FISHERIES

SCIENCE

2003;

69

: 1001–1009

Blackwell Science, LtdOxford, UKFISFisheries Science0919-92682003 Blackwell Science Asia Pty Ltd695October 2003719Dietary vitamins C, E on lipid metabolismH JI et al.10.1046/j.0919-9268.2003.00719.xOriginal Article10011009BEES SGML

*Corresponding author: Tel: 81-824-24-7989. Fax: 81-824-24-7989. Email: jh2000jp@hotmail.com

Received 21 October 2002. Accepted 13 May 2003.

Effect of dietary vitamins C and E fortification on lipid metabolism in red sea bream

Pagrus major

and black sea bream

Acanthopagrus schlegeli

Hong

JI,

1

*

Ahmad Daud

OM,

1

Takao

YOSHIMATSU,

2

Masahiro

HAYASHI,

3

Tetsuya

UMINO,

1

Heisuke

NAKAGAWA,

1

Masaya

ASANO

4

AND

Atsushi

NAKAGAWA

4

1

Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima 739-8528,

2

Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Fukuoka 812-8581,

3

Faculty of Agriculture, Miyazaki University, Miyazaki, Miyazaki 889-2192 and

4

Research Laboratories, Aquaculture Center, Kyowa Hakko Kogyo Co. Ltd, Ube, Yamaguchi 755-8501, Japan

ABSTRACT:

To determine the effect of vitamins C and E on lipid metabolism and interactionsbetween them, L-ascorbyl-2-monophosphate-Mg (APM) and

a

-tocopherol acetate (TA) were forti-fied to a commercially based diet and fed to 0-year red sea bream

Pagrus major

and 1-year blacksea bream

Acanthopagrus schlegeli

. Fortification of APM and TA, respectively, increased ascorbate(ASC) and

a

-tocopherol (

a

-Toc) contents in the organs. In addition, APM fortification increased

a

-Toc accumulation in both fishes, although TA fortification did not significantly affect the ASC con-tent. Fortification of APM caused a depression in lipid accumulation in the intraperitoneal fat bodyand liver in red sea bream. Furthermore, a decrease in the serum thiobarbituric acid value in blacksea bream and a reduction of the adipocyte diameter in the APM-fortified groups of both fisheswere observed. However, fortification of TA did not affect these parameters as significantly as didfortification of APM. The shortest recovery time to air-dipping was found in the APM

+

TA-fortifiedgroup, followed by the APM-fortified group in red sea bream. The results implied an effect ofvitamin C on lipid metabolism, and acceleration of vitamin E absorption and/or suppression ofvitamin E degradation.

KEY WORDS:

ascorbate, black sea bream, lipid metabolism, red sea bream, aaaa

-tocopherol.

INTRODUCTION

Vitamin C has a protective effect on lipidperoxidation

1

and improves lipid metabolism,such as lipolysis, by participating in carnitinesynthesis.

2,3

Dietary ascorbic acid also influencesplasma lipid levels in rainbow trout.

4

Catechinand

Spirulina

as feed additives protect dietaryvitamin C degradation and, consequently, resultin an improvement of vitamin C metabolism andactivation of lipolysis.

5,6

In addition, in juvenileJapanese flounder

Paralichthys olivaceus

, therewas a significant interaction of ascorbate and n-3highly unsaturated fatty acid (HUFA) in weightgain, feed conversion efficiency and concentra-

tion of ascorbic acid in the liver.

7

Our previousexperiments also showed vitamin C-activatedlipolysis in juvenile black sea bream, as well asHUFA.

8,9

In contrast, vitamin E limits lipid peroxidation.

10

However, its effect on lipid metabolism in fishremains unclear. Interactions between ascorbicacid and tocopherol were studied in mammals

11,12

and fishes.

13–15

Vitamin C might regenerate vitaminE from the vitamin E radical. Some researchers alsostudied the effect of dietary vitamin C and vitaminE in milkfish broodstock,

16

and fresh water walleyelarvae.

17

Murata

et al

.

18

compared the vitamin Cand vitamin E status in wild and cultured yellowtail, in relation to lipid peroxidation.

