed by 2.5 mL of 0.5 M HCI in methanol. The tubes were incubated at $O°C for 1.5 h. Methyl esters of FA were extracted twice with 3 mL of hexane and a final time with 1.5 mL. The combined hexane extracts were evaporated to dryness and methyl esters were redissolved in 50 pL of hexane for analysis by GLC.

Rta l trig kyeeride The concentration of plasma total triglyceride was deter- mined spectrophotometrically (SP 6550 UVIVIS PYE UNICAM) using an enzymatic procedure (Sigma Chemical, St. Louis, Mo.). Plasma (10 pL) was placed in a cuvet with 8.8 mL of triglyceride working reagent (triglyceride (GPO trinder) reagent A and triglyceride reagent B in a proportion of 4: 1). Triglyceride standards (Sigma Chemical, St. Louis, Mo.) at concentrations of 50, 75, 100, 115, and 130 p g - r n ~ - ' were used to generate a calibration curve. Concentrations in micrograms per millilitre were converted to moles using an average moIecular weight for TAG calcu- lated from the %;FA composition of plasma TAG deter- mined by GLC (930 for seabass and 925 for sea bream).

Tissues

Extractiora of lipids from liver and muscle After fish were killed by decapitation, and samples of their antemdorsal white muscle, liver, and adipose tissue (mesen- teric fat, sea bream only) taken immediately, approximately 2 g was placed in Folch solution (chloroform-methanol (2:l)) at a volume of 20 mL-g tissue-' (Folch et al. 1957). Tissues were homogenized using a Polytron homogenizer (PT OD 10) that was rinsed after each sample with 10 mL of Folch 2:1. The rinse solution was added to the homogenate. Homogenates were left at room temperature for about 1 h to ensure that all lipids were extracted. Each solution was then filtered and the tubes rinsed with 10 mL of chloroform-methanol (2: 1). Aqueous 0.25% KC1 solu- tion was then added to the filtrate at a volume one quarter that of the final volume of Folch solution (17.5 mL in most cases) after which the tube contents were mixed. The phases were separated by placing the tubes in a heated water bath (50°C) for several minutes; the top phase con- taining water, methanol, and water-soluble nonlipid con- taminants was removed and discarded. The proportion of chlorofom-methanol was restored to 2: 1 by the addition of 23 mL (one third total volume) of methanol and a second wash was carried out by the addition of 17.5 mL of distilled water (one quarter total volume). Again the layers were separated and the top layer was discarded.

The lipid extracts were transferred to a tared 258-mL evaporating flask and the solvent was evaporated at 60°C under vacuum using a Wotovap apparatus (Buchi). A sec- ond evaporation was performed after the addition of 10 mL of 99% ethanol used to eliminate any remaining water by forming an azeotropic mixture. The flask contents were dried a final time by using a high-vacuum oil pump and allowed to equilibrate to room temperature before weighing. Lipid weight is expressed as a percentage of wet weight of the fish tissue. Lipids were redissolved in 5 mL of ben- zene-methanol (2: I ) , 50 FL of which was taken for muscle and 25 FL for liver to determine total lipid phosphorus.

The remaining extracts were transferred to Teflon screw- capped glass culture tubes for storage at -30°C.

iPbtal lipid, triglyceride, arad PL FA copndposr'tion An aliquot of tissue lipid extract (approximately 1 mg of total lipids) was methylated directly with a HC1-methanol mixture (1 M) to determine total lipid FA composition. Triglycerides and PL were separated by depositing approx- imately 1-2 mg of total lipids on a TLC plate md using iso- propyl ether as the mobile phase to separate polar lipids from neutral lipids. Free cholesterol was also separated, remaining between PL and triglycerides on the silica gel. The sections of the silica gel containing TAG and PL were removed, placed in glass culture tubes with Teflon-lined screw-caps, and methylated as described earlier. Total lipid methylated FA were redissolved in 208 pL of hexane, and TAG and PL FA methyl esters were redissolved in 100 pL for analysis by gas chromatography.

