Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00904.x

2011 17; 585–594

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Department of Biology, University of Bergen, Bergen, Norway; (NIFES), Bergen, Norway

Vitamin K belongs to the lipid soluble vitamins, and occurs naturally as phylloquinone (vitamin K1) and menaquinone (vitamin K2). In addition, there is a synthetic provitamin, menadione (vitamin K3), primarily used as a vitamin K source in animal feed. Menadione is unstable during feed processing and storage and the dietary content may reach critically low levels. Recent publications also question the availability of menadione in feed for salmonids. Vitamin K plays vital roles in blood coagulation and bone mineralization in fish, but the suggested minimum requirement varies considerably depending on the vitamin K source used. Vitamin K deficiency is characterized by mortality, anaemia, increased blood clotting time and histopathological changes in liver and gills. However, one should assess both inherent and supplemented forms of vitamin K in feeds for exact determinations, as relevant novel feed ingredients of plant origin may be sufficient to meet the requirement for vitamin K. The current review gives an overview of the biochemical role of vitamin K, and discusses vitamin K requirement in fish in light of updated literature, with special emphasis on salmonids. key words: fish, menadione, menaquinone, phylloquinone, requirement, vitamin K Received 31 January 2011; accepted 28 July 2011 Correspondence: Christel Krossøy, Department of Biology, University of Bergen, PBox 7803, N-5020 Bergen, Norway. E-mail: Christel.Krossoy@ bio.uib.no

Fish, like all other animals, need a certain amount of vitamins for optimal growth and proper health that vary

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Ó 2011 Blackwell Publishing Ltd

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National Institute of Nutrition and Seafood Research

according to factors like nutritional status, external stressors, age and health status. Vitamin requirements published by the NRC (1993) usually designate minimum requirements as the vitamin level required to avoid clinical deficiency signs and support normal growth (Woodward 1994). There is a distinction between minimum requirement and requirement for optimal growth or optimal health, which could lead to the definition of higher requirement or recommendation levels adapted to a specific function or to certain conditions. In intensive commercial fish farming, the last decade has brought with it changes in genetics, husbandry and diet composition leading to increased growth rates and subsequently changes in the minimal requirement of micronutrients (Waagbø 2008). However, detailed evaluations of the nutrient requirements for fish have not kept pace with the changes as most of the vitamin requirements of salmonids were determined more than 30 years ago. It is thus unclear if the given requirements are appropriate for modern diet formulations. The earliest requirement studies on fish were performed in an effort to increase the survival of the stock in juvenile stages. Test diets and growth rates were not comparable to commercial rearing, and the response criteria used were mostly survival, weight gain, absence of deficiency signs and maximum tissue storage. The latter resulted in relatively high requirement estimates, but the cost of adding too high levels of vitamins were lower than the cost of suffering high mortalities. As commercial farming became more efficient, more sensitive response criteria for vitamins were used, some measuring metabolically active forms and specific enzyme activity. This lowered the recommendations for most vitamins (Woodward 1994). Historically, vitamin K is best known for its essential role in blood coagulation (Olson 1999), being responsible for the posttranslational modification and activation of the vitamin K-dependent (VKD) proteins (Knapen et al. 1993; Luo et al. 1997; Boskey et al. 1998; Lee et al. 2007), and the first VKD

proteins identified were those involved in vitamin K haemostasis (Nelsestuen et al. 1974; Stenflo et al. 1974; Ferland 1998). In the past few decades, it has become clear that vitamin K plays an important role in other biological processes, such as bone metabolism and growth control (Price 1988; Manfioletti et al. 1993). The diverse range of functions of VKD proteins implicates a broad biological impact of vitamin K (Berkner 2008), but the exact roles of vitamin K and VKD proteins have been difficult to assess, and the physiological consequences of non-carboxylated and undercarboxylated proteins are unknown. Estimates of dietary vitamin K requirement differ widely among fish species, and the quantitative requirement of vitamin K for most fish is still unknown (NRC 1993). In the current review, we will give an overview of the biochemical role of vitamin K, and discuss vitamin K requirement in fish in light of updated literature with special emphasis on salmonids. However, differences in experimental design, fish species, developmental stage, biomarkers, as well as inclusion level and forms makes the published studies challenging to compare. Overall, the minimum requirement of vitamin K has been difficult to estimate owing to natural occurrence in feed ingredients, feed processing and storage stability of inherent and added vitamin K, vitamin leaching, variable feed intakes and variable bioavailability of the different K vitamers.

