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 (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 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 a necessity for their biological role (Suttie and Jackson, 1977). Four other vitamin K-dependent proteins have been more recently 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). Function for proteins M and Z is presently unknown. 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.
Continuing research has revealed that vitamin K-dependent reactions are present in most tissues and not just blood, and that a reasonably large number of proteins are subjected to this post-translational carboxylation of specific glutamate residues to gamma-carboxyglutamate residues (Vermeer, 1986). Atherocalcin is a vitamin K-dependent protein in atherosclerotic tissue. A vitamin K-dependent carboxylase system has been identified in skin, which may be related to calcium metabolism in skin (de Boer-van den Berg et al., 1986). 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 D. 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 (Price, 1993). 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 under 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.
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 within 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.
Figure 5-1: The Mechanism of Blood Clotting
Adapted from Scott et al., (1982) and McDowell (1989)
Vitamin K requirements of ruminants are met by a combination of dietary intake and microbial biosynthesis in the rumen and intestines that may involve intestinal microorganisms such as Escherichia coli. Ruminal microorganisms in particular synthesize large amounts of vitamin K, which explains why ruminants do not appear to need a dietary source of the vitamin except during coumarin toxicity. The ruminant can absorb considerable amounts of ruminally synthesized vitamin K in the small intestine via active transport. In dairy calves fed milk replacer, there was no evidence of vitamin K deficiency based on prothrombin times, but the authors noted that supplementary vitamin K was observed to have health benefits in pre-ruminant calves, warranting further study (Nestor, Jr. and Conrad, 1990). Additionally liver, spleen and intestinal tissue levels of menaquinone-4 were increased significantly by feeding 8 or 16 mg menadione sodium bisulfate complex (MSBC) per kg (3.6 or 7.3 mg/lb) of diet dry matter (Nestor, Jr. and Conrad, 1990). Due to rumen and intestinal microbial synthesis, quantifying a precise vitamin K requirement for ruminants is difficult. Rapid rate of passage (e.g., diarrhea) through the digestive tract may also influence vitamin K synthesis in cattle. The daily requirement of most species falls in a range of 2 to 200 µg vitamin K per kg (0.91 to 91 µg per lb) body weight. It should be remembered that this requirement can be altered by age, sex, stress, anti-vitamin K factors such as coumarin or T-2 toxin, disease conditions and any condition which impairs lipid absorption, alters intestinal flora or interferes with liver function.
The two primary natural sources of vitamin K are phylloquinone (vitamin K1) from plants and menaquinones (vitamin K2) produced by bacterial flora. Vitamin K derived from bacteria would be considered the most important source for ruminants because large quantities of vitamin K are normally available from rumen synthesis.
Vitamin K is present in fish and, dark-green vegetation, especially leaves. It is abundant in pasture and green roughages, thus providing high quantities of vitamin K to grazing livestock. Feedlot animals would, however, receive little vitamin K from finishing diets which are based on grains and oilseed meals.
Sunlight is important for vitamin K formation. Non-chlorophyll-producing plant components contain little vitamin K, but the natural loss of chlorophyll from leaves in the fall does not cause a loss of vitamin K activity. Alfalfa meal is a good plant source of vitamin K, while liver and fish meal are good animal sources. All animal by-products, including fish meal and fish liver oils, are much higher in vitamin K after they have undergone extensive bacterial putrefaction.
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 (Table 5-1). Pure menadione is not used because of poor stability and handling characteristics. Water-soluble derivatives of menadione, including MSB, MSBC and MPB, are the principal forms of vitamin K included in commercial diets. The menadione concentration is 50% for MSB, 45.5% for MPB and 33% for MSBC (Table 5-1) (Schneider, 1986). A more recently developed source of vitamin K is a complex of menadione and nicotinamide- menadione nicotinamide bisulphate (MNB)- that is 43.7% menadione and 31.2% nicotinamide.
