Vitamins C and E concentrations in muscle of elasmobranch and teleost fishes

Comparative Biochemistry and Physiology, Part A 170 (2014) 26–30

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Vitamins C and E concentrations in muscle of elasmobranch and teleost fishes Marcela Vélez-Alavez a, Lía C. Méndez-Rodriguez a, Juan A. De Anda Montañez a, C. Humberto Mejía a, Felipe Galván-Magaña b, Tania Zenteno-Savín a,⁎ a b

Centro de Investigaciones Biológicas del Noroeste, S.C., Instituto Politécnico Nacional 195, Playa Palo de Santa Rita Sur, La Paz, Baja California Sur, C.P. 23096, Mexico Centro Interdisciplinario de Ciencias Marinas, Av. Instituto Politécnico Nacional s/n, Colonia Playa Palo de Santa Rita, Apartado Postal 592, La Paz, Baja California Sur, C.P. 23096, Mexico

a r t i c l e

i n f o

Article history: Received 11 October 2013 Received in revised form 20 January 2014 Accepted 21 January 2014 Available online 25 January 2014 Keywords: Antioxidants Elasmobranchs Teleosts Vitamin C Vitamin E

a b s t r a c t In fish, vitamins are part of the first line of the antioxidant defense, they are directly related to stress and disease, and they are involved in the maintenance of various physiological processes and metabolic reactions. In general, fish are unable to synthesize vitamin C due to a deficiency in gulonolactone oxidase (GLO), the enzyme responsible for its de novo synthesis. Vitamin E is involved in the immune response and perhaps one of its main physiological functions is to protect membranes from oxidative damage (lipid peroxidation) associated with free radical production. In fish muscle, vitamin E has an important role as an antioxidant in vivo and its content is highly related to the stability of lipids and fats. The aim of this study was to determine the content of vitamins C and E in muscle from different species of elasmobranch and teleost fishes. The concentrations of vitamins C and E were determined by high performance liquid chromatography (HPLC). The concentration of vitamin C found for the group of elasmobranchs was lower (p = 0.001) than that for teleosts. For Mustelus henlei vitamin C was found in only one individual; in Tetrapturus audax and Totoaba macdonaldi vitamin C concentration was below the detection limit. The concentration of vitamin E was lower in the group of elasmobranchs (p = 0.03) compared with that of teleosts. The main differences in the antioxidant system between teleosts and elasmobranchs appear to be the specific type and levels of antioxidant compounds, as well as the synergistic interactions among the antioxidants present in their tissues. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The antioxidant system, composed of nutrients, enzymes and small molecular weight molecules, differs between tissues, is related to the lifestyle and depends on the evolutionary history of a species; for instance, differences in the antioxidant system between elasmobranchs and teleosts have been reported (Wilhelm-Filho and Boveris, 1993; Wilhelm-Filho et al., 1993; López-Cruz et al., 2010; Faramarzi, 2012). In fish, vitamins are part of the first line of the antioxidant defense, are directly related to stress and disease, and are involved in the maintenance of various physiological processes and metabolic reactions (Martínez-Álvarez et al., 2005; Kumari and Sahoo, 2005; LoperaBarrero and Poveda-Parra, 2009). Vitamins are organic compounds obtained mainly through the diet because of either a lack of key enzymes involved in their synthesis, or failure to produce them in sufficient amounts (Weber, 1995; Drouin et al., 2011). Although the specific requirements are not equal for all species, vitamins A, D, E, K and C are essential nutrients in the fish diet, and insufficient intake of vitamins C ⁎ Corresponding author. Tel.: +52 612 123 8502; fax: +52 612 125 3625. E-mail addresses: [email protected] (M. Vélez-Alavez), [email protected] (L.C. Méndez-Rodriguez), [email protected] (J.A. De Anda Montañez), [email protected] (F. Galván-Magaña), [email protected] (T. Zenteno-Savín). 1095-6433/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2014.01.010