Lipid soluble vitamin K was first discovered by the Danish scientist Henrik Dam in 1929 as an antihaemorrhagic factor in chicks (Olson 1999). The factor was later shown to be related to the absence of prothrombin activity in plasma. For decades, it was believed that the only function of vitamin K was in the coagulation cascade, but several vitamin K dependent proteins have now been isolated from bone, dentin, cartilage, kidney, atherosclerotic plaque and numerous soft tissues (Vermeer et al. 1995, 1996; Shearer et al. 1996; Booth 1997; Ferland 1998). Vitamin K refers to a family of compounds derived from quinone, that share a common 2-methyl-1,4-naphthoquinone ring, but differ in the side chain at the C3-position (Lambert & De Leenher 1992). All vitamers K are insoluble in water, slightly soluble in alcohol and readily soluble in non-polar organic solvents (Koivu-Tikkanen 2001). They have a relatively high thermostability (Lambert & De Leenher 1992), but are sensitive to light and alkaline conditions (KoivuTikkanen 2001). There are at least two naturally occurring forms of vitamin K, designated vitamin K1 and K2. Vitamin K1 (phylloquinone; 2-methyl-3-phytyl-1,4-naphthoquinone,

(a)

(b)

(c)

Figure 1 The chemical structures of (a) vitamin K1 (phylloquinone): 2-methyl-3-phytyl-1,4-naphthoquinone; (b) vitamin K2 (menaquinones): 2-methyl-3-(prenyl)n-1,4-naphthoquinone; and (c) vitamin K3 (menadione): 2-methyl-1,4-naphthoquinone.

Fig. 1a) is synthesized by plants, and is mainly found in green leafy vegetables (Booth & Suttie 1998). Phylloquinone has a phytyl group with one double bond in the side chain. Vitamin K2 (menaquinones; MK; 2-methyl-3-(prenyl)n-1,4-naphthoquinone, Fig. 1b), on the other hand, is primarily of microbial origin, and is found in fermented products and in foods of animal origin (Booth & Suttie 1998). Menaquinones include a range of vitamin K forms, named according to the number (n) of prenyl groups in the unsaturated side chain, thus designated MK-n, with n ranging from 2 to 14 (Lambert & De Leenher 1992). Menaquinone-4 (MK-4) and MK-7 are the most relevant nutritional menaquinones (Fodor et al. 2010). Of these, MK-4 is unique as it is the product of certain tissue-specific conversions directly from dietary phylloquinone (Thijssen & Drittij-Reijnders 1994; Ronden et al. 1998; Okano et al. 2008). Menaquinones may be synthesized by bacteria in the gut (Conly & Stein 1993), and the requirement of vitamin K in mammals is met by a combination of dietary intake and intestinal bacterial synthesis. Both diet composition and the use of antibiotics are known to affect intestinal production (Mathers et al. 1990). The quantitative significance and role of menaquinones produced by the intestinal microflora in maintaining vitamin K status is still unknown (Conly & Stein 1993; Suttie 1995; Vermeer et al. 1995), but bacterially derived long-chain menaquinones have been found in human liver (Usui et al. 1989; Thijssen & DrittijReijnders 1996). However, the importance of intestinal production of vitamin K or the effect of antibiotics has not been established in fishes or crustaceans (Tan & Mai 2001). Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone, Fig. 1c) are chemically synthesized vitamin K compounds used in commercial feeds for domestic animals. It is a vitamin K derivate in the form of water soluble salts, like menadione

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Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd

sodium bisulphite (MSB) and menadione nicotinamide bisulphite (MNB). Menadione has no side chain, and is chemically unstable compared to the naturally occurring vitamin K forms (Marchetti et al. 1995, 1999). It is not itself biologically active and is easily excreted, but can at least be partly alkylated enzymatically to MK-4 in tissues when present in animal feeds (Dialameh et al. 1971; Udagawa 2000; Graff et al. 2002, 2010; Okano et al. 2008; Krossøy et al. 2009a).

