The generic term vitamin K is now used to describe not a single chemical entity, but a group of quinone compounds that have characteristic anti-hemorrhagic effects and are fat soluble. The basic molecule is naphthoquinone, and the various isomers differ in the nature and length of the side chain. Vitamin K extracted from plant material was named phylloquinone or vitamin K1 (Illus. 5-1). Vitamin K-active compounds from material that has undergone bacterial fermentation were named menaquinones or vitamin K2. The simplest form of vitamin K is synthetic menadione (K3), which has no side chain. Menaquinone is synthesized in liver from ingested menadione or changed to a biologically active menaquinone by intestinal microorganisms. Rumen microorganisms synthesize large quantities of vitamin K2, and the vitamin K content of cow’s milk is reportedly 10 times higher than that of monogastric species.
Vitamin K1 is a golden yellow, viscous oil. Naturally occurring sources of vitamin K are fat soluble; stable to heat; and labile to oxidation, alkali, strong acids, light and irradiation. Vitamin K1 is slowly degraded by atmospheric oxygen, but fairly rapidly destroyed by light. In contrast to naturally occurring sources of vitamin K, vitamin K3salts of menadione are water soluble.Vitamin K antagonists increase the need for this vitamin. Deficiency of this vitamin is produced by ingestion of the antagonist, dicumarol, or by feeding of sulfonamides (in monogastric species) at levels sufficient to inhibit intestinal synthesis of vitamin K.
Supplementation of vitamin K will overcome the anticoagulation effect of dicumarol. Mycotoxins are also antagonists that may cause vitamin K deficiency. Like all fat-soluble vitamins, vitamin K is absorbed in association with dietary fats and requires the presence of bile salts and pancreatic juice for adequate uptake from the alimentary tract. Absorption of vitamin K depends on its incorporation into mixed micelles, and optimal formation of these micellar structures requires the presence of both bile and pancreatic juice. Thus, any malfunction of the fat absorption mechanism (e.g., biliary obstruction) reduces availability of vitamin K (Ferland, 2006). Unlike phylloquinone and the menaquinones, menadione salts are relatively water soluble, and, therefore, are absorbed satisfactorily from low-fat diets. Male animals are more susceptible to dietary vitamin K deprivation than females, apparently as a result of a stimulation of phylloquinone absorption by estrogens; the administration of estrogens increases absorption in both male and female animals (Jolly et al., 1977).
The lymphatic system is the major route of transport of absorbed phylloquinone from the intestine. Shearer et al.(1970) demonstrated the association of phylloquinone with serum lipoproteins, but little is known of the existence of specific carrier proteins. Ingested phylloquinone is absorbed by an energy-dependent process from the proximal portion of the small intestine (Hollander, 1973). In contrast to the active transport of phylloquinone, menaquinone is absorbed from the small intestine by a passive noncarrier-mediated process.
The measured efficiency of vitamin K absorption ranges from 10% to 70%, depending on the form of the vitamin administered. Some reports have indicated that menadione is completely absorbed, but phylloquinone is absorbed only at a rate of 50%. The complete absorption of menadione may be due to aqueous solubility of the menadione salts. Rats were found to excrete about 60% of ingested phylloquinone in the feces within 24 hours of ingestion, but only 11% of ingested menadione (Griminger and Donis, 1960; Griminger, 1984). However, 38% of ingested menadione, but only a small amount of phylloquinone, was excreted via the kidneys during the same period. The conclusion was that although menadione is well absorbed, it is poorly retained, while the opposite is true for phylloquinone. Normal human subjects were found to excrete less than 20% of a large (1 mg) dose of phylloquinone in the feces, but as much as 70%-80% of the ingested phylloquinone was excreted unaltered in the feces of patients with impaired fat absorption caused by obstructive jaundice, pancreatic insufficiency, or adult celiac disease (Suttie, 2007).
Griminger and Brubacher (1966) showed that a major portion of the phylloquinone fed to chicks was absorbed and deposited in the liver intact and, as such, had equally as good biological activity upon prothrombin synthesis as menaquinone found in the chick’s liver following feeding of menadione. Therefore, menaquinone is most likely produced if menadione is fed or if the intestinal microorganisms degrade the dietary K1 or K2 to menadione. Formation of menaquinone is not required for metabolic activity of vitamin K, since phylloquinone is equally active in synthesis of the vitamin K-dependent, blood-clotting proteins (Scott et al., 1982).