and E can cause nutritional and growth deficiencies (Weber, 1995; Corredor and Landines, 2009; Lopera-Barrero and Poveda-Parra, 2009). Vitamin C (ascorbic acid or, under physiological conditions, ascorbate) is a water-soluble nutrient required for the synthesis of red blood cells and collagen, which is a component of connective tissues, blood vessels, bone matrix, cartilage and tissue repair (Fracalossi et al., 1998). Vitamin C is also involved in iron metabolism, in the formation of neurotransmitters such as serotonin, in the transformation of dopamine to noradrenaline and other hydroxylation reactions, as well as in the biosynthesis of catecholamines, which are part of the primary response to stressful situations (Verlhac and Gabaudan, 1997; Torres et al., 2002; Kumari and Sahoo, 2005; Corredor and Landines, 2009). In general, fish are unable to synthesize vitamin C due to a deficiency in gulonolactone oxidase (GLO; EC 1.1.3.8), the enzyme responsible for its de novo synthesis (Verlhac and Gabaudan, 1997; Fracalossi et al., 2001; Faramarzi, 2012). The majority of teleosts (bony fish) have no functional GLO genes; however, GLO activity was found in the kidney of several primitive non-teleost fishes, such as sharks, sturgeon and lamprey, suggesting that these species can synthesize vitamin C (Cho et al., 2007). Species that cannot synthesize this vitamin absorb it by a sodium (Na)-dependent active transport mechanism, which apparently acts at low micronutrient concentrations, whereas at higher concentrations absorption can occur via passive diffusion (Verlhac and

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Gabaudan, 1997). Physical and physiological effects of vitamin C deficiency are varied; including internal bleeding, immunosupression, increased susceptibility to bacterial infections, reduced growth, skeletal muscle injuries, structural deformities, abnormal pigmentation and poor reproductive performance, among others (Alava et al., 1993; Verlhac and Gabaudan, 1997; Fracalossi et al., 1998; Mæland and Waagbø, 1998; Fracalossi et al., 2001; Kumari and Sahoo, 2005; Cho et al., 2007). The term vitamin E includes two families of compounds, tocopherols and tocotrienols, which have similar structure and differ only in their saturation; α-tocopherol is the most active and abundant isomer in organelles and cell membranes of vertebrates (Torres et al., 2002; Wilhelm-Filho, 2007; Faramarzi, 2012). Vitamin E is a fat-soluble vitamin that is absorbed in the small intestine by passive diffusion; it is stored primarily in the liver, from which it can be mobilized quickly, but can also deposited in adipose tissue and muscle where it shows a slow turnover (Torres et al., 2002). Vitamin E is involved in the immune response and perhaps one of its main physiological functions is to protect membranes from oxidative damage (lipid peroxidation) associated with free radical production and/or with an imbalance between antioxidant and prooxidant molecules (Weber, 1995; Wilhelm-Filho, 2007). In fish muscle, vitamin E has an important role as an antioxidant in vivo and its content is highly related to the stability of lipids and fats (Pazos et al., 2005). Both vitamin C and vitamin E can act as antioxidants in a synergistic manner. Vitamin C acts as a terminal element in the protection against tissue damage caused by free radicals, but when both vitamins are present, the major function of vitamin C is restoration of vitamin E (Verlhac and Gabaudan, 1997; Torres et al., 2002). The requirements of antioxidant vitamins intake are directly related to the levels of highly unsaturated fatty acids (HUFA), given their susceptibility to oxidation (Faramarzi, 2012). Tissues and organs with an elevated unsaturated lipid content, as is the case of fish tissues, are expected to have elevated antioxidant vitamin content. Given the lifestyle and evolutionary history, elasmobranch fishes are expected to have higher antioxidant vitamin content than teleost fishes (WilhelmFilho and Boveris, 1993; Wilhelm-Filho et al., 1993; López-Cruz et al., 2010; Faramarzi, 2012). Despite the importance of fish as part of the aquatic food chains, studies of the antioxidants, including vitamin E and vitamin C, in fish are scarce. The aim of this study was to determine and compare the content of vitamins C and E in muscle from elasmobranch and teleost fishes. Three species of each group (elasmobranch and teleost) were selected based on their ecological relevance. 2. Materials and methods 2.1. Sample collection White muscle samples (approximately 5 g) were collected from the caudal area of six different elasmobranch and teleost fish species. The sampled elasmobranch species include Mustelus henlei (Carcharhiniformes, Triakidae) a species that lives mainly in the intertidal zone and continental shelf where it feeds mostly on crustaceans; Prionace glauca (Carcharhiniformes, Carcharhinidae), an oceanic and pelagic shark, which feeds on squid and other smaller pelagic fishes, and Isurus oxyrinchus (Lamniformes, Lamnidae), a coastal and oceanic shark, feeding primarily on other fishes and squid. Sampled teleosts included Totoaba macdonaldi (Perciformes, Sciaenidae), which primarily inhabits coastal waters and rocky bottoms and feeds mainly on crustaceans; Coryphaena hippurus (Perciformes, Coryphaenidae) is an oceanic pelagic species, whose feeding habits are based on crustaceans, squid and other fish, and Kajikia audax (Perciformes, Istiophoridae), an oceanic and epipelagic species that feeds on fish, crustaceans and cephalopods (Fischer et al., 1995; Compagno, 2002). Data of the sampled species are summarized in Table 1. Samples were collected at fishing camps (San Lazaro, Punta Lobos), in sportfishing docks (Cabo San Lucas) and during scientific sampling trips (Ensenada de Muertos, El Sargento, Upper Gulf of