Most of the work within vitamin K research has been conducted on humans and laboratory animals. It was long thought that the role of vitamin K was limited to the synthesis of factors within the coagulation system, but the discovery of vitamin K as a cofactor and the identification of additional VKD proteins, significantly expanded the understanding of its physiological roles (Stenflo et al. 1974; Suttie 1992; Ferland 1998; Vermeer et al. 1998). Key VKD proteins include coagulation proteins, anticoagulation proteins and bone proteins, in addition to the VKD growth factor growtharrest-specific-6 (Gas6, Table 1; Suttie 1992; Ferland 1998). Calcium binding is essential for the activation of the seven VKD proteins that mediate blood coagulation and anticoagulation. Coagulation factors II (prothrombin), VII, IX and X make up core actors of the coagulation cascade, while proteins C, S and Z belong to the anticoagulation proteins. With the exception of protein S, which is also synthesized by osteoblasts, these proteins are produced exclusively in the liver (Ferland 1998). Blood clotting follows the same fundamental pattern in both mammals and teleosts, generating thrombin by pathways involving VKD factors (see Hanumanthaiah et al. 2002 and Jiang & Doolittle 2003; and references cited therein). In addition to protein S, the VKD proteins found in bone are bone Gla-protein (BGP; synonym for osteocalcin) and matrix Gla-protein, MGP (Vermeer

Table 1 Vitamin K-dependent (VKD) proteins Coagulation proteins

Anticoagulation proteins

Bone proteins Other proteins

Prothrombin (Factor II) Factor VII Factor IX Factor X Protein C Protein S Protein Z Bone Gla Protein Matrix Gla Protein Growth-arrest-specific-6 Gla-rich Protein

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et al. 1995; Ferland 1998). Although the exact role of BGP is not clear, it is suggested to function as a regulator of bone formation and bone mineral maturation (Ducy et al. 1996; Boskey et al. 1998). BGP is produced by osteoblasts and odontoblasts only (Dimuzio et al. 1983). The protein was originally isolated from bovine bone where it was shown to inhibit the formation of hydroxyapatite (Price et al. 1976). Vitamin K is involved in the posttranslational modification of VKD proteins and acts as a cofactor for the enzyme c-glutamylcarboxylase (GGCX). GGCX catalyses the carboxylation of glutamic acid (Glu) residues in VKD proteins resulting in its conversion to c-carboxyglutamic acid (Gla) residues (Stenflo et al. 1974). Although VKD c-carboxylation occurs only on specific Glu-residues in a small number of proteins, it is critical for the functionality of these proteins (Suttie 1992). Both phylloquinone and menaquinones act as co-factors in the GGCX mediated carboxylation (Buitenhuis et al. 1990), where the naphthoquinone ring is the active site for the carboxylation reaction (Shea & Booth 2008). As a first step, vitamin K is reduced to vitamin K hydroquinone (KH2; Fig. 2). The KH2 provides the energy to drive the carboxylation reaction, leading to formation of Gla residues and vitamin K epoxide (KO). KO is subsequently reduced by KO-reductase to vitamin K, in a process commonly called the vitamin K cycle (Ferland 1998; Berkner 2000; Stafford 2005) which conserves the available vitamin K very efficiently. The resulting Gla domain formed from the carboxylation is a calcium-binding amino acid moiety required for the function of VKD proteins. In the presence of calcium ions, these proteins undergo a structural transition leading to the exposure of a phospholipid (membrane) binding site. Vitamin K deficiency leads to the occurrence of undercarboxylated proteins with Glu-residues, and are most often biologically inactive. Lower VKD enzymatic activities or degree of VKD protein carboxylation can be used as markers for suboptimal vitamin K nutrition (Ferland 1998; Vermeer et al. 1998; Furie et al. 1999). Bone Gla-protein missing one or more Gla residues is termed under-carboxylated osteocalcin (ucOC), and the ratio between fully carboxylated and ucOC has been suggested as a sensitive marker for vitamin K deficiency (Vermeer et al. 1995; Ferland 1998). In humans, a correlation between osteoporosis and ucOC has been found (Szulc et al. 1994, 1996). When supplemented with vitamin K, the level of ucOC, bone resorption and urinary calcium secretion is reduced, while bone formation increases (Braam et al. 2003). MGP, originally purified from mammalian bone (Price & Williamson 1985), is a small VKD protein synthesized by osteoblasts and a wide variety of other cells, like

