Effect of Dietary Catechin and Spirulina on Vitamin C Metabolism in Red Sea Bream

FISHERIES SCIENCE 2000; 66: 321–326

Original Article

Effect of dietary catechin and Spirulina on vitamin C metabolism in red sea bream

Heisuke NAKAGAWA,1,* Md Ghulam MUSTAFA,1 Kenji TAKII,2 Tetsuya UMINO1 AND Hidemi KUMAI2

1Faculty of Applied Biological Science, Hiroshima University, Higashi-hiroshima, Hiroshima 739-7989 and 2Fisheries Laboratory, Kinki University, Uragami, Wakayama 649-5145, Japan

SUMMARY: The zero-year red sea bream Pagrus major were fed for 41 days on dry pellets containing 1% Teaflan (60% catechin) or Spirulina supplement. The effects of catechin and Spirulina on ascorbate absorption, lipid metabolism, and collagen synthesis were compared in relation to vitamin C nutrition. Total ascorbate concentrations in the serum and liver were increased significantly in the catechin-fed groups relative to the control, but Spirulina increased in value only in liver. Feeding with catechin and Spirulina depressed non-esterified fatty acid and total lipid in serum, respectively. Liver lipid was depressed by feeding catechin and Spirulina. Hepatic free carnitine and long-chain acylcarnitine contents were markedly higher in both catechin- and Spirulina-fed groups than in the controls. Hepatic carnitine palmitoyltransferase activity was markedly elevated in the Spirulina-fed group. In contrast, glucose-6-phosphate dehydrogenase and fatty acid synthase activities were not different among the groups. The collagen fraction soluble at 20¡C was lower and the insoluble collagen fraction (not soluble at 70¡C) was higher in catechin- and Spirulina-fed groups. These results suggest that dietary supplementation with catechin, as well as Spirulina, improved vitamin C metabolism in young red sea bream.

KEY WORDS: catechin, collagen, lipid mobilization, Pagrus major, Spirulina, vitamin C.

INTRODUCTION vitamin C metabolism in channel catfish Ictalurus punctatus, respectively, have been reported.6,7 Algae as feed additives have been reported to improve Vitamin C is an important co-factor to lipid metabo- and elevate growth, feed efficiency, body constituents, lism and collagen synthesis. Therefore, we examined the carcass quality, physiological characteristics, stress effect of catechin supplementation on vitamin C nutri- responses, and disease resistance in several species of tion such as ascorbate absorption, lipid mobilization, and fish.1,2 Spirulina has already been shown to increase ascor- collagen synthesis in young red sea bream Pagrus major, bate absorption in red sea bream.3 These effects may be and the effects were compared with those of Spirulina. partly due to an improvement of vitamin C metabolism and suggest that it is due to polyphenol-like effects. As a feed supplement, catechin has been reported to MATERIALS AND METHODS be an anti-oxidative and hepato-protective agent that improves liver function and lipid metabolism in rats.4,5 Fish and rearing conditions Improvements by Tochu leaf powder and rutin (flavonoid) of carcass quality in eel Anguilla japonica and Zero-year red sea bream (body weight 18 g) produced in the Fisheries Laboratory of Kinki University, Wakayama, Japan, were divided into three dietary groups and reared for 41 days at the Fisheries Laboratory of Kinki Univer- *Corresponding author: Tel: 81-824-24-7989. Fax: 81-824-22-7059. sity. Two replicates of each dietary group were raised in Email: [email protected] 0.3 ton plastic tanks, each containing 15 fish. Water Received 4 August 1999. temperature during rearing period was 25.0 ± 1.1°C. 322 FISHERIES SCIENCE H Nakagawa et al.