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California) from April to December 2011. All samples were stored in the dark and frozen at −80°C, for a period not exceeding six months from the time samples were collected, until analyzed. 2.2. Vitamin C concentration Concentrations of vitamin C were quantified by high performance liquid chromatography (HPLC) (Waters Model 2695; Milford, MA, USA) following the methods of Carvajal et al. (1997), Ledezma-Gairard (2004) and Agilent Technologies Company (2001). The extraction of water-soluble compounds was carried out using a solution of metaphosphoric acid (3%), acetic acid (8%), and EDTA (0.01 M). Following homogenization (Polytron 3100, Switzerland), samples were centrifuged at 23895 g at 4°C for 10 min. The supernatant was filtered using a cellulose membrane (0.45 μm). The extract was separated in a Hypersil BDS C8 column (5 μm, 250 mm in length, 4.6 mm internal diameter; Bellefonte, PA, USA) by a gradient separation system with two mobile phases, deionized water (pH 2.4) and acetonitrile (100%). Vitamin C was detected at 245 nm wavelength with a retention time of 6.7 min (PAD UV detector; Waters). Vitamin C content in samples was derived from a calibration curve of L-ascorbic acid (1, 5, 10, 25, 50, 100, 150 and 200 ng μL−1). When the concentrations were below the minimum detection limit (MDL, 0.01 ng μL−1) a manual integration based on the retention time and absorption spectrum of vitamin C was performed. Results are expressed as μg g−1 of wet tissue. 2.3. Vitamin E concentration Concentrations of vitamin E in fish muscle samples were quantified by HPLC (Waters) according to the methods of Alava et al. (1993) and Supelco (2003/2004). Samples were homogenized with anhydrous sodium sulfate (0.5 g), hexane, ethanol and distilled water (7:3:2 v/v). Subsequently, butylated hydroxytoluene (BHT, 504 mM) was added, and the sample was mixed and centrifuged at 944 g at 10 °C for 10 min. The supernatant was recovered and evaporated between 35 and 45°C. The precipitate was again subjected to extraction by adding hexane (98.5%), centrifuged and the recovered supernatant was mixed with the first extraction product. The extracted mixture was completely evaporated with nitrogen gas. Each concentrate was reconstituted in ethanol, pyrogallol (1%) and potassium hydroxide (50%). Tubes were placed at 65°C for 25 min. Tubes were cooled in a mixture of BHT (504 mM), hexane and distilled water, shaken, and the hexane phase was recovered; this step was repeated twice. The mixture was filtered through cellulose acetate filters (0.45 μm) and completely evaporated with nitrogen gas. Finally, the lipidic extract was diluted in acetonitrile (100%) and separated in a Supelcosil LC18 column (3 μm, 150 mm in length, 4.6 mm internal diameter) by a gradient separation system with mobile phase consisting of acetonitrile:water (9:1 v/v). Vitamin E was detected at 285 nm wavelength with a retention time of 13 min (Waters PAD detector UV). Vitamin E concentration in samples was derived from a calibration curve of DL-α-tocopherol acetate (1, 6.25, 12.5, 25, 50 and 100 ng μL−1). When the concentrations were below the MDL (0.001 ng μL−1), a manual integration based on the retention time and absorption spectrum of vitamin E was performed. Results are expressed as μg g−1 of wet tissue. 2.4. Statistical analyses Because the data did not meet the criteria for normality and homoscedasticity, non-parametric statistical analyses were performed. In two species, vitamin C concentration was found to be below the detection limit precluding statistical analyses within teleosts. In samples where vitamin concentrations were below the MDL the methodology of simple value replacement reported by Helsel (1990) was applied in order to run the statistical analyses. Kruskal–Wallis and Mann–Whitney tests were applied to probe for significant differences in the concentrations