Therefore, we examined the effect of dietaryvitamins C and E on lipid metabolism and theinteraction between them in biological and bio-chemical parameters with reference to incorpora-tion of these vitamins in 0-year red sea bream and1-year black sea bream.

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MATERIALS AND METHODS

Fish and rearing conditions

Zero-year red sea bream

Pagrus major

(bodyweight 0.3 g) and 1-year black sea bream

Acantho-pagrus schlegeli

(body weight 25 g) were producedin Tsuyazaki Marine Station of Kyushu Universityfrom captive broodstock. The experiment was con-ducted in duplicate. A total of 640 red sea breamand 160 black sea bream were divided equally intoeight rectangular indoor aquaria (capacity 150 L).The water temperature ranged between 23.7 and28.5

∞

C and the water flow rate was maintained at1.8–4.7 L/min.

The fish were fed with a test diet formulatedsimilar to a commercial diet (types A and C; KyowaHakko Kogyo Co. Ltd, Ube, Japan) in the control

group. The other three diets were fortified with500 mg L-ascorbyl-2-monophosphate-Mg (APM),100 mg

a

-tocopherol acetate (TA) and a mixture of500 mg APM and 100 mg TA in 1 kg of diet, respec-tively. The diets were kept at

-

20

∞

C until feeding.Proximate compositions of experimental diets areshown in Tables 1 and 2. The red sea bream werehand fed three times daily (9.00 h, 13.00 h, 17.00 h)for 47 days and the black sea bream were hand fedtwo times daily (10.00 h and 16.00 h) six days aweek for 49 days at a rate of 3–6% of body weight.

Biological measurements

At the end of the feeding experiment, all the fish ineach replicate treatment group were weighed indi-vidually. Fifteen red sea bream and 10 black sea

Table 1

Dietary composition and proximate analysis of diets for red sea bream

Dietary group

Control VC VE VC

+

VE

Dietary compositionCommercially based diet (g) 100 100 100 100APM (g) 0 0.05 0 0.05

a

-Tocopherol acetate (g) 0 0 0.01 0.01

Proximate composition (%)Moisture 4.0 10.4 10.6 10.3Crude protein 59.1 51.0 51.8 52.0Lipid 13.7 12.0 12.0 12.7Ash 12.6 13.7 13.7 13.7

Ascorbate (mg/100 g wet basis) 9.2 20.6 2.2 23.9APM (mg/100 g wet basis) 0.0 30.6 0.0 35.9

a

-Tocopherol (mg/100 g wet basis) 40.0 41.8 66.6 56.2

APM, L-ascorbyl-2-monophosphate-Mg.

Table 2

Dietary composition and proximate analysis of diets for black sea bream

Dietary group

Control VC VE VC

+

VE

Dietary compositionCommercially based diet (g) 100 100 100 100APM (g) 0 0.05 0 0.05

a

-Tocopherol acetate (g) 0 0 0.01 0.01

Proximate composition (%)Moisture 3.8 11.1 11.1 10.9Crude protein 57.0 48.6 49.0 53.0Lipid 13.9 12.9 13.1 13.5Ash 13.8 13.7 13.8 13.8

Ascorbate (mg/100 g wet basis) 6.5 34.4 5.1 20.8APM (mg/100 g wet basis) 0.0 50.9 0.0 34.3

a

-Tocopherol (mg/100 g wet basis) 49.3 45.7 62.4 62.4

APM, L-ascorbyl-2-monophosphate-Mg.

Dietary vitamins C, E on lipid metabolism

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bream were used for biological measurements.Whole muscle (fillet), liver and intraperitoneal fatbody (IPF) were obtained from 20 fish in eachgroup. The IPF was scraped out from the intraperi-toneal cavity and weighed. The biological parame-ters were defined as follows.

Muscle ratio (%)

=

(muscle weight/body weight)

¥

100

Hepatosomatic index (HSI;%)

=

(liver weight/body weight)

¥

100

IPF ratio (%)

=

(IPF weight/body weight)

¥

100

Feed efficiency (%)

=

(body weight gain/diet given)

¥

100

Protein efficiency ratio

=

body weight gain/diet protein given

Measurement of adipocyte diameter

Fish samples fixed in Bouin’s solution were embed-ded in paraffin and cut transversely into 10

m

m-thick serial sections. The adipocytes were stainedwith hematoxylin and eosin, and the diameter wasmicroscopically measured in 50 cells.