The concentration of PL in both tissue and plasma Hipids was determined by the Bartlett (1959) method as modi- fied by Portoukaliwn et al. (1978). The total phosphorus concentration was calculated by taking the lipid phospho- rus, expressed in milligrams per 100 g of tissue, and multi- plying by 27 (coefficient previously determined by assaying the fish lipid phosphorus of PL fractions separated by col- umn liquid chromatography).

Gas chromatography Methyl esters of FA were analyzed using a Chromopack gas chromatograph model CP 9000 equipped with a flame ionization detector (FID). Analysis was performed on an 80-m capillary column coated with a liquid phase con- sisting of a film of CP Sil 88 (100% cyanopropyl- polysiloxane). The column was held at 190°C for the first 5 min of each run and was then raised at 2 "~ -min - ' for 17.5 min to 22S0C. The column was held at this tempera- ture for 5 min before increasing to the final temperature of 235'C at a rate of 10"~-min- ' . The oven temperature remained at 235°C until component elution was complete. Injection port and detector temperatures were 250 and 275"C, respectively. Nitrogen was used as the carrier gas. The gas chromatograph was calibrated with FA standards PUFA 1, PUFA 2, rapeseed oil, and GC 98 (Supelco).

Statisties Means for individual NNEFA were compared using a one-way ANOVA. Percentages were arcsine transformed. If test for normality failed, then a nonparametric Kruskal-Wallis one- way ANBVA on ranks was used. All values presented are means 2 $EM.

Results

Dietary FA The FA composition of the dietary lipids appears in Table 1. The feed was high in both saturated, monounsaturated, and polyunsaturated FA, especially l6:8 at l6%, 18:l(n - 9) at 15%, 20:5(n - 3) at 11%, and 22:6(n - 3) at 17% (Table 1).

Plasma NEFA, TAG, and PL Concentrations of NEFA in plasma are similar in the two species at 2790 2 42 and 2223 k 32 nmol-m~- ' in seabass

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Table 1. FA composition (%) of csmmercial feed lipids (Aqualirn, Nersac, France) fed to seabass and sea bream.

B4:O 16:O 16: B (n-7) 18:O 18: 1 (n-9) 18:2(n-6) 18:3(n-3) 20: 1 (n-9) 20:4(n-6) 22: I (pa-9) 20:5(n-3) 22:4(n-6) 22:5(n-3) %2:6(n-3)

Saturated Monoeane Diem Triene PUFA n-3 n-6 pa-3/n-6

and sea bream, respectively (p > 0.05). Seabass, however, have much higher total TAG concentrations, 12 260 2

90 vs. 9670 k 580 nmo1.m~-I in sea bream (p = 0.002; Table 2).

Plasma NEFA composition as well as FA composition for TAG and PL fractions for both seabass and sea bream appear in Table 2. The two species have similar FA com- positions in their plasma NEFA, TAG, and PL fractions. All three fractions in both species have high proportions of palmitate (16:0), giving high total saturated FA values (24-3096). MEFA and TAG in both species are also high in oleate (18: B(n - 9)), while PL is significantly lower in this FA (p < 0.05, ANBVA). All fractions are high in rnonoenes (B5-45%), and polyunsaturated FA (24-58%), particularly re - 3 (28-55%). NEFA and PL in both species are high in docosahexaenoate (22:6(n - 3)), but TAG is lower in this FA (gs < 0.05).