Table1 Formulation and composition of diets vanillin method, a NEFA C test kit, and a Lipoperoxide Ingredients (%) Dietary group test kit (Wako Pure Chem. Ltd, Osaka, Japan), respec- Control Catechin Spirulina tively. Brown fish meal 63 63 63 Wheat gluten 10 10 10 Biochemical analyses Starch (cooked) 10 10 10 Sardine oil 5 5 5 Vitamin mixture1 55 5Crude protein was measured by the Kjeldahl method. Mineral mixture2 44 4Crude lipid in the diet was determined by the Soxlet Cellulose 3 2 method. Lipids in muscle, liver, and intraperitoneal fat Teaflan3 1 body (IPF) were extracted with methanol-chloroform Spirulina meal 3 according to Bligh and Dyer.8 Crude sugar in diet was Proximate composition (%) measured by phenol-sulfuric acid method. Moisture 5.8 4.7 4.3 Immediately after killing, muscle and liver were frozen Crude protein 49.3 50.3 50.7 in liquid nitrogen and stored at -80°C. The samples were Crude lipid 9.6 10.0 9.8 submitted for analyses within 2 weeks of killing. Liver Ash 13.3 13.8 13.4 glycogen was determined by the method of Carroll et al.9 Crude sugar 11.0 11.3 11.4 Total L-ascorbic acid level of serum, muscle, and liver was determined by high performance liquid chromato- 1 Halver’s vitamin mixture. 2 graphy (HPLC; Hitachi 655A-11, Tokyo, Japan) as Salt mixture No. 2 (ICN Nutritional Biochemical). 10 3 Contains 60% catechin. described by Rose and Nahrwold. A LiChrospher 100 NH2, 5 mm (Cica-Merck, Tokyo, Japan) and a mobile phase 30% of 0.05 M KH2PO4 in acetonitrile (pH 5.8) at a flow rate of 0.5 mL/min were used. The total ascorbic Diet acid was a sum of both ascorbic acid and dehydroascorbic acid which were detected by 254 nm and 210 nm, The control group was fed with dry diet consisting of respectively, by a UV monitor (Hitachi 655A-21). brown fish meal, wheat gluten, cooked starch, sardine oil, Muscle and liver were used for carnitine analysis. Free vitamins, minerals, and cellulose, as presented in Table1. carnitine, acid-soluble total carnitine, and long-chain In the catechin and Spirulina groups, 1% and 3% of acylcarnitine were measured by HPLC according to the cellulose were replaced by Teaflan (containing 60% Miyasaki et al.11 using a LiChrospher 100 RM-8, 5 mm catechin; Ito-en Co., Tokyo, Japan) or pulverized Spir- (Cica-Merck) and 190 mM KH2PO4:CH3OH as a solvent ulina (Dainippon Ink and Chemical Co. Ltd, Tokyo, (87:13 v/v) at a flow rate of 0.7 mL/min. Samples were Japan), respectively. The Spirulina meal was composed of injected into the HPLC column immediately after the 62.5% crude protein, 7.5% fat, 17.5% carbohydrate, 7% enzymatic reaction and detected at 254 nm against ash, and 3% crude fiber. Ascorbate was not found in Spir- authentic L-carnitine standard (Sigma Chemical Co., ulina nor in Teaflan, and the level in the diet after prepa- Tokyo, Japan). ration was between 102 and 111 mg in 100 g diet. The The livers of six fish from each group were subjected proximate compositions of the diets are shown in to enzyme assay. The liver was homogenized with nine Table1. Fish were fed to satiation about 4% of body volumes of 0.25 M sucrose buffer (pH 7.4) containing weight twice every day. 3 mM HEPES and 1 mM Na-EDTA for 1 min and centrifuged at 500 g for 10 min at 0–4°C; the superna- tant was then cooled in an ice bath and analyzed for car- Blood analyses nitine palmitoyltransferase (CPT, EC 2.3.1.23) according to the method of Bieber et al.12 For determination Blood was drawn from the caudal vein with a syringe, of glucose-6-phosphate dehydrogenase (G6PDH, EC and hematocrit was measured using the micro- 1.1.1.49), fatty acid synthase (FAS), and arginase (EC hematocrit method. The serum was separated by cen- 3.5.3.1) activities, the liver was homogenized with trifugation at 1100 g for 10 min. Then a part of the serum 0.14 M KCl buffer and centrifuged at 15 850 g for was immediately subjected to lipoperoxide analysis. The 20 min. These enzyme activities were then determined rest of serum was stored at -20°C until analysis of other according to the method of Glock and McLean,13 Martin serum parameters. Serum total protein, glucose, aspartate et al.14 and Schwartz,15 respectively. All enzyme assays aminotransferase (AST, EC 2.6.1.1), and alanine amino- were carried out at 30°C. Enzyme activities were transferase (ALT, EC 2.6.1.2) were determined by an expressed as mmol of substrate or coenzyme converted per auto-analyzer (Abbott vision system, Abbott Park, min per 100 g body weight and g liver. USA). Total lipid, non-esterified fatty acids (NEFA), For collagen analysis, the muscle was fractionated into and lipoperoxide were determined by the Sulfo-phospho- 20°C soluble, 70°C soluble, and insoluble collagen frac- Dietary catechin on vitamin C nutrition FISHERIES SCIENCE 323