Biochemical measurements

The fish were immediately frozen at

-

20

∞

C aftersampling until biochemical analysis. The muscle,liver and IPF were taken and analyzed separately.Crude protein was determined by the Kjeldhalmethod. Lipids extracted with methanol-chloroform according to the method of Bligh andDyer

19

were subjected to quantitative and qualita-tive analyses.

Ascorbate and tocopherol analyses

Fish organs, such as the liver, muscle, heart, brainand eye, were preserved in a deep freezer at

-

80

∞

Cuntil they were analysed. Diets were maintainedbelow

-

20

∞

C until analysis. For the ascorbate anal-ysis, sample preparation and determination weredescribed previously.

8

The organs were pooled andsubmitted for analysis. L (

+

)-ascorbic acid (KantoChem., Tokyo, Japan) and APM (Wako Pure Chem.Ltd, Osaka, Japan) were used as standards.

Tocopherol analysis was conducted accordingto the method of Bai and Gatlin III with modifica-tion,

20

and carried out by high-performance liquidchromatography (HPLC; Waters Associates Inc.,Milford, MA, USA) with a reversed-phase column

(Cosmosil 5SL-II; Nacalai Tesque Inc., Kyoto,Japan), a model 515 HPLC pump and a model 474scanning fluorescence detector. The analyticalconditions were as follows: wavelength, excitation295 nm, emission 340 nm; column temperature,40

∞

C; eluent, 0.7% isopropylalcohol in n-hexane;flow rate, 1.5 mL/min. DL-

a

-tocopherol (NacalaiTesque Inc.) was used as standard. Seven to 10muscles and four to 10 livers of each group wereused for tocopherol analysis in black sea bream.The brain and eye for the analysis were pooledin each tank. The organs of red sea bream werepooled and submitted to the analysis.

Thiobarbituric acid value

The blood withdrawn from the caudal vein wascentrifuged at 900

¥

g for 10 min and the serumwas submitted to thiobarbituric acid (TBA) analy-sis with a kit (Wako Pure Chemical Co., Osaka,Japan) and the value was expressed as mg of mal-onaldehyde/mL serum.

Air-dipping test

Twenty red sea bream from each treatment groupwere exposed to air on a net as the air-dipping testfor 5 min and returned to oxygen-saturated sea-water to determine the vitality of the fish. Therecovery time from the succumbed condition wasrecorded. The water temperature during the treat-ment was 24.8

∞

C.

Statistical analysis

Data on growth performance, biological and bio-chemical parameters were examined by

ANOVA

.When the

F

-ratio for the

ANOVA

was statistically sig-nificant (

P

<

0.05), these values were further com-pared using Fisher’s Protected Least SignificantDifference test. The air-dipping test was examinedby log–rank test (Mantel-Cox test).

RESULTS

Moisture content was higher in the APM and TAfortified groups than in the control group. As thecommercially based diet was supplemented withcalcium ascorbate as a vitamin C source, theascorbate (ASC) levels were not consistent in thediets of the control and vitamin E (VE) groupsbecause of partial degradation of calcium ASCduring preservation.

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Red sea bream

Survival was not significantly different amongcontrol and APM-fortified groups. Although bodyweight was lower in APM-fortified groups, bio-mass gain among groups was not significantlydifferent (Table 3). Fortification of APM and TAsignificantly depressed HSI and increased themuscle ratio. The reserved lipids in muscle, liverand IPF are shown in Fig. 1. Fortification of APMmarkedly depressed lipid accumulation in theliver and IPF. A decrease in the IPF ratio by APMfortification was accompanied by a reduction inthe adipocyte diameter.

Table 4 shows proximate composition of mus-cle and liver. The proximate composition of mus-cle was not affected by dietary APM or TA. Liverlipid was decreased by APM fortification, butnot by TA. The fatty acid compositions of the

liver, muscle and IPF were not significantly differ-ent among the groups. Percentage of docosa-hexaenoid acid (DHA) ranged from 10.6 to 13.6%in the liver, 22.0 to 26.5% in the muscle and 8.3 to9.8% in the IPF.