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Table 1 Morphometric data from the analyzed elasmobranch and teleost species. Species

N

Sex (n) F

Elasmobranch fishes Mustelus henlei (Triakidae) Brown smooth hound Prionace glauca (Carcharhinidae) Blue shark Isurus oxyrinchus (Laminidae) Mako shark Teleost fishes Totoaba macdonaldi (Sciaenidae) Totoaba Coryphaena hippurus (Coryphaenidae) Common dolphinfish Kajikia audax (Istiophoridae) Striped marlin

M

Size range (total length, cm)

u/s

Maturity stage (according to size)

15

10

5

–

72–98

30

17

11

2

114–267

20

12

7

1

83–190

Juvenile

33

14

10

9

61–86

Juvenile

27

18

7

2

47–135

Adults

30

9

20

1

161–221⁎

Adults

Adults

Juvenile and adults

F, females; M, males; u/s, unknown sex. ⁎ Jaw-fork length.

of vitamins C and E between species and between groups (Durán et al., 2003). Statistical analyses were performed using Statistica v.7. (StatSoft, Inc., 2007). Statistical significance was considered when p b 0.05.

0.040

A

b

0.035

3. Results

Within the elasmobranch species analyzed in this study, blue sharks (P. glauca) had higher vitamin C concentration than mako sharks (I. oxyrinchus) (p = 0.001) (Fig. 1A). In muscle samples from the brown smooth-hound shark (M. henlei) vitamin C was found to be above the MDL in only one individual (0.016 μg g−1). Among teleosts, vitamin C concentrations were below the MDL in muscle samples of striped marlin (K. audax) and totoaba (T. macdonaldi). In the common dolphinfish (C. hippurus) muscle samples, vitamin C concentrations were within the range of 0.013 to 0.042 μg g−1 (Fig. 1B). As a group, teleosts had a wider range of vitamin C concentrations. In general, vitamin C concentrations in muscle samples of elasmobranchs were significantly lower than those of teleosts (p = 0.001).