Table2 Effects of feeding with catechin and Spirulina on biological and cellular parameters in red sea bream Dietary group Control Catechin Spirulina Feed efﬁciency (%) 66.6 58.9 53.0 Survival (%) 100 97 84 Body weight (g) 32.7 ± 8.6 31.4 ± 7.2 29.7 ± 6.0 Hepatosomatic index (%) 2.81 ± 0.80a 2.29 ± 0.45b 2.66 ± 0.12ab Intraperitoneal fat body ratio (%) 2.05 ± 0.66 1.67 ± 1.04 2.58 ± 0.11 Muscle ratio (%) 39.6 ± 4.1 40.9 ± 3.8 38.3 ± 3.6

Mean ± SD (n = 10–30). Different letters following the values of the same row indicate signiﬁcant difference (P < 0.05).

Table3 Effects of feeding with catechin and Spirulina on serum properties in red sea bream Dietary group Control Catechin Spirulina Hematocrit (%) 36.4 ± 5.8a 30.1 ± 5.1b 34.7 ± 5.4a (23) (20) (18) Total protein (g/100mL) 3.85 ± 0.65 3.59 ± 0.47 3.59 ± 0.77 (10) (10) (7) Total lipid (mg/100mL) 807 ± 194a 600 ± 135ab 439 ± 211b (5) (5) (5) Non-esteriﬁed fatty acid (mEq/L) 0.68 ± 0.21a 0.35 ± 0.18b 0.56 ± 0.26ab (5) (5) (5) Glucose (mg/100 mL) 65.3 ± 17.3a 94.0 ± 30.9b 64.9 ± 13.1a (10) (10) (7) Aspartate aminotransferase (IU/L) 102 ± 72 132 ± 104 74 ± 44 (8) (10) (7) Alanine aminotransferase (IU/L) 35.5 ± 18.9 31.0 ± 14.8 25.0 ± 8.8 (7) (10) (8) Lipoperoxide (nmol/mL) 19.7 ± 5.7 20.6 ± 4.8 19.1 ± 9.4 (10) (10) (9)

Mean ± SD (number of fish in parentheses). Different letters following the values of the same row indicate significant difference (P < 0.05). tions according to Hatae et al.16 Hydroxyproline levels in except the hepatosomatic index which was lower in the each fraction were determined according to the method catechin-fed group. of Bergman and Loxley,17 after hydrolysis of each fraction Serological parameters are shown in Table3. Lower with 6 N HCl at 116°C for 16 h. The collagen contents hematocrit and higher glucose level were observed in were calculated according to Sato et al.18 the catechin-fed group. Total protein, AST, ALT, and lipoperoxide were not different among the groups. Serum total lipid and NEFA concentration were lower in the Statistical analyses Spirulina-fed group and the catechin-fed group, respectively, than the control group. The data were analyzed for significance using Duncan’s The proximate compositions of muscle, liver, and IPF multiple range test after examination by analysis of were not different among the groups except hepatic lipid variance. Probabilities of 0.05 or less were considered content, which was significantly lower in both catechin- statistically significant. and Spirulina-fed groups than in controls (Table4). Total ascorbate concentration in the serum, liver, and RESULTS muscle is presented in Fig. 1. Serum ascorbate level of the catechin group was significantly higher than that of the The effects of feeding catechin and Spirulina on biologi- control group. The ascorbate level in the liver was con- cal parameters are shown in Table2. The body weight, siderably higher in the experimental groups than in the survival, and feed efficiency were not different among control group. However, muscle ascorbate level was not the groups. Biological parameters were not different different among the groups. 324 FISHERIES SCIENCE H Nakagawa et al.

