DSM in Animal Nutrition & Health
Properties and Metabolism
Vitamin B12 is now considered by nutritionists as the generic name for a group of compounds having vitamin B12activity. These compounds have very complex structures (Illus. 10-1). The empirical formula of vitamin B12 is C63H88O14N14PCo, and among its unusual features is the content of 4.5% cobalt. The name cobalamins is used for compounds where the cobalt atom is in the center of the corrin nucleus. Adenosylcobalamin and methylcobalamin are naturally occurring forms of vitamin B12 in feedstuffs and in animal tissues. Cyanocobalamin is not a naturally occurring form of the vitamin, but is the most widely used form of cobalamin in clinical practice because of its relative availability and stability.
Vitamin B12 is a dark-red crystalline hygroscopic substance, freely soluble in water and alcohol but insoluble in acetone, chloroform and ether. Cyanocobalamin has a molecular weight of 1,355 and is the most complex structure and heaviest compound of all vitamins. Oxidizing and reducing agents and exposure to sunlight tend to destroy its activity. Losses of vitamin B12 during feed processing are usually not excessive, because vitamin B12is stable at temperatures as high as 250°C.Of the total vitamin B12 produced by rumen microorganisms, only 1% to 3% is absorbed. As in most species, the absorptive site for ruminants is the lower portion of the small intestine. Substantial amounts of B12 are secreted into the duodenum and then reabsorbed in the ileum.
Passage of vitamin B12 through the intestinal wall is a complex procedure and requires intervention of certain carrier compounds able to bind the vitamin molecule (McDowell, 2000). For vitamin B12 absorption in most species studied, the following is required: (a) adequate quantities of dietary vitamin B12 or cobalt; (b) functional abomasum (true stomach) for breakdown of food proteins for release of vitamin B12; (c) functional abomasum for production of intrinsic factor for absorption of vitamin B12 through the ileum; (d) functional pancreas (trypsin secretion) required for release of bound vitamin B12 prior to combining the vitamin with the intrinsic factor; and (e) functional ileum with receptor and absorption sites. Gastric juice defects are responsible for most cases of food-vitamin B12 malabsorption in monogastrics (Carmel, 1994), but for ruminants the lack of cobalt for vitamin B12 synthesis is the major problem (McDowell, 1997).
Low blood vitamin B12 (hypocobalamanemia) in cats has often been identified with diseases of the alimentary tract (including the liver and pancreas) (Ruaux et al., 2001; 2005; 2009; Simpson et al., 2001; Reed et al., 2007). Factors that diminish vitamin B12 absorption include deficiencies of protein, iron and vitamin B6; thyroid removal; and dietary tannic acid (Hoffmann-La Roche, 1984). The absorption of vitamin B12 is limited by the number of intrinsic factor-vitamin B12 binding sites in the ileal mucosa, so that not more than about 1 to 1.5 µg of a single oral dose of the vitamin in humans can be absorbed (Bender, 1992). The absorption is also slow; peak blood concentrations of the vitamin are not achieved for some 6 to 8 hours after an oral dose.
Intrinsic factor concentrates prepared from the stomach of one animal species do not in all cases increase B12absorption in other animal species or in humans. There are structural differences in the vitamin B12 intrinsic factors among species. Similarly, there are species differences for vitamin B12 transport proteins (Polak et al., 1979). Intrinsic factor has been demonstrated in the human, monkey, pig, rat, cow, ferret, rabbit, hamster, fox, lion, tiger and leopard. It has not, at present, been detected in the guinea pig, horse, sheep, chicken and various other species. It is now established that the dog stomach produces only small amounts of intrinsic factor, with larger amounts produced by the pancreas (Batt et al., 1991).