Contents of ASC and

a

-Toc in the organs areshown in Table 5. Whereas ASC was markedlyaccumulated in the brain,

a

-Toc was highest in theliver. Dietary APM was well incorporated intomuscle, liver and heart, but did not significantlyaffect ASC contents in the brain and eye. DietaryTA showed little influence on the ASC content inthe organs. Nevertheless,

a

-Toc content was sig-nificantly increased in muscle and brain by fortifi-cation with APM, as seen in the groups fortifiedwith TA.

Figure 2 shows the recovery time in the air-dipping test as the vitality test. Compared with thecontrol group, the other three treatment groupsshowed a markedly reduced impact of air-dipping,and the 50% recovery time of the vitamin C (VC)-and VE-fortified groups was half compared withthe control group, although no statistical differ-ence was found among groups. The shortest recov-ery time was found in the VC

+

VE group and therecovery time of the VC group was shorter than thatof the VE group.

Black sea bream

Table 6 shows the growth performance and biolog-ical parameters in 1-year black sea bream. DietaryAPM affected muscle ratio, HSI and adipocytediameter. The control group and the VE group werenot different from each other with regard to theseparameters.

Table 3

Effect of ascorbate and tocopherol fortification on growth and biological parameters in red sea bream

n

Dietary group

Control VC VE VC

+

VE

Total diet given (g, dry basis) 2 238 239 239 239Biomass increased (g) 2 391 379 389 382Survival (%) 2 60.0

ab

72.6

ab

62.6

a

76.9

b

Feed efficiency (%) 2 165 159 163 160Protein efficiency ratio (%) 2 2.67 2.77 2.81 2.76

Body weight (g) 100 7.60

±

3.10

a

6.80

±

2.00

b

7.60

±

2.50

a

6.30

±

1.70

b

Body length (mm) 100 61.4

±

7.9 62.3

±

7.3 63.2

±

7.3 60.3

±

6.4Muscle ratio (%) 20 28.7

±

3.0a 31.4 ± 2.7b 32.1 ± 3.2b 30.4 ± 3.8a

Hepatosomatic index (%) 20 1.17 ± 0.42a 0.89 ± 0.24b 0.85 ± 0.16b 0.74 ± 0.23b

IPF* ratio (%) 20 0.77 ± 0.40a 0.42 ± 0.32b 0.62 ± 0.45a 0.41 ± 0.26b

Adipocyte diameter (mm) 300 65.2 ± 17.3a 51.7 ± 15.4b 67.7 ± 17.5a 52.3 ± 16.0b

IPF, intraperitoneal fat body.Values are means and SD.Values in a row with different superscript letters are significantly different (P < 0.05).

Fig. 1 Effect of ascorbate and tocopherol fortificationon lipid accumulation in red sea bream.

Dietary vitamins C, E on lipid metabolism FISHERIES SCIENCE 1005

The proximate composition of muscle and livershowed no significant difference among the groups(Table 7). The significant difference in the liverlipid content in 0-year red sea bream was notfound in 1-year black sea bream. However, the

serum TBA value was significantly lower in the VCgroup, while fortification of TA did not decrease theTBA value. However, the value in the VC + VE groupshowed no significant difference from that of thecontrol and VE groups. As seen in red sea bream,fatty acid composition of the liver, muscle, IPF,brain, eyes and heart was not significantly differentamong groups. Percentage of DHA in total fattyacid ranged between 14.7 and 15.6% in the liver,13.7 and 16.4% in the muscle, 9.3 and 10.2% in theIPF, 18.6 and 20.6% in the brain, 11.2 and 13.0% inthe eye, and 10.3 and 15.9% in the heart.

With regard to incorporation of dietary APM andTA, black sea bream showed similar results to thoseobserved for red sea bream (Table 8), except in theeyes in which dietary APM was well incorporated.However, liver a-Toc content, but not muscle a-Toccontent, of the VC group increased, and muscle a-Toc content of the VC + VE group showed the high-est level among the groups. Fortification with APMaccelerated to incorporate Toc. Furthermore, anincrease in the ASC content in the organs was littleaffected by dietary TA.