in turbot, Scophthalmus maximus (Roberto et al. 2009). In Atlantic salmon (Salmo salar L.), BGP and MGP are expressed in vertebrae, as well as in fin, gills and scales, confirming the presence of vitamin K in bone, and suggesting involvement of vitamin K in bone metabolism of Atlantic salmon (Krossøy et al. 2009b). The latest addition to the VKD family, is Gla-rich protein isolated from sturgeon (Acipenser nacarii) cartilage. This VKD protein is highly expressed in chondroblasts, chondrocytes, osteoblasts and osteocytes, and is suggested to regulate calcium in the extracellular environment (Viegas et al. 2008).

Figure 2 The vitamin K cycle: The vitamin K-dependent (VKD) c-carboxylation system consists of the vitamin K-dependent enzyme c-glutamylcarboxylase (GGCX) which requires the reduced vitamin K form, vitamin K hydroquinone (KH2), as a cofactor and the enzyme vitamin K 2,3-epoxide reductase (KO-reductase). Vitamin K is reduced to KH2 by KO-reductase. The GGCX converts glutamic acid (Glu) residues in VKD proteins to c-carboxyglutamic acid (Gla) residues by adding CO2 to newly synthesized proteins, using KH2 as a cofactor for the posttranslational reaction. The conversion of KH2 to vitamin K 2,3-epoxide (KO) coincide with the c-carboxylation. The epoxide is subsequently reduced back to vitamin K by KOreductase, ready to enter another cycle. (Enzyme nomenclature adapted from http://www.chem.qmul.ac.uk/iupac/iupac.html).

chondrocytes and vascular smooth muscle cells. It contains five Glu-residues that need modification to Gla for its activation (Schurgers et al. 2007). Animal studies suggest that MGP is a physiological inhibitor of tissue calcification (Luo et al. 1997; Lee et al. 2007; Schurgers et al. 2007), and its gene structure, amino acid sequence and tissue distribution are similar among examined animal species (Laize´ et al. 2005). MGP is also important in chondrocyte differentiation and maturation, regulating endochondral and intramembranous ossification (Luo et al. 1997; Newman et al. 2001). As in mammals, studies have shown that MGP expression and function is associated with regulation of mineralization

Bone and spinal deformities represent a recurring problem for commercial fish farming, and have raised ethical concerns in animal welfare issues in recent years. Suggested risk factors are nutrition, genetics, environment, vaccination and fast growth (Waagbø et al. 2005; Waagbø 2008). The importance of vitamin K in bone health has been established in mammals (Vermeer et al. 1995, 1996; Shearer et al. 1996; Booth 1997; Ferland 1998), and the interest in vitamin K requirement for normal bone development in fish has recognized that the vitamin K supply may be suboptimal for bone but sufficient to maintain normal growth and prevent mortality (Udagawa 2000). To date, there is no information on the form or the levels of vitamin K required to achieve optimal bone health neither in humans nor in fish. Only a few reports have dealt with the impact of vitamin K deficiency on fish bone health (Udagawa 2001, 2004; Graff et al. 2002; Roy & Lall 2007; Krossøy et al. 2009a). Studies on mummichog (Fundulus heteroclitus) larvae have shown that diets without vitamin K supplementation caused a higher incidence of deformities in the vertebrae and caudal skeleton (Udagawa 2001). Further, the effect of parental vitamin K deficiency on bone structure was examined in the developing mummichog larvae (Udagawa 2004). The author concluded that the offspring from fish fed a vitamin K deficient diet had abnormal vertebral formation 5 days posthatching compared to larvae from fish fed a vitamin K rich diet with significantly lower incidences of malformations. More specifically, vitamin K deficiency caused the formation of thin and weak bone, and induces bone structure abnormalities such as vertebral fusion and row irregularity, both in early development and during later growth in mummichog (Udagawa 2001, 2004). Radiological and histological findings in haddock (Melanogrammus aeglefinus L.), however, showed that vitamin K deficiency decreased bone mineralization and increased the occurrence of bone deformities, without affecting the number of