A number of studies have shown that phylloquinone is specifically concentrated and retained but menadione is poorly retained in the liver. Menadione is found to be widely distributed in all tissues and to be very rapidly excreted. Although phylloquinone is rapidly concentrated in liver, it does not have a long retention time in this organ (Thierry et al., 1970). The inability to rapidly develop a vitamin K deficiency in most species results, therefore, from the difficulty in preventing absorption of the vitamin from the diet or from intestinal synthesis rather than from a significant storage of the vitamin. Some breakdown products of vitamin K are excreted in the urine. One of the principal excretory products is a chain-shortened and oxidized derivative of vitamin K, which forms gamma-lactone and is probably excreted as a glucuronide.
Vitamin K dependent proteins can be identified by γ-carboxyglutamic acid residues (Gla). This amino acid is common to all vitamin K proteins. Discovery of this new amino acid clarified the role of vitamin K in blood coagulation and led to the discovery of additional vitamin K-dependent proteins (e.g., bone proteins) (Ferland, 2006; Suttie, 2007). Coagulation time of blood is increased when vitamin K is deficient because the vitamin is required for the synthesis of prothrombin (factor II). Plasma clotting factors VII (proconvertin), IX (Christmas factor) and X (Stuart-Prower factor) also depend on vitamin K for their synthesis. These four blood-clotting proteins are synthesized in the liver in inactive precursor forms and then converted to biologically active proteins by the action of vitamin K (Suttie and Jackson, 1977). In deficiency, administration of vitamin K brings about a prompt response in four to six hours. In the absence of the liver, this response does not occur. Bleeding disorders result from an inability of a liver microsomal enzyme, currently called the vitamin K-dependent carboxylase, to carry out the normal post-translational conversion of specific glutamyl residues (Suttie, 2007). The result of insufficient vitamin K to serve as a cofactor for this enzyme is, therefore, a decrease in the rate of thrombin generation.
Two of the best characterized vitamin K-dependent proteins not involved in hemostasis are osteocalcin or bone Gla protein (BGP) and matrix Gla protein, which were initially discovered in bone. Osteocalcin is a protein containing three Gla residues that give the protein its mineral-binding properties. Osteocalcin appears in embryonic chick bone and rat bone matrix at the beginning of mineralization of the bone (Gallop et al., 1980). It accounts for 15% to 20% of the non-collagen protein in the bone of most vertebrates and is one of the most abundant proteins in the body. Osteocalcin is produced by osteoblasts, with synthesis controlled by 1,25-dihydroxy vitamin D3, about 20 percent of the newly synthesized protein is released in the circulation and can be used as a measure of bone formation. Matrix Gla protein-deficient mice have abnormal calcification leading to osteopenia, fractures and premature death owing to arterial calcification (Booth and Mayer, 1997).
As is true for other non-blood vitamin K-dependent proteins, the physiological role of osteocalcin remains largely unknown. However, reduced osteocalcin content of cortical bone (Vanderschueren et al., 1990) and alteration of osteocalcin distribution within osteons (Ingram et al., 1994) are associated with aging. It remains unknown whether any of these findings are related to the age-related increased risk of fracture. Osteocalcin may play a role in the control of bone remodeling because it has been reported to be a chemoattractant for monocytes, the precursors of osteoclasts. Serum osteocalcin has been shown to be a good predictor of bone turnover in pigs (Carter et al., 1996). This suggests a possible role for osteocalcin in bone resorption (Binkley and Suttie, 1995). Several reports have indicated that warfarin treatment alters the functional properties of bone particles prepared from rats. However, vitamin K-deficient chick embryos were able to mobilize sufficient quantities of calcium for normal skeletal development, although they exhibited severe reduction in blood clotting and bone osteocalcin concentration (Lavelle et al., 1994).
Observations that vitamin K could be involved in the pathogenesis of bone mineral loss have been summarized (Binkley and Suttie, 1995; Cashman and O’Connor, 2008): (1) low blood vitamin K in patients with bone fractures; (2) concentration of circulating gamma-carboxylated osteocalcin associated with age, low bone mineral density and hip fracture risk; (3) anticoagulant therapy associated with decreased bone density and (4) decreased bone loss and calcium excretion with vitamin K supplementation.