Table 4 Effect of ascorbate and tocopherol fortification on proximate composition (%) of red sea bream

n

Dietary group

Control VC VE VC + VE

MuscleMoisture 4 77.9 ± 0.1 78.7 ± 0.3 78.9 ± 0.3 78.7 ± 0.8Ash 4 1.6 ± 0.0 1.6 ± 0.0 1.6 ± 0.1 1.6 ± 0.1Crude protein 4 19.0 ± 0.1 17.4 ± 0.8 17.2 ± 1.3 17.8 ± 0.4Lipid 4 1.5 ± 0.3 1.4 ± 0.1 1.7 ± 0.1 1.3 ± 0.0

LiverLipid 4 14.5 ± 2.3a 9.0 ± 0.4bc 12.0 ± 0.6ac 7.9 ± 2.4bc

Values are means and SD.Values in a row with different superscript letters are significantly different (P < 0.05).

Table 5 Effect of ascorbate and tocopherol fortification on their contents in organs of red sea bream

n

Dietary group

Control VC VE VC + VE

Ascorbate content (mg/g)Muscle 2 3.6 ± 0.6a 6.1 ± 1.6b 2.9 ± 1.5a 6.2 ± 1.5b

Liver 2 36.7 ± 5.5a 59.0 ± 4.9b 24.5 ± 3.0a 61.4 ± 6.3b

Brain 2 302.0 ± 20.5 295.0 ± 27.6 200.0 ± 2.2 266.0 ± 20.8Heart 2 23.6 ± 5.9a 43.90 ± 3.1b 21.60 ± 5.0a 42.9 ± 5.8b

Eye 2 32.0 ± 5.9 42.60 ± 9.2 19.0 ± 7.9 30.0 ± 2.1

a-Tocopherol content (mg/g)Muscle 2 2.2 ± 0.1a 4.5 ± 0.1b 4.9 ± 0.5b 4.9 ± 0.2b

Liver 2 127.0 ± 7.9a 213.0 ± 39.0ab 260.0 ± 17.2b 231.0 ± 23.0b

Brain 2 10.9 ± 0.5a 15.80 ± 0.9b 15.1 ± 2.3ab 17.9 ± 0.4b

Eye 2 5.6 ± 0.6 9.6 ± 3.3 8.7 ± 2.1 6.7 ± 1.3

Values are means and SD.Values in a row with different superscript letters are significantly different (P < 0.05).

Fig. 2 Effect of ascorbate and tocopherol fortificationon recovery time after air-dipping in red sea bream.

1006 FISHERIES SCIENCE H JI et al.

Table 8 Effect of ascorbate and tocopherol fortification on their contents in organs of black sea bream

n

Dietary group

Control VC VE VC + VE

Ascorbate content (mg/g)Muscle 2 3.6 ± 2.1a 13.9 ± 0.1b 3.2 ± 0.7a 11.1 ± 1.7b

Liver 2 19.2 ± 7.1a 62.9 ± 2.1b 20.4 ± 7.9a 52.3 ± 10.5b

Brain 2 208.0 ± 24.0 230.0 ± 4.3 213.0 ± 4.4 220.0 ± 43.3Heart 2 10.9 ± 5.7a 47.9 ± 10.7b 12.2 ± 5.4ab 43.1 ± 27.1ab

Eye 2 13.1 ± 4.4a 32.7 ± 0.7b 12.9 ± 6.8ab 25.0 ± 1.4a

a-Tocopherol content (mg/g)Muscle 7–10 1.8 ± 0.6a 2.6 ± 0.1ac 3.1 ± 0.3bc 4.5 ± 0.3d

Liver 4–10 134.0 ± 30.7a 211.0 ± 9.3b 216.0 ± 10.7b 238.0 ± 24.0b

Brain 2 12.1 ± 1.3a 15.5 ± 0.6bc 15.0 ± 0.5ac 16.6 ± 1.6bc

Eye 2 10.7 ± 1.0 9.5 ± 0.7 10.0 ± 2.9 10.7 ± 0.9

Values are means and SD.Values in a row with different superscript letters are significantly different (P < 0.05).