Vitamin C (µg g-1)

0.030

3.1. Vitamin C concentration

0.025 0.020

0.010 0.005 0.000

All organisms have the ability to produce molecules, such as enzymes, glutathione and some hormones, which contribute to the endogenous antioxidant defense system. In addition, the consumption

P. glauca

M. henlei

0.045

3.2. Vitamin E concentration

4. Discussion

I. oxyrinchus

Elasmobranch fishes

0.040

B

0.035

Vitamin C (µg g-1)

Among the shark species analyzed in this study, vitamin E concentrations were higher in muscle samples from M. henlei (0.017–0.038 μg g − 1) than those from I. oxyrinchus (0.011 to 0.014 μg g−1; p = 0.01) and P. glauca (0.012–0.034 μg g−1; p = 0.001) (Fig. 2A). Within bony fish, vitamin E concentration in muscle samples from C. hippurus was higher than that in samples from T. macdonaldi (p = 0.004) (Fig. 2B). Although the highest vitamin E concentration was found in a sample from K. audax, no significant differences were observed between species. Teleosts, as a group, had the wider range of vitamin E concentrations. Overall, vitamin E concentrations were lower in the elasmobranchs than in the teleosts analyzed in this study (p = 0.03).

a

0.015

0.030 0.025 0.020 0.015 0.010 0.005 0.000

K. audax

C. hippurus

T. macdonaldi

Teleost fishes Fig. 1. Concentrations of vitamin C (μg g−1 wet tissue) in muscle samples from A) elasmobranch fishes (mako shark, Isurus oxyrinchus; blue shark, Prionace glauca; brown smoothhound shark, Mustelus henlei), and B) teleost fishes (striped marlin, Kajikia audax; common dolphinfish, Coryphaena hippurus; totoaba, Totoaba macdonaldi). Data are shown as median, percentile ranks (25%–75%), minimum and maximum values. Different letters denote differences between species; p b 0.05.

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0.24 0.22

a

A

0.20

Vitamin E (µg g-1)

0.18 0.16

a

0.14 0.12 0.10 0.08 0.06 b

0.04 0.02 0.00 -0.02

I. oxyrinchus

P. glauca

M. henlei

Elasmobranch fishes 0.6

B

ab

0.5

Vitamin E (µg g-1)

a

0.4 0.3 0.2

b

0.1 0.0 -0.1 K. audax

C. hippurus

T. macdonaldi

Teleost fishes Fig. 2. Concentrations of vitamin E (μg g−1 wet tissue) in muscle samples from A) elasmobranch fishes (mako shark, Isurus oxyrinchus; blue shark, Prionace glauca; brown smoothhound shark, Mustelus henlei), and B) teleost fishes (striped marlin, Kajikia audax; common dolphinfish, Coryphaena hippurus; totoaba, Totoaba macdonaldi). Data are shown as median, percentile ranks (25%–75%), minimum and maximum values. Different letters denote differences between species; p b 0.05.

of nutritional antioxidants in the diet aids in protecting against various oxidative processes (Bhadra et al., 2004). Vitamin E is strictly obtained as a nutritional antioxidant in vertebrates and invertebrates, while vitamin C is synthesized in few fish species (Weber, 1995; Drouin et al., 2011). It has been suggested that in elasmobranch fishes the antioxidant system relies mostly on non-enzymatic molecules, while teleost fishes have higher antioxidant enzyme activities (Wilhelm-Filho and Boveris, 1993; Wilhelm-Filho et al., 1993; López-Cruz et al., 2012). In the present study, the concentrations of vitamins C and E were determined in fish and shark species, including top predators (mako shark, blue shark, striped marlin, and common dolphinfish) and secondary consumers (brown smooth-hound shark and totoaba), for some of which no information is available. Vitamin C is typically found in high concentrations in various endocrine tissues compared to what can be found in the liver, muscle, intestine and skin. In teleost fishes, such as some salmonids, the highest concentration of ascorbate is found in the anterior kidney (Moreau and Dabrowski, 1996). Concentrations of vitamin C in several species of fish are lower in muscle as compared to other tissues, such as the liver (43–78 μg g−1) (Bhadra et al., 2004). This is consistent with the assumption that fish muscle usually has low concentrations of vitamin C, at levels that can be considered negligible (Bragadóttir, 2001), as in the case of the observed concentrations in elasmobranch and teleost species analyzed in this study.