Another vitamin K-dependent protein is Gas 6; the function of this protein has a possible role in nervous system function, vascular cell function and platelet activation (Suttie, 2007). Both in vitro and in vivo studies to date suggest a role of vitamin K in the regulation of multiple enzymes involved in sphingolipid metabolism with the myelin-rich regions in the brain (Denisova and Booth, 2005). The brain is enriched with sphingolipids, which are important membrane constituents and major lipid signaling molecules that have a role in motor and cognitive behavior.
The action of converting inactive precursor proteins to biologically active forms involves the carboxylation of glutamic acid residues in the inactive molecules. The action of converting inactive precursor proteins to biologically active forms involves the carboxylation of glutamic acid residues in the inactive molecules. Carboxylation allows prothrombin and the other procoagulant proteins to participate in a specific protein-calcium phospholipid interaction that is necessary for their biological role (Suttie and Jackson, 1977). Four other vitamin K-dependent proteins have also been identified in plasma (i.e., proteins C, S, Z and M). Protein C and protein S play an anticoagulant rather than a procoagulant role in normal hemostasis (Suttie and Olson, 1990). Protein C inhibits coagulation, and stimulated by protein S, it promotes fibrinolysis. Also, a protein C-S complex can partially hydrolyze the activated factors V and VIII and thus inactivate them. Protein S also has the potential to be involved in the regulation of bone turnover (Binkley and Suttie, 1995). Functions for proteins M and Z are unclear. Protein Z has been shown to have an anticoagulant function under some conditions (Suttie, 2007). Menaquinone, when given to humans at pharmacological doses, appears to protect against fracture risk and bone loss at the spine (Shea and Booth, 2008).
Two of the best characterized vitamin K-dependent proteins not involved in hemostasis are osteocalcin or bone Gla protein (BGP) and matrix Gla protein, which were initially discovered in bone. Osteocalcin is a protein containing three Gla residues that give the protein its mineral-binding properties. Osteocalcin appears in embryonic chick bone and rat bone matrix at the beginning of mineralization of the bone (Gallop et al., 1980). It accounts for 15% to 20% of the non-collagen protein in the bone of most vertebrates and is one of the most abundant proteins in the body. Osteocalcin is produced by osteoblasts, with synthesis controlled by 1,25-dihydroxy vitamin D3, about 20 percent of the newly synthesized protein is released in the circulation and can be used as a measure of bone formation. Matrix Gla protein-deficient mice have abnormal calcification leading to osteopenia, fractures and premature death due to arterial calcification (Booth and Mayer, 1997).
As is true for other non-blood vitamin K-dependent proteins, the physiological role of osteocalcin remains largely unknown. However, reduced osteocalcin content of cortical bone (Vanderschueren et al., 1990) and alteration of osteocalcin distribution within osteons (Ingram et al., 1994) are associated with aging. It remains unknown whether any of these findings are associated to the age-related increase in fracture risk. Osteocalcin may play a role in the control of bone remodeling because it has been reported to be a chemoattractant for monocytes, the precursors of osteoclasts. Serum osteocalcin has been shown to be a good predictor of bone turnover in pigs (Carter et al., 1996). This suggests a possible role for osteocalcin in bone resorption (Binkley and Suttie, 1995). Several reports have indicated that warfarin treatment alters the functional properties of bone particles prepared from rats. However, vitamin K-deficient chick embryos were able to mobilize sufficient quantities of calcium for normal skeletal development, although they exhibited severe reduction in blood clotting and bone osteocalcin concentration (Lavelle et al., 1994).
Bleeding disorders result from an inability of a liver microsomal enzyme, currently called the vitamin K-dependent carboxylase (Esmon et al., 1975), to carry out the normal post-translational conversion of specific glutamyl residues in the vitamin K-dependent plasma proteins to gamma-carboxyglutamyl residues (Nelsestuen et al., 1974). The result of insufficient vitamin K to serve as a cofactor for this enzyme is, therefore, a decrease in the rate of thrombin generation.