Table 6 Effect of ascorbate and tocopherol fortification on growth and biological parameters in black sea bream

n

Dietary group

Control VC VE VC + VE

Total diet given (g, dry basis) 2 795 793 798 791Biomass increased (g) 2 492 483 517 503Survival (%) 2 100 100 100 100Feed efficiency (%) 2 61.8 61.0 64.8 63.5Protein efficiency ratio (%) 2 1.04 1.12 1.18 1.07

Body weight (g) 40 49.3 ± 6.8 48.9 ± 5.8 50.6 ± 7.1 49.9 ± 6.9Body length (mm) 40 115 ± 5 112 ± 23 119 ± 3 116 ± 5Muscle ratio (%) 20 34.8 ± 3.2a 37.1 ± 3.3bc 36.5 ± 3.5ac 37.1 ± 3.2bc

Hepatosomatic index (%) 20 1.43 ± 0.28a 1.21 ± 0.34bc 1.43 ± 0.36ac 1.25 ± 0.30ac

IPF ratio (%) 20 2.13 ± 1.20 2.41 ± 1.04 2.06 ± 0.93 1.92 ± 0.80Adipocyte diameter (mm) 300 70.9 ± 23.0a 51.5 ± 13.9b 71.9 ± 17.2a 64.4 ± 18.7c

IPF, intraperitoneal fat body ratio.Values are means and SD.Values in a row with different superscript letters are significantly different (P < 0.05).

Table 7 Effect of ascorbate and tocopherol fortification on the proximate composition and serum thiobarbituric acid(TBA) in black sea bream

n

Dietary group

Control VC VE VC + VE

MuscleMoisture (%) 4 76.3 ± 0.4 75.8 ± 0.1 75.5 ± 0.3 75.9 ± 0.6Ash (%) 4 1.4 ± 0.0 1.4 ± 0.0 1.4 ± 0.0 1.5 ± 0.0Crude protein (%) 4 20.0 ± 0.4 20.3 ± 0.4 20.4 ± 0.6 19.7 ± 0.7Lipid (%) 4 3.1 ± 0.3 3.2 ± 0.1 3.0 ± 0.4 3.0 ± 0.2

LiverMoisture (%) 4 69.9 ± 2.0 66.6 ± 4.2 66.8 ± 2.1 68.4 ± 1.6Lipid (%) 4 10.5 ± 3.6 11.9 ± 1.6 7.1 ± 1.2 7.9 ± 1.0Serum TBA 12 7.2 ± 0.2a 4.8 ± 0.7bc 7.8 ± 1.1a 6.2 ± 0.8ac

Values are means and SD.Values in a row with different superscript letters are significantly different (P < 0.05).

Dietary vitamins C, E on lipid metabolism FISHERIES SCIENCE 1007

DISCUSSION

The optimal dietary level of ASC has been reportedas 50–100 mg/kg of diet in channel catfish,21 rain-bow trout22 and Korean rockfish23 in order to sus-tain normal growth performance and to preventdeficiency symptoms. The optimum level of vita-min E has been reported to be 50–100 mg/kg in thediet with 5% lipid in tilipia24 and 45 mg/kg in juve-nile Korean rockfish.25 The fortification levels ofAPM and TA were determined according to fishrequirements and our previous studies.8,21–25 Thecommercially based diet used in the present exper-iment included 20–90 mg ASC in 1 kg diet and 400–500 mg of a-Toc.

Histological study showed a significant reduc-tion in the adipocyte diameter of IPF by APM for-tification. A reduction in the adipocyte diameterwas previously found by dietary ascorbate8 andHUFA.9 The lower efficiency of dietary a-Toc in adi-pocyte growth could be confirmed by the presentresults. Dietary ASC suppressed adipocyte growthand activated lipolysis in black sea bream juve-niles.8 Adipocyte diameter was closely related tolipolysis activity in rats and humans.26,27 The adipo-cytes of wild ayu are relatively smaller than thoseof cultured ayu, suggesting that adipocyte diame-ter may be a useful indicator to evaluate lipolysisactivity.28

Ascorbic acid indirectly stimulates fatty acidutilization in cultured guinea-pig hematocytes.29

Lipid content in the eviscerated whole body wassignificantly decreased by ASC supplementationduring starvation in rainbow trout.3 Fatty acid pro-file was not changed by dietary APM in Japaneseflounder juveniles.7 Our previous study showed ahigher IPF ratio and a lower DHA level after starva-tion in the ASC-fortified group.8 These inconsistentresults might be caused by the difference in fishspecies and experimental conditions.