Storage of vitamin B12 is found principally in the liver. Other storage sites include the kidney, heart, spleen and brain.Andrews et al. (1960) reported that the proportion of liver cobalt that occurs as vitamin B12 varied with the cobalt animal status. Under grazing conditions with adequate cobalt in the pasture, most liver cobalt can be accounted for as vitamin B12, but in cobalt deficiency only about one-third of the liver cobalt is present in this form. This indicates that in cobalt deficiency, liver vitamin B12 is depleted faster than other forms of cobalt.
Even though vitamin B12 is water soluble, Kominato (1971) reported a tissue half-life of 32 days, indicating considerable tissue storage. Cattle and sheep with normal liver stores can receive a cobalt-deficient diet for months without showing vitamin B12 deficiency signs.
In ruminants, both cobalt and vitamin B12 are mainly excreted in feces, although variable amounts are excreted in urine (Smith and Marston, 1970). Lactating cows on a normal diet excrete 86% to 87.5% of all excreted cobalt in the feces (mainly with the bile), 0.9% to 1.0% with urine, and 11.5% to 12.5% with milk.
Vitamin B12 is an essential part of several enzyme systems that carry out a number of basic metabolic functions. Most reactions involve transfer or synthesis of one-carbon units, such as methyl groups. Vitamin B12 is metabolically related to other essential nutrients, such as choline, methionine and folic acid. (Savage and Lindenbaum, 1995; Stabler, 2006). Although the most important tasks of vitamin B12 concern metabolism of nucleic acids and proteins, it also functions in metabolism of fats and carbohydrates. A summary of vitamin B12 functions includes: (a) purine and pyrimidine synthesis; (b) transfer of methyl groups; (c) formation of proteins from amino acids; and (d) carbohydrate and fat metabolism. General functions of vitamin B12 are to promote red blood cell synthesis and to maintain nervous system integrity, which are functions noticeably affected in the deficient state. Overall synthesis of protein is impaired in vitamin B12-deficient animals (Friesecke, 1980; McDowell, 2000). Gluconeogenesis and hemopoiesis are critically affected by cobalt deficiency; and carbohydrate, lipid, and nucleic acid metabolism are all dependent on adequate B12and folic acid metabolism. An additional function of vitamin B12 relates to immune function. In mice, vitamin B12deficiency was found to affect immunoglobulin production and cytokine levels (Funada et al., 2001).Vitamin B12 is necessary in reduction of the one-carbon compounds of formate and formaldehyde, and in this way it participates with folic acid in biosynthesis of labile methyl groups. Formation of labile methyl groups is necessary for biosynthesis of purine and pyrimidine bases, which represent essential constituents of nucleic acids. For humans, interference with one-carbon metabolism enhances the risk of colorectal cancer because one-carbon metabolism has critical functions in biological methylation reactions in DNA synthesis and repair. In studies with rats, a vitamin B12–deficient diet, which was of insufficient severity to cause anemia or illness, created aberrations in both base substitution and methylation of colonic DNA, which might increase susceptibility to carcinogenesis (Choi et al., 2004).
Deficiency of vitamin B12 will induce folic acid deficiency by blocking utilization of folic acid derivatives. An enzyme containing vitamin B12 removes the methyl group from methylfolate, thereby regenerating tetrahydrofolate (THF), used in producing the 5,20-methylene THF required for thymidylate synthesis.
In animal metabolism, propionate of dietary or metabolic origin is converted into succinate, which then enters the tricarboxylic acid (Krebs) cycle. Methylmalonyl-CoA isomerase (mutase) is a vitamin B12-requiring enzyme (5´-deoxyadenosylcobalamin) that catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA. Flavin and Ochoa (1957) established that for succinate production the following steps are involved:
Propionate + ATP + CoA → propionyl-CoAPropionyl-CoA + CO2 + ATP → methylmalonyl-CoA (a)
Methylmalonyl-CoA (a) → methylmalonyl-CoA (b)
Methylmalonyl-CoA (b) → succinyl-CoA
Methylmalonyl-CoA (a) is an inactive isomer. Its active form, (b), is converted into succinyl-CoA by a methylmalonyl-CoA isomerase (methylmalonyl-CoA mutase) (fourth reaction). Vitamin B12 is a metabolic essential for animal species studied, and vitamin B12 deficiency can be induced with the addition of high dietary levels of propionic acid. However, metabolism of propionic acid is of special interest in ruminant nutrition because large quantities are produced during carbohydrate fermentation in the rumen. Propionate production proceeds normally, but in cobalt or vitamin B12 deficiency, its rate of clearance from blood is depressed and methylmalonyl-CoA accumulates. This results in an increased urinary excretion of methylmalonic acid and also loss of appetite because impaired propionate metabolism leads to higher blood propionate levels, which are inversely correlated to voluntary feed intake (MacPherson, 1982).