The blood clotting mechanism can apparently be stimulated by either an intrinsic system, in which all the factors are in the plasma, or an extrinsic system. In the extrinsic system of coagulation, injury to the skin or other tissue frees tissue thromboplastin that in the presence of various factors and calcium changes prothrombin in the blood to thrombin. The enzyme thrombin facilitates the conversion of the soluble fibrinogen into insoluble fibrin. Fibrin polymerizes into strands and enmeshes the formed elements of the blood, especially the red blood cells, to form the blood clot (Griminger, 1984). The final active component in both the intrinsic and extrinsic systems appears to activate the Stuart factor, which in turn leads to activation of prothrombin. The various steps involved in blood clotting are presented in Figure 5-1. The action of vitamin K is required at four different sites in these reactions.
The vitamin K requirement of mammals is met by a combination of dietary intake and microbial biosynthesis in the gut, which may involve intestinal microorganisms (such as Escherichia coli). Animals that practice some degree of coprophagy, such as the pig, can utilize much of the vitamin K that is eliminated in the feces. In rats, the majority of menaquinone absorbed resulted from fecal ingestion compared to dietary sources or from direct synthesis and absorption from the intestine (Kindberg et al., 1987). Because of microbial synthesis, a precise expression of vitamin K requirements is not feasible. However, attempts to determine the contribution of microbial synthesis have been made. In conventional rats, the vitamin requirement is 0.05 to 0.10 mg per kg (0.023 to 0.045 mg per lb) of diet, whereas in germ-free rats the requirement is more than doubled to about 0.25 mg per kg (0.11 mg per lb) (Suttie and Olson, 1984). Kindberg and Suttie (1989) reported that more than 500 mg of phylloquinone per kg (227 mg per lb) of diet was required to prevent one of the most sensitive signs of vitamin K-deficiency, activity of liver vitamin K-dependent carboxylase. Animal feces contain substantial amounts of vitamin K even when none is present in feed. Despite the intestinal synthesis, animals can be rendered deficient when fed vitamin K-free diets and coprophagy is prevented (e.g., germ-free animals) or if a vitamin K antagonist is given. Difficulties in demonstrating dietary requirement in many species include the varying degrees to which they utilize vitamin K synthesized by intestinal bacteria and the degree to which different species practice coprophagy.
Rapid rate of food passage through the digestive tract may also influence vitamin K synthesis in the pig. Swine are able to obtain more benefit from vitamin K intestinal synthesis than are poultry. First defecation in pigs, for a specific portion of diet, may occur about 15 hours after feeding, but most of a given meal will be retained in the tract appreciably longer. A comparable time period for chickens would be approximately three hours (Griminger, 1984b); consequently, less vitamin K synthesis and absorption would be expected.
The daily requirement for most species falls in a range of 2 to 200 µg vitamin K per kg (0.91 to 91 µg per lb) of body weight. Schendel and Johnson (1962) established a daily dietary vitamin K requirement of 5 µg of menadione sodium phosphate per kg (2.3 µg per lb) of body weight in young pigs fed a purified diet containing a high level of sulfathiazole to preclude gut synthesis of vitamin K2. Hall et al. (1986) reported that the dietary vitamin K requirement of growing pigs was estimated not to be greater than 2 mg per kg. In a later study, Campbell and Combs (1988) reported no improvement on starter pig performance when 3 mg per kg (1.4 mg/lb) of vitamin K was added. The Agricultural Research Council (ARC, 1981) estimated the dietary vitamin K requirement of menadione to be 200 to 300 mg per kg (91 to 136 mg per lb) of diet dry matter. It should be remembered that this requirement can be altered by sex, age, strain, anti-vitamin K factors, disease conditions and any condition influencing lipid absorption or altering intestinal flora. The dietary vitamin K requirement estimated for all classes of swine is 0.50 mg per kg (0.23 mg per lb) of diet (NRC, 1998). Studies have not been conducted to determine whether a supplemental source of vitamin K is beneficial for the breeding herd (NRC, 1998).
There are two major sources of vitamin K: phylloquinones (vitamin K1) in plant sources and manaquinones (vitamin K2) produced by bacterial flora.
Vitamin K is present in fresh dark-green vegetables. Most feedstuffs of plant origin containing very high levels of vitamin K are not usually fed to swine in confinement. They are receiving diets based on grains and oilseed meals. Alfalfa leaf meal contains a small amount of vitamin K, while liver and fish meal are good animal sources of the vitamin. All by-product feedstuffs of animal origin, including fish meal and fish liver oils, are much higher in vitamin K after they have undergone extensive bacterial putrefaction.