Tissue TI,, TAG, and PL TL, TAG, and PL FA compositions for liver, muscle, and adipose tissue, the main lipid storage organs, appear in Tables 3, 4, and 5. Liver is a major lipid storage organ in the seabass, with 34.0 & 1.4% of its wet weight made up sf lipid (3 1 .Q k 2.1 exists as TAG), and makes up on average 2.1 2 1% of their body weight. In comparison, sea bream liver is only 4.8 k 0.2% lipid per wet weight and makes up a lower percentage sf their total body weight at 1.7 f 0.2% (Table 3). All fractions were high in saturated FA, especially 16:O. Monoenes were high in the TL fraction of both species and the TAG fraction of sea bream, mainly due to

high oleate values. PUFA were How in TL in seabass hav- ing much Hower 22:$(n - 3) levels when compared with PL CgB < 0.05) or sea bream TL values. The 22:6(rt - 3) values were comparable in the seabass TL and sea bream TAG. PUFA values were relatively high in seabass PL, and in all three fractions (TL, PL, and TAG) in sea bream, n - 3 PUFA values were again high (Table 3). There were, however, differences between these species in many sf the individual FA levels of PL Heading to differences in dienes and trienes.

The major storage site for lipid in bsth these species is adipose tissue. Sea bream adipose tissue ranges from 1.5 to 2.5% of body weight, depending on the season, and is 8 1% lipid by wet weight (Table 3). Seabass are also high in adipose tissue, with adiposity (adipose tissue/body weight) ranging from 3.5 + 0.8 to 6.5 k 0.6% body weight, again depending on the season. Their adipose tissue is, on average, 81.3 9 4.2% TL per tissue wet weight, with 81% of this existing as neutral lipid (mostly TAG, unpublished data). Adipose tissue TL in sea bream was high in palmitate (16:0) at B3%, oleate (18:l(n - 9)) at 27%, helping to give high monoene levels, and docosahexaenoate (22:6(n - 3)) at 13%, leading to high PUFA values. These are close to values for dietary NEFA (see Table 31).

Both species stored about the same amount sf lipid in their muscles (3.05 k 0.35 and 3.17 2 0.53% lipid by wet weight in seabass and sea bream, respectively; Table 5) . As seen in the liver, dl lipid fractions were high in satmated FA, especially l6rO. The most abundant FA in TL fractions of bsth species was IS: 1 (n - 9) which produced high t s td monoene levels. Both species had high levels of palmitate and oleate in TAG, but sea bream had higher levels of 22:6(n - 3). BL had significantly lower levels of oleate in both fish (p < 0.05). PL was, however, much higher in 22:6(n - 3) (p < 0.05) giving them higher PUFA and sz-3 fatty acid levels. Again there were some differences between the two species in individual FA of PL (Table 5).

The results show that two species with similar low-activity life-styles store lipids primarily outside of Hocornotory muscle, in liver anddor adipose tissue. Two species fed the same commercial diet have similar FA compositions in all plasma lipid fractions, except PL, as well as TAG and TL fractions in liver and muscle. There were, however, dif- ferences in individual FA of the PL fractions between these two species and their diet.

Lipid storage These species have about the same mass and similar life- styles. Both are benthic and live at a depth of 50-98 rn near the bottom of the Mediterranean Sea (UNESCO 1986). They resemble related North American fish species Moro~ke saxatdlis and Stemtornus chrysops considered to be only moderately active owing to their gill surface area to body mass ratio (Gray 1954). As hypothesized for less active fish (Weber and Zwingelstein 1994), the fish we have stud- ied store their lipids primarily in organs other than muscle: liver and adipose tissue for seabass and mainly adipose tissue for sea bream (Tables 3 and 4).

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McClelland et al.

Table 2. Plasma FA composition (%) of NEFA, TAG, and PL in seabass and sea bream (az = 5). Values are means + SEM. ND = not detectable.