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osteoblasts (measured by histomorphometry) in the vertebrae. This indicates that vitamin K is necessary for bone mineralization in haddock (Roy & Lall 2007). Investigations of bone health, performed by mechanical testing and radiological and/or visual examination, revealed no signs of vertebral deformities in juvenile Atlantic salmon (Krossøy et al. 2009a) and Atlantic salmon smolts (Graff et al. 2002) given an un-supplemented diet. Moreover, neither phylloquinone nor MK-4 were detected in samples of vertebrae (Graff et al. 2010), but both bgp and mgp were expressed in vertebrae, gills and pectoral fin as analysed by in situ hybridization and qPCR (Krossøy et al. 2009a,b). Furthermore, gene expression of ggcx was found in vertebrae, scales, operculum and fin of adult Atlantic salmon, indicating GGCX activity in bony tissues of Atlantic salmon (Krossøy et al. 2010). Although the exact role of the VKD bone proteins BGP and MGP remains unknown, they may be important in regulation of bone growth (Dimuzio et al. 1983; Boskey et al. 1998). Together these latter results suggested the involvement of vitamin K in bone metabolism of Atlantic salmon (Krossøy et al. 2009b).

Lately, studies in mammals have proposed multiple roles of vitamin K beyond coagulation that are both dependent and independent of its classical role as an enzyme cofactor, as reviewed by Booth (2009). A novel mechanism of vitamin K function in transcriptional regulation of osteoblastic cells was demonstrated by Tabb et al. (2003), showing that menaquinone is a transcriptional regulator of bone markers, such as alkaline phosphatase and MGP in osteoblastic cells. It has been shown that menaquinone is a ligand for the nuclear pregnance X receptor (PXR; also known as steroid xenobiotic receptor or SXR), suggesting a role of menaquinone in regulation of bone homoeostasis (Tabb et al. 2003; Zhou et al. 2009) and collagen formation (Ichikawa et al. 2006). Menaquinones potentially contribute to improved bone quality by gene regulation (Ichikawa et al. 2006; Horie-Inoue & Inoue 2008) in addition to its role as an enzymatic co-factor. Gas6 is involved in regulating cell survival and proliferation, and protecting against cellular apoptosis (see review by Hafizi & Dahlba¨ck 2006). Gas6 is found throughout the nervous system, as well in the heart, lungs, stomach, kidneys and cartilage (Ferland 1998; Hafizi & Dahlba¨ck 2006). It affects vascular smooth muscle cell movement and apoptosis (Danziger 2008), and appears to play important physiological roles in inflammation, energy metabolism, renal disease, sepsis and neoplasia (Manfioletti et al. 1993; Arai et al. 2008; Booth 2009).

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Lastly, a role of vitamin K in prevention of oxidative damage of the brain and sphingolipid synthesis has been suggested, as reviewed by Shearer & Newman (2008).