The menaquinones (vitamin K2) are produced by the bacterial flora in animals and are especially important in providing the vitamin K requirements of mammals. Type of diet, independent of vitamin K concentration, will influence total menaquinone synthesis. Rats fed a diet based on boiled white rice had less menaquinone production and more severe vitamin K deficiency than animals consuming diets based on autoclaved black-eye beans (Mathers et al., 1990). In non-ruminants, site of synthesis is in the lower gut, an area of poor absorption, and thus availability to the host is limited unless the animal practices coprophagy, in which case the synthesized vitamin K is highly available.
Vitamin K1 is not utilized by the feed industry, due to cost and lack of a stabilized form. Instead water-soluble menadione (vitamin K3) salts are used to provide vitamin K activity in feeds. Because of poor stability, menadione is not used as the pure vitamin but is produced as water-soluble salts. Water-soluble derivates of menadione, including menadione sodium bisulfite (MSB), menadione sodium bisulfite complex (MSBC) and menadione dimethylpyrimidinol bisulfite (MPB), are the principal forms of vitamin K included in commercial diets. The menadione concentration is 50% for MSB followed by 45.4% for MPB and 33% for MSBC (Schneider, 1986). Sometimes MSB is coated with gelatin to increase stability, resulting in a 25% menadione activity.
In considering vitamin K deficiency, Kindberg and Suttie (1989) noted that vitamin K deficiency is strongly dependent upon the assay used. The authors indicated that direct measurements of prothrombin concentration or alterations in liver vitamin K-dependent carboxylase activity are more sensitive and more readily quantitated criteria of sufficiency than one-stage prothrombin times.The major clinical sign of vitamin K deficiency in all species is impairment of blood coagulation. Other clinical signs include low prothrombin levels, increased clotting time and hemorrhaging. In its most severe form, a lack of vitamin K will cause subcutaneous and internal hemorrhages, which can be fatal. Vitamin K deficiency can result from dietary deficiency, lack of microbial synthesis within the gut, inadequate intestinal absorption or inability of the liver to use the available vitamin K.Schendel and Johnson (1962) were able to produce a vitamin K deficiency in the baby pig by using a sulfa drug and an antibiotic and by carefully minimizing coprophagy by cleaning the feces from wire bottom cages where pigs were housed. Likewise, hemorrhagic disease occurred in piglets weaned at five to six weeks of age within two weeks after transfer onto flat decks (Hoppe, 1987). This vitamin K-responsive outbreak had occurred because of (1) the diet being virtually free of vitamin K; (2) dietary inclusion of sulfonamide, a known vitamin K antagonist; and (3) flat deckhousing, which precludes intake of microbial vitamin K from feces or litter.
Hall et al. (1991) accidentally produced vitamin K deficiency characterized by increased blood clotting times and high death losses when feeding excess calcium (2.7%) without supplemental vitamin K. Either supplemental vitamin K or reduced dietary calcium prevented the disease condition.
Clinical and subclinical signs of vitamin K deficiency include both increased prothrombin and blood-clotting time, internal hemorrhage, and anemia due to blood loss (Seerley et al., 1976; Cunha, 1977; Newsholme et al., 1985). Newborn pigs may be pale with loss of blood from the umbilical cord. Until recently, vitamin K deficiency under natural conditions was not expected, as it was thought that the pig synthesized most if not all of the vitamin that was required. However, in the late 1960s and early 1970s there were prevalent reports of a bleeding disease of young pigs on commercial diets that was successfully overcome by vitamin K supplementation. Observations from Australia and New Zealand were of hemorrhaging in the navel of newborn pigs (Cunha, 1977).