The origin of vitamin B12 in nature appears to be microbial synthesis. It is synthesized by many bacteria but apparently not by yeasts or by most fungi. There is little evidence that the vitamin is produced in tissues of higher plants or animals. A few reports have suggested limited B12 synthesis by a few plants, but in insignificant quantities in relation to animal requirements. Synthesis of this vitamin in the alimentary tract is of considerable importance for animals (McDowell, 2000). However, for some species (e.g., poultry) intestinal synthesis would be insignificant due to the short intestinal tract and rapid rate of feed movement through the gastrointestinal tract. Vitamin B12 requirements are exceedingly small. An adequate allowance is only a few µg per kg of feed. The vitamin B12 requirements of various species depend upon the levels of several other nutrients in the diet. Excess protein increases the need for vitamin B12as does performance level. The vitamin B12 requirement seems to depend on the levels of choline, methionine and folic acid in the diet; vitamin B12 is also interrelated with ascorbic acid metabolism (Scott et al., 1982). The requirements for both vitamin B12 and folic acid are reduced when the diet contains an abundance of compounds that can supply methyl groups (Dyer et al., 1949). Sewell et al. (1952) showed that vitamin B12 has a sparing effect on the methionine needs of the pig. A reciprocal relationship occurs between vitamin B12 and pantothenic acid in chick nutrition, with pantothenic acid having a sparing effect on the vitamin B12 requirement. Dietary ingredients may also affect the requirement, as wheat bran has been shown to reduce availability of vitamin B12 in humans (Lewis et al., 1986).
Dietary need depends on intestinal synthesis and tissue reserves at birth. Intestinal synthesis probably explains frequent failures to produce a vitamin B12 deficiency in pigs and rats on diets designed to be vitamin B12 free. The deficiency can be readily produced in rats, however, when coprophagy is prevented completely (Barnes and Fiala, 1958). The dog’s inclination toward coprophagy will supply part of the vitamin B12 requirement. Both dogs and cats likely also obtain some vitamin B12 by direct absorption of the vitamin produced by bacterial synthesis in the intestine. However, the amount from this source is not reliable. Some intestinal microorganisms reportedly may compete with the intrinsic factor for vitamin B12, thus preventing absorption of the vitamin in sufficient quantities (Giannella et al., 1971; 1972). In the dog, hookworm infections can also increase vitamin B12 requirements (Corbin and Kronfeld, 1972).
Genetic factors have been shown to increase the vitamin B12 requirements of both dogs and cats. A diagnosis of hereditary selective vitamin B12 malabsorption in dogs has been reported in border collies, giant schnauzers, Australian shepherd dogs, beagles and Chinese shar peis (Fordyce et al., 2000; Battersby et al., 2005; Grϋtzner et al.,2010). Grϋtzner et al. (2010) observed that the region of chromosome 13 should be mapped and closely examined for potential mutations associated with this disease in the shar peis. Fyfe et al. (1989; 1991a) described a family of dogs with the clinical, genetic and laboratory features of selective intestinal vitamin B12 malabsorption seen in humans. The malabsorption was caused by inefficient brush-border expression of intrinsic factor-vitamin B12 receptor due to a mutation of this complex and its retention (Fyfe et al., 1991b). Vitamin B12 deficiency associated with methylmalonic acidemia has been demonstrated in cats (Vaden et al., 1992). Apparently, this defect in vitamin B12 absorption is the result of an inborn error of metabolism.