The major clinical sign of vitamin K deficiency in all species is impaired blood coagulation (Griminger, 1984b). Other clinical signs include low plasma 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. Microorganisms in the rumen synthesize large amounts of vitamin K, and a deficiency is seen only in the presence of a metabolic antagonist, such as dicumarol from moldy sweet clover (Melilotus officinalis; M. alba). Dicumarol is a fungal metabolite produced from substrates in sweet clover hay, which is common in the Northern Plains of the United States and in Canada. The coumarins in fresh sweet clover are not active because they are bound to glycosides. They are activated when sweet clover is improperly cured (Vermeer, 1984). This condition, referred to as “sweet clover poisoning” or “hemorrhagic sweet clover disease,” has been responsible for a large number of animal deaths. Affected animals can die from hemorrhage following a minor injury, or even from apparently spontaneous bleeding. Dicumarol passes through the placenta in pregnant animals, and newborn animals may become affected immediately after birth. All species of animals studied have been shown to be susceptible, but cases of poisoning have involved mainly cattle and, to a very limited extent, sheep. Anti-vitamin K toxicity has been observed in sheep fed Ferula communis brevifolia powder (Tligui et al., 1994), and in cattle fed sweet vernal (Anthoxanthum odoratum) hay (Pritchard et al., 1983). A low-coumarin variety of sweet clover (Melilotus dentata) is available for use as forage.Clinical signs of dicumarol poisoning relate to the hemorrhages caused by failure of blood coagulation. First appearance of clinical disease varies greatly and depends to a large extent on dicumarol content of the particular sweet clover fed and animal age. If dietary dicumarol is low or variable, animals may consume the forage for months before signs of disease appear. In an experiment with calves, dicumarol poisoning was produced by feeding naturally spoiled sweet clover hay that contained a minimum of 90 mg per kg (40.8 mg/lb) dicumarol (Alstad et al., 1985). The minimum time required to develop clinical signs of vitamin K deficiency in these calves was three weeks. A case of sweet clover poisoning in dairy cattle in California (Puschner et al., 1998) was caused by feeding sweet clover silage that contained dicumarol produced by mold infestation. Symptoms included subcutaneous hemorrhage, bleeding from the reproductive tract, weakness and death. Other reported symptoms are subcutaneous hemorrhage and clotting in the brisket, neck and hips; stiffness and lameness; dull, listless behavior and pale mucous membranes. Dicumarol has been reported to cause reproductive failure when fed at sub-clinically toxic levels. Dicumarol poisoning can be reversed by administration of vitamin K. Parenteral vitamin K1 was an effective treatment for calves at rates of 1.1, 2.2 and 3.3 mg per kg (.5, 1 and 1.5 mg per lb) body weight. Other researchers have reported that vitamin K1 injections were effective in treating sweet clover poisoning in cattle, but that vitamin K3 (menadione) injections were not (Casper et al., 1989). Pritchard et al. (1983) reported that large oral doses of vitamin K1 were effective in treatment of sweet vernal poisoning of cattle, but that vitamin K3gave less consistent results in terms of prothrombin time. This may reflect a greater antagonism of dicumoral against menadione. Another common cause of induced vitamin K deficiency in veterinary practice is the accidental poisoning of animals with warfarin (a synthetic coumarin used as a rodent poison). Initial clinical signs may be stiffness and lameness caused by bleeding into the muscles and joints. Hematomas, epistaxis or gastrointestinal bleeding may be observed. Death may occur suddenly with little preliminary evidence of disease and is caused by spontaneous massive hemorrhage or bleeding after injury, surgery or parturition. DeHoogh (1989) reported that two possible early embryonic deaths occurred and one cow aborted from sweet clover poisoning.
Measurement of clotting time or prothrombin time has been used to evaluate vitamin K status and is considered a fairly good measure of vitamin K deficiency. Prolongation of the clotting time in the absence of liver disease indicates vitamin K deficiency. Further clarification of a deficiency can be provided by assays for specific vitamin K-dependent factors, or by the rapid response to administration of vitamin K. Currently vitamin K status is assessed by measurement of the plasma concentration of one or more of the vitamin K-dependent clotting factors, prothrombin (factor II), factor VII, factor IX or factor X (Suttie, 1991). More recently, plasma osteocalcin has been proposed as the most sensitive index of vitamin K status in animals and humans (Vermeer et al., 1995).