In contrast, dietary a-Toc exerted no apprecia-ble effects on the lipid content of whole bodies,muscle and hepatopancreas in carp,30 while anexcess dose of a-Toc increased lipid accumulationin the liver of rainbow trout.31 It was found that thefatty acid composition of tissues is independent ofdietary a-Toc content once fish have satisfied theirminimum requirement.25,30,32 Barja et al.33 reporteddecreases in the percentage of unsaturated mem-brane fatty acids at both too low and too highdietary vitamin E doses in the guinea-pig liver invivo. No effect of a-Toc fortification on lipid accu-mulation and fatty acid composition was found inthe present study.

The difference of a-Toc content in the organsamong control and VC-fortified groups might bedue to the interactive effect of ASC on suppression

of a-Toc consumption. As a result, ASC fortificationmight contribute to an increase in Toc accumula-tion.

Vitamins E and C interact, and each can exertsparing effects on the other in inherent scorbuticrats11 and juvenile hybrid striped bass.15 ASC andToc are biologically active as anti-oxidants in thewater and lipid phases, respectively. Hamre et al.14

mentioned that there were two different interac-tion mechanisms in juvenile Atlantic salmon: asynergistic effect of simultaneous protection of thewater and lipid phases against oxidation, andregeneration of vitamin E from the vitamin E radi-cal by ascorbic acid. Furthermore, vitamin C defi-ciency was accompanied by a large drop in liver a-Toc concentration. Shiau and Hsu34 reported thathigher gill vitamin E was found in juvenile hybridtilapia fish fed the vitamin E-deficient diet supple-mented with high vitamin C than in the fish fed adiet supplemented with an adequate level of vita-min C. In the present study, higher a-Toc contentswere found in the VC-fortified group. This agreeswith the findings of Liu and Lee12 where raising thedietary vitamin C level caused higher a-Toc con-centrations in guinea-pigs fed oxidized frying oil.Moreover, the present study showed the highest a-Toc content in the muscle of the VC + VE group inblack sea bream. The reason for this is not known.The present study also demonstrated that vitaminC has a sparing effect on vitamin E in the fish.

In the present study, the VC group showed lowerserum TBA values, but in the VC + VE group, thevalue appeared to be close to that of the controland VE groups. Tanaka et al.11 found that a defi-ciency of vitamin C in inherent scorbutic ratscaused a larger increase in oxidative stress thana deficiency of vitamin E. Some studies alsosuggested the pro-oxidant effect of high dose a-tocopherol.14,15 Thus, the present results maysuggest that ASC is more effective as a lipid anti-oxidant than a-Toc and there is a possible pro-oxidant effect of high-dose Toc.

Ascorbate is preliminarily accumulated in thebrain, as described by Ikeda et al.35 Koshio et al.36

suggested that vitamin C might act as a neu-rotransmitter modulator, which implied a relationbetween brain vitamin C and behavior in ayu Plec-oglossus altivelis. The largely independent brainpool of ascorbic acid is assumed to be of particularimportance in stressful situations.37 The currentresults of the vitality test partly confirmed that vita-mins C and E could synergistically improve fishvitality.

In conclusion, it might be partly confirmed thatvitamin C, but not vitamin E, influences lipidmetabolism. Meanwhile, vitamin C interacted toaccelerate vitamin E absorption and/or prevent

1008 FISHERIES SCIENCE H JI et al.

vitamin E oxidation. The ability to behave as a lipidanti-oxidant appeared to be stronger in vitaminC than in vitamin E and there might be a pro-oxidation effect of high-dose Toc in black seabream. In addition, vitamins C and E could syner-gistically improve fish vitality.

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2. Ginter E, Bobek P. The influence of vitamin C on lipidmetabolism. In: Counsell JN, Hornig DH (eds). Vitamin C/Ascorbic Acid. Applied Science Publishers, London. 1981;299–347.

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