Seabass Sea bream

NEFA TAG PL NEFA TAG PL

14:O 1 6 9 16: 1(u-7) 18:O 18: 1 (n-9) 18:2(n-6) 18:3(n-6) 18:3(n-3) 20: 1 (n-9) 18:4(n-3) 20:2(n-6) 20:3(n-6) 20:4(n-6) 22: 1 (n-9) 20:5(~z-3) 22:4(n-6) 24: 1 (n-9) 22:5(n-6) 22:5(n-3) 22166~~-3)

Saturated Monoene Diene Triene PUFA n-3 n-6 n-3/n-6

wrnsal.mL-'

Seabass store six times more lipid in their liver per wet weight than sea bream, but they also have large adipose tissue stores, as much as 6.5% body weight depending on the season (6. Brichon, unpublished data). Sea bream have on average 2.8 & 0.3 g of adipose tissue, roughly 1% of their body mass but, depending on the season, this can be as great as 2.5%. At 80.6 & 3.5% lipid per tissue wet weight or 874 pmol-g tissue-' it is their major site of stor- age (Table 4). This value is similar to that found for TAG in adipose tissue of salmonids (800 pmol-g tissue-'; Leger et al. 1981)- Subdermal fat is quite reduced in these two species, particularly during late summer (unpublished obser- vation), and is not included here as adipose tissue.

Both species have similar lipid content in their white muscle (3.05 & 0.35, and 3.17 k 0.53% wet weight in seabass and sea bream, respectively) or approximately 25 pmol-g tissue-' (Table 5). If we assume that in fish the muscle represents approximately 3040% body mass (Bone 1978), whole-animal lipid stores in muscle would range from 7500 to 15 000 ymol-kg- ' in these fish.

Expressed in similar units, the adipose tissue TAG stored in sea bream is 8157 Fmolskg-' and total liver stores are approximately 457 yrnol.kg-' in sea bream and about 3333 pmol.kg-' in seabass. Muscle can therefore be a large lipid storage depot when total tissue mass is con- sidered. These numbers are higher in more active fish such as rainbow trout (Oncorhynchus mykiss) with muscle lipid content of about 7% wet weight (Henderson and Tocher 1987). Red muscle fibres, although not studied here, are important users of FA during low-intensity prolonged swim- ming (Moyes et al. 1992; Kiessling and Kiessling 1993). Although lipid content is higher than in white muscle on a per gram basis (40 pmol-g-I) red muscle does not represent a major lipid storage site (about 3 mmolskg body masss' in most species; Moyes and West 1994) because of its small size.

Plasma concentration of NEFA is relatively high in both species when compared with other teleosts (1 100-2 110 nmol-mL- ' ; Zammit and Newsholme H 9759, including levels previously measured for seabass (Zammit

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Table 3. Liver FA composition (9%) and lipid content per wet weight of TL, TAG, and BE in seabass (re = 5 ) and sea bream (PO = 4).

- - -

Seabass Sea bream

TLa PE TL TAG PE

Saturated Monoerne Diene Triene PUFA m-3 m-6 n-3/n-6

% lipid-wet wt.-' - - -

"TL exists as TAG in seabass.

and Newsholme 1979). They have much higher levels than osteichthyans (758 nmol-ml-', Singer and Ballantyne 1991) and over an order sf magnitude higher than most elasmobranchs (150 nmol-ml-" Zammit and Newsholme 1979) (Table 2). Low plasma NEFA levels in elasmo- branchs are not surprising due to the absence of a NEFA carrier protein, while the presence sf an albumin-like pro- tein in telessts allows them to maintain much higher NEFA concentrations (Zammit and Newsholme 1979; Mailloka and Nimrno 1993).

If these species are assumed to have the same cardiac output (Q) (17.6 mlkg-'vrnin-' at rest and 52.6 rnL-kg-'. min-I at high swimming speed (UCri,) in trout; Kiceniuk and Jones 1977) and hematocrit similar to that in other teleosts (chinook salmon (O~~corhynch~~$ t?ih~p~?grtsckEa) at low work rates (23.8%) and at U,,, (24.5%); Thorarensen et al. 1993), they can be expected to have NEFA 4eliver-y rates higher than most teleosts. Plasma flow (Q( l -Hct) or Q,,,,,a) and NEFA concentration can be used to calculate NEFA delivery rates (McClelland et al. 1994). QP1,,,, in