Fig. 1 Effects of feeding with catechin and Spirulina on L - Fig. 2 Effects of feeding with catechin and Spirulina on car- ascorbic acid level of serum (in 100 mL), liver (in 1 g), and nitine components in red sea bream. The values are means of muscle (in 1 g) in red sea bream. The values are means of two two determinations of pooled samples consisting of three fish determinations of pooled samples consisting of three fish from from each replicate group. Different letters on the bar repre- each replicate group. Different letters on the bar represent sig- sent significant differences (P < 0.05). nificant differences (P < 0.05).

Table4 Effects of catechin and Spirulina on proximate composition (%)* in red sea bream Dietary group Control Catechin Spirulina Muscle Moisture 74.1 74.2 74.4 Crude protein 21.8 22.6 20.2 Lipid 4.4 4.1 5.7 Ash 1.6 1.6 1.5 Liver Protein 10.9 10.3 11.5 Lipid 16.5a 12.5b 12.7b Glycogen 5.1 5.0 6.1 Fig. 3 Effects of feeding with catechin and Spirulina on muscle Intraperitoneal fat body collagen contents in red sea bream. Vertical lines indicate stan- Lipid 75.9 73.3 75.1 dard error of six observations.

* Means of two determinations of pooled samples consisting of three fish from each replicate group. Different letters following the values of the same row indicate sig- Muscle collagen contents are shown in Fig. 3. Total nificant difference (P < 0.05). collagen which was calculated from hydroxyproline content was higher in the catechin- and Spirulina-fed groups, but not significantly. The muscle pooled from Figure 2 shows muscle and liver carnitine concentra- three fish from each replicate group was fractionated into tions. While muscle carnitine was not different among three categories; the level of collagen soluble at 20°C was the groups, liver free carnitine and long-chain acylcar- lower and that of insoluble collagen (which was not nitine concentrations were significantly higher in the soluble even at 70°C) was higher in the catechin- and catechin- and Spirulina-fed groups than in the controls. Spirulina-fed groups than the controls. Nevertheless, Table5 shows the hepatic enzyme activities. The these differences were not statistically significant. activity of CPT, which is related to fatty acid oxidation, was considerably elevated in the Spirulina-fed group. In contrast, the activities of the lipogenic enzymes G6PDH DISCUSSION and FAS, which are involved in fatty acid synthesis, were not different among the groups. Activities of the amino In the present study, supplementation of the diet with acid degrading enzyme arginase seemed to be depressed catechin did not produce any adverse effects on feeding by feeding with catechin, but not significantly. activity. The growth, feed efficiency, and survival rate Dietary catechin on vitamin C nutrition FISHERIES SCIENCE 325

Table5 Hepatic enzyme activities of catechin- and Spirulina-fed red sea bream Dietary group Control Catechin Spirulina Carnitine palmitoyltransferase (mmol/min per 100 g body weight) 1.96 ± 0.47a 1.60 ± 0.29a 3.19 ± 0.75b (mmol/min per g liver) 0.73 ± 0.32a 0.66 ± 0.15a 1.15 ± 0.16b Glucose 6-phosphate dehydrogenase (mmol/min per 100 g body weight) 149 ± 42 129 ± 28 177 ± 42 (mmol/min per g liver) 49.3 ± 1.7 52.7 ± 9.1 61.2 ± 17.0 Fatty acid synthase (mmol/min per 100 g body weight) 0.49 ± 0.18 0.77 ± 0.59 0.78 ± 0.27 (mmol/min per g liver) 0.18 ± 0.07 0.29 ± 0.12 0.31 ± 0.23 Arginase (mmol/min per 100 g body weight) 16.9 ± 5.1 11.6 ± 2.3 14.0 ± 2.5 (mmol/min per g liver) 5.67 ± 0.79 4.74 ± 0.59 5.19 ± 0.78