Research has shown that vitamin B12 is needed by dogs and cats but a quantitative requirement has not been determined in detail. The stated requirements are based on some research conducted in dogs and cats, and on data from other animals.
A. Requirements for Dogs
Because a vitamin B12 deficiency is not common in dogs, no minimal requirement value is known as yet, but the NRC (2006) suggests a recommended requirement for vitamin B12as 35 µg per kg (15.9 µg per lb) of diet for all classes of dogs. It is more difficult to make an accurate recommendation of the additional requirements during pregnancy and lactation. The requirement for bitches fed diets based on soy protein may be greater during pregnancy than the above recommendation, as Woodward and Newberne (1966) reported hydrocephaly in rat pups from female rats fed a soy-based diet. This condition was prevented by providing 50 µg vitamin B12 per kg (22.7 µg per lb) of diet. On a feed basis, the Association of American Feed Control Officials (AAFCO, 2007) recommended 22 µg vitamin B12 per kg (10 µg per lb) for all classes of dogs.
B. Requirements for Cats
Teeter et al. (1977) found that feeding cats a purified diet containing 20 µg vitamin B12 per kg (9.1 µg per lb) of dry diet was sufficient for good health. A cat diet containing 20 µg vitamin B12 per kg (9.1 µg per lb) supported pregnancy, lactation, growth in kittens and normal hemoglobin. According to the NRC (2006) the estimated vitamin B12 requirement for all classes of cats is 22.5 µg per kg (10.2 µg per lb) of diet. On a feed basis, AAFCO (2007) recommends 20 µg vitamin B12 per kg (9.1 µg per lb) for all classes of cats.
Feedstuffs of animal origin are reasonably good sources of vitamin B12– meat, liver, kidney, milk, eggs and fish. Kidney and liver are excellent sources, and these organs are richer in vitamin B12 from ruminants than from most nonruminants. The presence of vitamin B12 in animal tissue is due to the ingestion of vitamin B12 in animal feeds or from intestinal or ruminal synthesis. Among the richest sources are fermentation residues, activated sewage sludge, and manure. Microbial synthesis of this vitamin in the alimentary tract is of considerable importance for animals. Typical commercially prepared dog and cat food diets that contain, meat, milk, fish or other animal products should contain ample quantities of vitamin B12. Also, most commercial pet foods are supplemented with stable B12 (Hand et al., 2010).
Evidence suggests that the amounts and types of roughages affect rumen synthesis of vitamin B12, even when adequate cobalt is supplied. Roughage restriction reportedly increased rumen fluid levels, elevated serum levels, reduced milk and liver levels of vitamin B12, and increased urinary excretion of this vitamin in cows compared to cows fed roughage ad libitum (Walker and Elliot, 1972). Vitamin B12 rumen levels of dairy heifers fed corn silage were 1.4 to 2.7 times higher than those fed chopped or pelleted hay (Dryden and Hartman, 1970).
Plant products are practically devoid of B12. The vitamin B12 reported in higher plants in small amounts may result from synthesis by soil microorganisms, excretion of the vitamin onto soil, with subsequent absorption by the plant. Root nodules of certain legumes contain small quantities of vitamin B12. Certain species of seaweed (algae) have been reported to contain appreciable quantities of vitamin B12 (up to 1 µg per gram or 0.45 µg per lb of solids). Seaweed does not synthesize vitamin B12. It is synthesized by the bacteria associated with seaweed and then concentrated by the seaweed (Scott et al., 1982). Dagnelie et al. (1991) reported that vitamin B12 from algae is largely unavailable. Providing algae to B12-deficient children was ineffective in elevating blood parameters compared to B12from fish sources. Vitamin B12 was found to be more available in pasteurized milk (1.91 pmol per ml) than in raw milk (1.54 pmol per ml), sterilized milk (1.25 pmol per ml), and dried milk (1.27 pmol per ml), indicating that heat treatment probably affect the bioavailability (Fie et al., 1994). Commercial sources of vitamin B12 are produced from fermentation products, and it is available as cyanocobalamin. Little is known about the bioavailability of orally ingested B12 in food and feeds.