teleosts at rest should be around 13.4 rnlkg-'+rnin-' and at high work rates around 39.7 rnl-kg-'amin-'. NEFA delivery rates in seabass, therefore, might be in the range of 29.8 and 88.3 pmol.kg-l-min-l and in sea bream about 37.4 and 110.8 pmol-kg-l-min-l at rest and at high work rates, respectively. These values can be considered as an underestimate of real in vivo rates because NEFA con- centrations will most likely be much higher in both s% these species during exercise (BBisetskaya 8980). 'These rates are, however, about an order of magnitude lower than those found in even the most sedentary mammals (McClellanad et al. 1994) but could support 100% s f total energy expenditure (from oxygen consumption; Kiceniuk and Jones 1977) at high swimming speeds in trout.

High NEFA concentrations, and possibly flux rate, in these species may be the result of storing lipid outside of muscle in liver and adipose tissue where it must first be mobilized and then transported by the circulation before it can be used by working muscles. Unfortunately, there are no data on NEFA flux rates and little on the storage

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of lipid to make any generalizations for all teleosts. Also there is considerable variability among studies in measur- ing NEFA concentrations in fish owing to different, and often inaccurate, techniques (Singer et al. 1990; Hallaq et al. 1993).

Plasma total TAG in both species fall into the high end of the range of values measured in fish (1080-12 000 nmol. mL-I; Weber and Zwingelstein 1994). Sea bream values are higher than those previously measured in a related species, the red sea bream (Chry~ophrys major; Sakamoto and Yone 1980). Levels of TAG in fish are much higher than that in the resting fed state of humans (1250 nmol- m ~ - ' ) and rats (2080 nmol-ml-') or even in mammals considered to be endurance adapted (dogs, 1400 nmo1.m~-') (Terjung et al. 1983). These high levels of TAG in fish may be related to the fact that teleosts may rely heavily on circulatory lipids as a fuel source, using glucose and lactate to a much lower extent than mammals. Turnover of glucose in fish (with the exception of tuna and eel) is 1/20 to 1/100 and lactate 1/26 to 11290 that in mammals of equivalent size (Weber et al. 1986; Wekr and Zwingelstein 1994). Even during sustained exercise, estimated oxida- tion rates of glucose account for only about 10% of aero- bic metabolism in trout heart and red muscle (West et al. 1993). Fish have been shown to have a high capacity to oxidize fat, especially in aerobic tissues like red muscle (Moyes et al. 1992; Kiessling and Kiessling 1993). The fish used in this study were not postabsorptive and because fish absorb dietary lipids as NEFA and TAG directly into the bloodstream (Sheridan 1988), this may lead to high plasma levels of both metabolites. TAG may be an impor- tant fuel source for exercising fish because, along with PL, it can represent 10-40 times the energy stored in cir- culatory NEFA (Weber and Zwingelstein 1994). NEFA are thought to be only 5-10% of total plasma lipids (Ballantyne et al. 8993). Plasma in seabass has previously been mea- sured to be 2.3% lipid per wet weight, and 1.6% (70% total plasma lipid) of that was TAG (Brichon 8984). Evi- dence from mammalian studies show that, at least in the fed state, TAG can supply energy to working muscles in dogs (Terjung et al. 1982). Little work has been done on the potential importance of TAG as a fuel source in fish, but it has been shown in migrating salmon that mobilization of fat fuels is important for long-term swimming, as plasma and whole-body TAG levels decrease over the migration route (Idler and Clemens 1959; French et al. 1983).

FA c ~ m p o s i t i ~ n Dietary lipids not only affect concentration but also FA composition of plasma and tissue lipids (Henderson and Tocher 1987). The FA composition of the diet was reflected in plasma NEFA and TAG as well as liver and adipose tissue TL and TAG (Tables 1, 2, 3, and 4). This suggests that lipids are absorbed in fish as FFA and TAG with little modification to FA composition. Absorption takes place primarily in the anterior intestine and the pyloric caeca, and digestibility increases with degree of unsaturation (those of 18-22 C length being the easiest) and water tem- perature (Henderson and Tocher 1987). This is reflected in the high proportion of 18:1(n - 9) and 22:6(n - 3) in all fractions. 22:6(n - 3) is thought to be the principal FA

Table 4. Adipose FA composition (9%) and lipid content per wet weight of adipose tissue in sea bream ( n = 4).