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It has been suggested that, unlike bony fish, elasmobranch fishes are able to synthesize vitamin C; however, the reports include only some species of rays and sharks of the Squalidae, Triakidae and Scyliorhinidae (Drouin et al., 2011). Activity of the enzyme GLO was analyzed under saturating conditions and reported in the kidney of Mustelus manazo with a maximum production rate of 14 μg h−1 mg−1 protein, and for Squalus acanthias with a maximum production rate of 513 ± 224 μg h−1 g−1 tissue (Mæland and Waagbø, 1998). M. henlei, analyzed in this study, belongs to the Triakidae, and the genus Mustelus is listed among those that perhaps can synthesize vitamin C. However, in this study, vitamin C concentrations were below the detection limit in muscle samples of M. henlei. Further studies on the vitamin C concentrations and GLO activity are needed to determine if the ability to produce and store vitamin C is comparable among species within the genus Mustelus and the Triakiadae. Factors such as the phylogenetic position may be more important than the dietary habits of the fishes in their ability to synthesize vitamin C (Corredor and Landines, 2009). Most teleost fishes cannot synthesize vitamin C; therefore, it has been suggested that the synthesis of vitamin C is an ancestral feature of vertebrates and that it was lost in the common ancestor of teleost fishes (Drouin et al., 2011). Drouin et al. (2011) suggested that loss of the capacity to synthesize vitamin C can be advantageous since the synthesis process facilitates the formation of hydrogen peroxide and depletion of glutathione, a major endogenous antioxidant; this suggestion remains to be confirmed. Vitamin E concentrations in elasmobranch and teleost fishes analyzed in this study were within the range of 0.011–0.038 μg g−1. Few data have been reported on the concentration of vitamin E in different species of fish. The vitamin E concentrations reported for Sardina pilchardus (0.016 μg g−1), Engraulis encrasicolas (0.0002 μg g− 1), Salmo salar (0.02 μg g−1), Merluccius merluccius (0.0035 μg g−1), Solea vulgaris (0.01 μg g−1) and Pagellus bogaraveo (0.015 μg g−1) (FalderRivero, 2005) are similar to those found in the species included in this study, except for C. hippurus (0.002–0.424 μg g−1) and K. audax (0.008–0.508 μg g−1) in which the vitamin E concentration ranges were higher. The concentrations of vitamin E in muscle from common dolphinfish and striped marlin found in this study are lower than those reported in the liver of Scophthalmus maximus (160 μg g−1), Hippoglossus hippoglossus (71 μg g−1) and Sparus aurata (54 μg g−1) (Tocher et al., 2002), and are also lower than those reported in plasma for Parapercis colias (14 μM) and Notolabrus fucicola (18.9 μM) (Gieseg et al., 2000). Vitamin E content in tissues such as the liver, muscle and blood of different species of fish is influenced by its dietary intake and, to a lesser extent, by lipid intake (Stephan et al., 1995). In most fish, the liver is the main storage site of vitamin E when there is an abundance of this nutrient in the diet (Tocher et al., 2002). Levels of vitamin E are related to the level of polyunsaturated fatty acids (PUFA) in the fish, probably as a protection mechanism against peroxidation of these molecules highly susceptible to oxidative damage (Stephan et al., 1995; Halliwell and Gutteridge, 2002). The concentration of vitamin E is also related to the environment inhabited by different species of fish, as was determined in two species of Antarctic fish having a concentration of up to six times more than temperate species (Wilhelm-Filho, 2007). A less common component of vitamin E, α-tocomonoenol, is found in Antarctic fish; therefore, it is likely that the concentration of various forms of vitamin E differs between species (Wilhelm-Filho, 2007). The combination of vitamins C and E in fish contributes to improve growth, immune response, metabolism of nutrients and resistance to stress; vitamin C aids in the process of absorption of vitamin E and metabolism of lipids (Corredor and Landines, 2009). Vitamin E concentration increased in the muscle, liver, heart and kidney of S. maximus (turbot) receiving diets high in α-tocopherol, while the effect on the concentration of ascorbate in muscle was the opposite (Ruff et al., 2003). It is possible that the main difference in the antioxidant capacity between teleost and elasmobranch fishes, and perhaps even mammals, is related to the specific type and levels of antioxidant compounds found