To supply ruminants with vitamin B12, the important consideration is dietary sources of cobalt. Although most feeds are adequate in cobalt, the element is deficient in forages for grazing ruminants in many parts of the world (McDowell, 1997, 2000). Concentration of cobalt in crops and forages is dependent on soil factors, plant species, stage of maturity, yield, pasture management, climate and soil pH. Soil containing less than 2 mg per kg (0.9 mg per lb) of cobalt is generally considered deficient for ruminants (Corrêa, 1957). Raising the pH by liming reduces the cobalt uptake by the plant and may increase the severity of the deficiency. Plants grown on a 15 ppm cobalt soil that is neutral or slightly acid may contain more cobalt than those grown on a 40 ppm cobalt alkaline soil (Latteur, 1962). High rainfall tends to leach cobalt from the topsoil. This problem is often aggravated by rapid growth of forage during the rainy season, which dilutes the cobalt content. Plants have varying degrees of affinity for cobalt, some being able to concentrate the element much more than others. Legumes, for example, generally have greater ability to concentrate cobalt than do grasses (Underwood, 1977). Cobalt is needed by the nitrogen-fixing bacteria in the root nodule of legumes.
The general signs of a vitamin B12 deficiency in most animal species are a loss of appetite, variable feed intake and a dramatic growth suppression. In addition, sometimes there is rough skin and hair coat, vomiting and diarrhea, voice failure, and a slight anemia (Catron et al., 1952). Clinical cases in dogs and cats proven to be due solely to vitamin B12deficiency have been limited. The megaloblastic anemia and the neurological symptoms observed in vitamin B12deficiency in pernicious anemia in humans have not been observed in most other animals, including dogs and cats.A positive diagnosis of vitamin B12 deficiency is usually made by the finding of subnormal serum and tissue B12concentrations. Low serum levels are associated with low body content of the vitamin. In addition to serum vitamin B12, elevations of methylmalonic acid and total homocysteine are very sensitive and specific in diagnosing vitamin B12deficiency and can be used to help differentiate vitamin B12 deficiency from folic acid deficiency (Stabler et al., 1996). Serum concentrations of vitamin B12 have been determined in dogs (Caprelli et al., 1994; Davenport et al., 1994) and cats (Dunn et al., 1984) to evaluate status of the vitamin. Vitamin B12 deficiency has been confirmed by elevated urinary methylmalonic acid in dogs (Williams et al., 1969; Chanarin et al., 1973) and cats (Vaden et al., 1992). A clinical test for vitamin B12 deficiency in dogs is to load the animal with a precursor of methylmalonic acid (e.g., valine) and measure urinary excretion of methylmalonic acid (Williams et al., 1969; Chanarin et al., 1973).