Pigs placed on a high-sugar diet suffered heart lesions and hemorrhagic syndrome, which could be prevented by provision of vitamin K (Brooks et al., 1973). A number of field trials in the United States have reported a hemorrhagic syndrome for growing pigs. In one study, hemorrhagic syndrome occurred nine days after pigs were fed a standard diet, while those receiving either 2.5% dehydrated alfalfa meal or supplemental vitamin K remained in good health (Fritschen et al., 1970). Gross visible signs for hemorrhagic syndrome include large subcutaneous hemorrhages, blood in the urine and abnormal breathing. Additional clinical signs from field observations are that some pigs will develop enlarged blood-filled joints and become lame, whereas others may have swellings along the body wall that are filled with unclotted blood. Hematomas (or blood swellings) in the ears also occur (Cunha, 1977). Hemorrhagic conditions in the growing pig have in some cases been associated with ingestion of molds, such as aspergillus or moldy materials, and have usually responded to vitamin K therapy.
The exact causes of more recent needs for vitamin K supplementation are not definitely known. Cunha (1977) and Scott et al. (1982) have summarized likely reasons for vitamin K deficiency under field conditions:
As long as natural dietary vitamin K sources (i.e., green leafy plants) are sufficiently high and (or) bacterial synthesis in the intestinal tract remains functional, the need for supplemental vitamin K is not required. However, rich sources of vitamin K such as green leafy plants are not usually fed to swine under today’s intensive confinement systems. An exception is alfalfa leaf meal, which sometimes is included in small amounts in swine feed. Therefore, a source of vitamin K needs to be added to the diets of swine since they are not getting sufficient fresh greens or their dried equivalent and may not synthesize sufficient amounts of this vitamin in their gastrointestinal tract. Vitamin K antagonists will increase the vitamin K needs of livestock. Even calcium can be an antagonist as excess concentrations of the mineral may increase the pig’s requirement for vitamin K (Hall et al., 1985). Data from Brooks et al.(1973) were reported to suggest a need for vitamin K supplementation in high-sugar starter diets. In adjusting dietary vitamin K fortification levels, an appropriate margin of safety is needed to prevent deficiency and allow optimum performance in swine. Vitamin K antagonists include the use of certain antibiotics and sulfa drugs. Sulfonamides and broad-spectrum antibiotic drugs can virtually sterilize the lumen of the intestine (McDowell, 2000).The level of supplemental vitamin K should be adequate to meet the requirements under the wide variety of stress conditions encountered in practical swine production. Squibb (1964) obtained increased prothrombin times, indicating a higher vitamin K requirement in chicks during the early stages of Newcastle disease. Field reports with swine indicate that hemorrhaging in stressed animals occurs at birth in the navel and following castration. Various reports indicate that levels of 2 to as high as 16 gm vitamin K per ton (2.2 to 17.6 mg per kg; 1.0 to 8.0 mg per lb) of feed were needed because lower levels were not effective under certain farm conditions (Cunha, 1977).
Stability of the naturally occurring sources of vitamin K is poor. However, stability of the water-soluble menadione salts (e.g. MSB or MSBC) is satisfactory in multivitamin premixes unless trace minerals are present (Frye, 1994). Basic pH conditions also accelerate the destruction of menadione salts, thus soluble or slightly soluble basic mineral substances should not be included in multivitamin premixes containing menadione. Stability of vitamin K3 derivatives is likewise impaired by moisture and choline chloride in feeds and premixes. Choline chloride is particularly destructive to vitamin K (Coelho, 1991). Heat, moisture and trace minerals increase the rate of destruction of menadione salts in both pressure-pelleted and extruded feeds (Hoffmann-La Roche, 1981). For these reasons, greater quantities of vitamin K3are recommended in premixes that contain large quantities of choline chloride and certain trace minerals and especially when premixes are exported or stored for an extended period of time (Schneider, 1986).
Toxic effects of the vitamin K family are manifest mainly as hematologic and circulatory derangements. Not only is species variation encountered, but profound differences are observed in the ability of the various vitamin K compounds to evoke a toxic response (Barash, 1978). The natural forms of vitamin K, phylloquinone and menaquinone, are nontoxic at very high dosage levels. The synthetic menadione compounds, however, have shown toxic effects when fed to humans, rabbits, dogs and mice in excessive amounts. The toxic dietary level of menadione is at least 1,000 times the dietary requirement (NRC, 1998). Menadione compounds can safely be used at low levels to prevent the development of a deficiency, but should not be used as a pharmacologic treatment for a hemorrhagic condition. In studies with various monogastric species the major effect of toxic levels of menadione was hemolytic anemia, with a high mortality rate (NRC, 1998).
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