Saturated Monoene Dierae Triene PUFA n-3 n-6 n-3In-6

in TL of most temperate and tropical fish species, with seawater species being highest in 20- and 22-42 unsatu- rated FA (Henderson and Tocher 1984). Fish are also capable of converting 20:5(n - 3) to 22:6(n - 3) (Cowey and Sargent 1979) which may explain instances where 20:5(s2 - 3) levels are lower than dietary levels (liver TL in both species and TAG in sea bream) and 22:6(n - 3) are higher (muscle and plasma PL in both species). Mono- enes, dienes, and PUFA were high in both species, but as expected, trienes were low (Henderson and Tocher 1987).

Both fish have similar PE content in their tissues, about 2% tissue wet weight in liver and 1 % in muscle. The pat- tern of FA in PL, in particular the PUFA fraction, does not follow that of diet, contrary to results found in fresh- water fish kept in captivity (Henderson and Tocher 1987). These results are in accordance with Atlantic salmon (SaHrn~ scalar) where PL FA composition was shown to be indepen- dant of dietary FA (Polvi and Ackman 1992). Seabass and sea bream had significantly lower levels of oleate in plasma, liver, and muscle than found in either the diet or in TAG (Tables 1, 2, 3, and 5). As in freshwater fish, PL are richer in PUFA but lower in monoenes than TAG, 22:6(n - 3)

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Table 5. White muscle FA composition (96) and lipid content per wet weight for Tk, TAG, and Bk in seabass and sea bream (n = 5).

Seabass Sea bream

TL TAG PL T L TAG PL

Saturated Monoene Diene Triene PLTFA n-3 ~e-6 n-3ln-6

% lipid-wet wt.-"

being the major PUPA (Henderson and Toeker 1987). This difference most likely reflects the fact that monoenes are preferred as a fuel source and therefore are stored in TAG readily mobilized as an energy source. Maintaining mem- brane fluidity in the face of high salinity or low tempera- tures 1s the primary role played by PUFA in membrane PL. High percentages of 16:0, 18: 1, and 2 2 5 are a reflection of PL preference for even-numbered carbon chains (Stryer 1988) and the preferential incorporation of 22:6(n - 3 ) by fish into PE (Cowey and Sargent 1979).

Marine lipids are thought to contain more n-3 PUFA leading to higher n-3/n-6 ratios, while freshwater Bipids are higher in n-6 FA (Henderson and Tocher 1987). Ratios in these fish ranged from 1.7 in seabass liver TL to 9.76 in seabass plasma PL (Tables 2 and 3). Ratios in PL for both species were slightly lower than those reported previously in marine fish (7.$-18.5) but higher than those found in freshwater fish (1.6-2.0) (Henderson and Tocher 1987; Ballantyne et al. 8993). High n-3 in marine fish has been attributed to high levels of n-3 in their diet, as seen here in a commercial diet (Table I), and in marine phytoplankton (Ballantyne et al. 1993).

Life-style may be a determining factor in where and how fish species store their Bipids. Seabass and sea bream are marine fish living in the Mediterranean with similar life-styles: both species are bottom-dwelling sedentary fish. Both store lipid primarily outside of locomotory mus- cle. Diet plays the primary role in determining tissue and plasma FA composition. There is a g o d comelation between dietary FA composition and that found in TAG and NEFA of plasma. The FA composition of the TAG and TL frac- tions in their storage tissues also closely reflected dietary FA composition, while FA composition of PL did not.

We thank Mohammed Elbabili for performing phosphorus measurements and for enlightening discussions. During this study, G. McClelland was supported by a grant from the French Ministry of External Affairs (C.I.E.S.).

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