A. Deficiency in Dogs
Normally, dogs have not shown any signs of vitamin B12 deficiency, yet they definitely have a requirement for this vitamin because clinical signs appear in dogs with certain disease conditions and when animals are fed low dietary concentrations of the vitamin (Ralston Purina, 1987). There are a number of clinical reports of vitamin B12 deficiency in dogs. These reports relate to either deficiencies induced by bacterial overgrowth of the intestine resulting in decreased availability of cobalamin to the dog, or genetic abnormalities of vitamin B12 metabolism (Hand et al., 2010).Lavrova (1969) found that dogs with internal biliary fistulas had vitamin B12 malabsorption and macrocytic anemia, as well as bone marrow abnormalities. The anemia was generally macrocytic hypochromic, macrocytic normochromic, normocytic hypochromic, or normocytic normochromic in type. The bone marrow erythropoietic centers appeared hypoplastic. Arnrich et al. (1952) observed that cocker spaniel puppies fed a 20% purified casein diet for 20 weeks grew better when 50 µg vitamin B12 per kg (22.7 µg per lb) diet was added to the diet, even though there were no signs of anemia. Campbell and Phillips (1953) reported impaired reproduction and also found that puppies grew better when their diets provided 22 µg per kg (10 µg per lb) vitamin B12.An inherited intestinal vitamin B12 malabsorption disorder has been reported in dogs as a result of a defective brush-border expression of the intrinsic factor-vitamin B12receptor (Fyfe et al., 1989; 1991b). Vitamin B12-deficient dogs developed chronic inappetence, lethargy, a chronic nonregenerative anemia and failure to thrive at 12 weeks of age. Vitamin B12-deficiency has been identified in 50% of German shepherds with degenerative myelopathy; however, the clinical signs were not responsive to vitamin B12treatment (Toennessen and Morin, 1995).
B. Deficiency in Cats
Vitamin B12-deficient kittens exhibited poor growth, lethargy, emaciation and a high level of methylmalonic acid excretion (Keesling and Morris, 1975; Vaden et al., 1992). Morris (1977) reported that kittens given a vitamin B12-deficient diet grew normally for three to four months, after which growth ceased. Subsequently, body weight was lost at an accelerating rate until supplementation was initiated with parenteral vitamin B12, which restored weight gain.
Vitamin B12 is produced by fermentation and is available commercially as cyanocobalamin for addition to feed. Vitamin B12 is only slightly sensitive to heat, oxygen, moisture and light (Gadient, 1986). Verbeeck (1975) reported vitamin B12 to have good stability in premixes with or without minerals, regardless of the source of the minerals. However, Yamada et al. (2008) reported degradation of supplemental vitamin B12. The vitamin was affected by storage time, light exposure, temperature and vitamin C. It was determined that some of the vitamin B12 might have been converted into vitamin B12 analogues. Scott (1966) indicated that there is apparently little effect of pelleting on vitamin B12content of feed.
Results of a large number of animal experiments are about equally divided between those reporting a positive response to dietary cyanocobalamin supplementation and those reporting little or no response. Variable responses may be due to several factors: initial body stores, environmental sources of the vitamin (such as molds, soil and animal excreta), microbial synthesis in the intestinal tract and adequacy or deficiency of other nutrients that influence B12 requirements.
The use of commercially available feeds for dogs and cats would normally provide sufficient amounts of dietary vitamin B12 to meet their requirements. Cats should receive ample vitamin B12 through fish products, which are rich in the vitamin. Dogs practicing coprophagy would receive a rich source of the vitamin. Special supplementation of vitamin B12would be needed for dogs and cats suspected or known to have genetic defects in the metabolism of the vitamin. Pancreas insufficiency would warrant B12 supplementation since this would be an important cause of vitamin B12malabsorption in dogs. The pancreas is the main source of intrinsic factor in this species. Dogs in heavy training need ample amounts of vitamin B12 to facilitate erythrocyte development and, in turn, increase the oxygen-carrying capacity of the blood. Some sled dogs reportedly have been injected with vitamin B12 several days before racing to increase plasma levels of erythrocytes (Corbin and Kronfeld, 1972).
Addition of vitamin B12 to feed in amounts far in excess of need or absorbability appears to be without hazard. Dietary levels of at least several hundred times the requirement are suggested as safe for the mouse (NRC, 1987). No data are available pertaining to vitamin B12safety in cats.Although frank vitamin B12 toxicity has not been described in the dog, in one report Pshonik and Gribanov (1961) noted disturbances of reflex activity in the form of reduction in size of vascular conditioned reflexes and exaggeration of unconditioned reflexes when vitamin B12 was injected subcutaneously in doses of 2 to 33 µg per kg (0.91 to 15 µg per lb) of body weight.