The minimum requirements given by NRC (1993) are primarily determined for small fish and the studies are performed under optimal experimental conditions, using purified, synthetic or semi-synthetic diets produced under conditions causing minimal losses. These studies and requirements are obviously not valid for commercial conditions using practical diets. Normally, most vitamins are supplemented at levels above the NRC minimum requirements to compensate for factors influencing the vitamin level. Thus, practical vitamin allowances correct for losses under feed production and storage (Marchetti et al. 1999), and should take into consideration the bioavailability of vitamin forms, challenging rearing conditions and the developmental stage of the fish (Hamre et al. 2010). Practical dietary vitamin K recommendations given for optimum health and productivity of farmed fish are therefore often several folds above the minimum requirement. In fish, typical vitamin K deficiency signs include increased blood coagulation time, reduced growth, anaemia, haemorrhages, loss of fin tissue, weak bones, and occurrence of spinal curvature, short tails and increased mortality (Taveekijakarn et al. 1996; Udagawa 2004; Lall & Lewis-McCrea 2007). The earliest vitamin K requirement studies in fin fish were based on increased blood coagulation time and mortality as the primary criteria (Kitamura et al. 1967; Poston 1976; Murai & Andrews 1977). Studies with vitamin K deficient feed caused no detectable deficiency symptoms in rainbow trout (Kitamura et al. 1967) and channel catfish, Ictalurus punctatus (Murai & Andrews 1977), but reduced growth and increased mortality in amago salmon, Oncorhynchus rhodurus (Taveekijakarn et al. 1996), and increased mortality in mummichog (Udagawa & Hirose 1998). Lately, more sensitive biomarkers have been used. As the major function of vitamin K is to act as co-factor for GGCX, the activity of this enzyme may provide a biomarker for deficiency. Results from recent studies in juvenile Atlantic salmon confirmed that GGCX activity is a sensitive marker for evaluating vitamin K status and intake (Krossøy et al. 2009a, 2010). However, altered enzyme activity does not necessarily represent a deficiency state and because there were no indications of deficiency in any of the other parameters measured, Krossøy et al. (2009a) concluded that the minimum requirement in salmon juveniles was at, or less

Table 2 Overview over published vitamin K requirement and recommendations in fish (1970–2011)

Fish species

Response criteria

Diet

K vitamer

Lake trout Salmonids Atlantic cod

Haematology, coagulatin time Growth Mortality, haematology, coagulatin time Growth, mortality Growth, mortality Growth Growth, bone health Growth, coagulation time, bone health

Semisynthetic Purified Practical

n.g. K1 n.g.

Semipurified/Practical Semipurified/Practical

K3 K3 n.g. K3 K1

Salmonids European seabass Salmonids Haddock Atlantic salmon

Semipurified Practical

Recommendations/ Requirement (mg kg)1 feed) 0.5–1 0.45 0.2 1.5 1.5 10* 20 0.1

Reference Poston (1976) Woodward (1994) Grahl-Madsen & Lie (1997) Kaushik et al. (1998) Kaushik et al. (1998) Halver (2002) Roy & Lall (2007) Krossøy et al. (2009b)

n.g.,not given; *, vitamin recommendation for growth.

than, the basal level of phylloquinone found in the diets (0.1 mg kg)1 feed). This is comparable to the study of Graff et al. (2002) where the basal level of vitamin K in the diet for Atlantic salmon was 0.06 mg phylloquinone kg)1 feed, and where no signs of deficiency were recorded. Current estimates of dietary vitamin K requirement differ in what is considered adequate levels in the feeds for fish (Table 2). In NRC (1993) recommendation, the minimum requirement for growing lake trout (Salvelinus namaycush) is 0.5–1 mg vitamin K kg)1 diet (based on Poston 1976), while in Halver (2002) the vitamin K recommendation for growth in trout and salmon is 10 mg kg)1 diet. A previous comprehensive review of vitamin requirement studies in fish suggested that vitamin K concentrations equivalent to 0.45 mg phylloquinone kg)1 feed might be sufficient for salmonid fish (Woodward 1994). In addition, Kaushik et al. (1998) showed that supplementation of practical diets with 1.5 mg menadione kg)1 was sufficient to maintain growth and prevent deficiency signs in juvenile rainbow trout (Oncorhynchus mykiss), Chinook salmon (Oncorhynchus tschawytscha) and European seabass (Dicentrachus labrax). In the same period, Grahl-Madsen & Lie (1997) suggested that