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in their tissues. In elasmobranch tissues the antioxidant properties of urea derivatives (hydroxyurea, dimethylurea, thiourea), as well as the specific levels of glutathione and vitamins C and E may contribute to their tolerance of higher levels of lipoperoxidation compared to tissues from other vertebrates (Wilhelm-Filho et al., 1993; Wang et al., 1999; López-Cruz et al., 2010, 2012). These antioxidant molecules appear to act synergistically to prevent further oxidative damage in tissues of elasmobranch fishes. Acknowledgments The authors acknowledge the fishing communities of San Lazaro and Punta Lobos for samples obtained, also the support of S. Ortega (CICIMAR), O. Lugo-Lugo and N.O. Olguín-Monroy who helped in the collection of samples and provided guidance and technical assistance in processing samples. Samples from T. macdonaldi were collected under scientific sampling permit SGPA/DGVS/05508/11. This study was funded by CONABIO (FB1508/HK050/10), SEP-CONACYT (2011-01/165376) and CIBNOR (PC2.0, PC0.10, PC0.05). FGM thanks Instituto Politécnico Nacional (COFAA, EDI) for fellowships provided. MVA is a recipient of a CONACYT scholarship (12949). Comments and suggestions by two anonymous reviewers helped to improve the manuscript. References Agilent Technologies Company, 2001. HPLC for Food Analysis. Publication Number 5988-3294EN (September 01). Alava, V., Kanazawa, A., Teshima, S., Koshio, S., 1993. Effects of dietary vitamins A, E, and C on the ovarian development of Panaeus japonicus. Nippon Susian Gakkaishi 59, 1235–1241. Bhadra, A., Yamaguchi, T., Takamura, H., Matoba, T., 2004. Radical-scavenging activity: role of antioxidative vitamins in some fish species. Food Sci. Technol. Res. 10, 264–267. Bragadóttir, M., 2001. Endogenous antioxidants in fish. Master of science thesisUniversity of Iceland (59 pp.). Carvajal, M., Remedios-Martínez, M., Martínez-Sánchez, F., Alcaraz, C., 1997. Effect of ascorbic acid addition to peppers on paprika quality. J. Sci. Food Agric. 75, 442–446. Cho, Y.S., Douglas, S.E., Gallant, J.W., Kim, K.Y., Kim, D.S., Nam, Y.K., 2007. Isolation and characterization of cDNA sequences of L-gulono-gamma-lactone oxidase, a key enzyme for biosynthesis of ascorbic acid, from extant primitive fish groups. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 147 (2), 178–190. Compagno, L., 2002. Sharks of the World. In: FAO (Ed.), Vol. II, Vol. II (269 p.). Corredor, A., Landines, M., 2009. Efecto del ácido ascórbico sobre la respuesta de los peces ante condiciones de estrés. Rev. Med. Vet. Zootec. 56, 53–66. Drouin, G., Godin, J.R., Pagé, B., 2011. The genetics of vitamin C loss in vertebrates. Curr. Genom. 12, 371–378. Durán, A., Cisneros, A., Vargas, A., 2003. Bioestadística. Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Iztacala, México 222. Falder-Rivero, A., 2005. Enciclopedia de los alimentos. Productos del mar y de las aguas continentales. Distrib. consum. 84, 103–121. Faramarzi, M., 2012. The influences of vitamins C and E on the growth factors and carcass composition of common carp. Glob. Vet. 8 (5), 498–502. Fischer, W., Krupp, F., Schneider, W., Sommer, C., Carpenter, K.E., Niem, V.H., 1995. Guía FAO para la identificacíon de especies para los fines de la pesca. In: FAO (Ed.), Pacífico Centro-Oriental. Vertebrados, vol. II (Roma. 1200 pp.).

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