In experimentally induced dicumarol poisoning, “hemorrhagic sweet clover disease,” Alstad et al. (1985) reported that normal prothrombin time is equal to or less than 20 seconds. Deficiency of vitamin K was characterized by prothrombin times of greater than 40 to 60 seconds, and with severe deficiency, prothrombin time can be as long as 5 to 6 minutes.
As long as natural vitamin K sources (i.e., green leafy plants) are sufficiently high in the diet and (or) bacterial synthesis in the rumen and intestinal tract remains functional, supplementary dietary vitamin K is not necessary to prevent deficiency (Perry et al., 1968). In addition to dicumarol, other vitamin K antagonists include certain sulfonamide antibiotics, mycotoxins (T-2 toxin) and warfarin. Sudden or severe alteration of rumen or intestinal microflora may result in the loss of an excellent source of vitamin K. Vitamin K supplementation is warranted when white or yellow sweet clover is a major forage source. Marks (1975) observed that the most common cause of vitamin K deficiency in veterinary practice is the accidental poisoning of domestic animals with warfarin. Vitamin K supplementation may be helpful in correcting vitamin K deficiency induced by mycotoxins, in particular T-2 toxin. Vitamin K antagonists will increase the vitamin K needs of livestock. In adjusting dietary vitamin K fortification levels, an appropriate margin of safetly is needed to prevent deficiency and allow optimum performance. Vitamin K antagonists include the use of certain antibiotics and sulfa drugs. Sulfonamides and bad-spectrum antibiotic drugs can virtually sterilize the lumen of the intestine (McDowell, 2000). Mycotoxins, such as aflatoxin, are toxic substances produced by molds. Supplemental vitamin K may be helpful in correcting vitamin K deficiency in aflatoxinosis. Nelson and Norris (1961) showed that the inclusion of 0.1% sulfaquinoxaline increased the chick’s need for supplemental vitamin K by four- to seven-fold. By altering the intestinal microflora, an excellent source of vitamin K is lost. Stability of the naturally occurring sources of vitamin K are poor. However, stability of the water-soluble menadione salts is satisfactory in multivitamin premixes without trace minerals (Frye, 1978). Basic pH conditions accelerate the destruction of menadione salts. Thus, soluble or slightly soluble basic mineral substances should not be included in multivitamin premixes containing menadione. Vitamin K in the form of MSB or MSBC is very sensitive to moisture and trace minerals, sensitive to light and basic pH and moderately sensitive to reduction and acid pH. Choline chloride is particularly destructive to vitamin K, with an average monthly loss of 34% to 38% for MSBC and MPB when stored in a vitamin premix with choline. Heat, moisture and trace minerals increase the rate of destruction of menadione salts in both pelleted and extruded feeds (Hoffmann-La Roche, 1981). For these reasons, greater quantities of vitamin K are recommended in premixes that contain large quantities of choline chloride and certain trace minerals, especially when premixes are exported or stored for an extended period of time (Schneider, 1986).
Toxic effects of the vitamin K family are manifested mainly as hematological 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 naturally occurring forms of vitamin K, phylloquinone and menaquinone, are nontoxic at very high dosage levels. The synthetic menadione compounds, however, have produced 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, 1987). Menadione compounds can safely be used at low levels to prevent the development of a deficiency but should not be used to treat a hemorrhagic condition. The parenteral LD50 of menadione or its derivatives is 200 to 500 mg per kg (91 to 227 mg per lb) of body weight in some species and dosages of 2 to 8 mg per kg (0.9 to 3.6 mg per lb) body weight have been reported to be lethal in horses. Such data are not available for ruminants (NRC, 1989).
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