Mean ± SD (n = 6). Different letters following the values of the same row indicate significant difference (P < 0.05). were slightly lower, but not significantly in catechin- and rutin, which is one of the flavonoids, induced a decrease Spirulina-fed groups. Other studies have shown that of 2-thiobarbituric acid values in channel catfish.7 growth and feed efficiency of red sea bream improved sig- L-Carnitine is involved in the b-oxidation of long- nificantly by supplementation of Spirulina,19,20 however, chain fatty acids. Biosynthesis of carnitine hydroxylation no improvements in growth or feed efficiency were found reactions require ascorbate as a co-factor. Miyazaki et al. by feeding Spirulina in the present experiment. found that vitamin C improved lipid and carnitine In the present study, absorption and assimilation of metabolism in rainbow trout.27 Higher liver free carnitine ascorbate were higher in the catechin- and Spirulina-fed and long-chain acylcarnitine levels in both catechin- red sea bream than in controls. Total ascorbate in serum and Spirulina-fed fish indicated the elevation of fatty acid and liver was significantly elevated by catechin supple- utilization as energy in fish. L-Carnitine supplementa- mentation. Tea leaf catechin showed marked synergistic tion of the diet promotes growth and lipid metabolism effects with ascorbate as an anti-oxidant.21 Protective in hatchery-reared sea bass Dicentrarchus labrax and red effect of tea catechin against ascorbate degradation in sea bream fingerlings.28,29 The activity of hepatic carni- fish feed was confirmed elsewhere.22 While dietary tine palmitoyltransferase, which is responsible for b- Spirulina also elevated ascorbate level in serum and liver oxidation of fatty acids in mitochondria, was markedly and protected ascorbate from degradation in red sea elevated in Spirulina-fed fish. Dietary catechin and bream,3 the current study showed high liver ascorbate in Spirulina may have indirectly increased carnitine syn- the Spirulina-fed group. Accordingly, it was confirmed thesis and consequently promoted lipolysis activity. that dietary catechin and Spirulina elevated ascorbate Activated lipid mobilization resulted in depression of level, and active carnitine syntheses followed the rise in lipid accumulation in fish. the ascorbate level. Vitamin C is well known as a co-factor in hydroxyla- The lower liver lipid in catechin- and Spirulina-fed tion of proline to hydroxyproline in collagen synthesis. groups may indicate effects on lipid metabolism and thus Muscle total collagen and its mature fraction which is lipid accumulation. Dietary catechin and Spirulina insoluble even at 70°C tended to increase by feeding decreased NEFA and total lipid in serum, respectively, with catechin and Spirulina. Fish containing higher col- suggesting an influence on lipid metabolism. The phe- lagen content and lower 20°C soluble collagen fraction nomena could be due to carnitine synthesis accelerated have firmer raw meat texture.11 Administration of Tochu under the presence of ascorbic acid. Improved lipid leaf powder increased muscle stroma fraction as well as metabolism has been reported in Ulva-fed red sea bream23 hydroxyproline content in cultured eel.6 In another and black sea bream Acanthopagrus schlegeli.24 In addition, study, Spirulina also increased stroma fraction in muscle, Chlorella-extract enhanced lipid mobilization in ayu which mainly consisted of collagen, in 1-year red sea Plecoglossus altivelis.25,26 Serum lipoperoxide value, an bream.30 indicator of lipid oxidation, was unaffected in the present The results suggested that catechin supplementation study. However, anti-oxidative effects have been to diet improved vitamin C metabolism in young red sea reported in catechin-fed rats,5 and feeding with Spirulina bream, and partly resembled those of Spirulina supple- depressed lipoperoxide in 1-year red sea bream.3 Dietary mentation. The improvement of vitamin C metabolism 326 FISHERIES SCIENCE H Nakagawa et al.

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