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DSM in Animal Nutrition & Health

Vitamin B12

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 feature 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.

 

Illustration 10-1

10Illustration_10-1_VitaminB12

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 B12are 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 B12synthesis is the major problem (McDowell, 1997). 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 always increase B12 absorption 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 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 animal’s cobolt 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.

 

Functions

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 (McDowell, 2000). Overall synthesis of protein is impaired in vitamin B12-deficient animals (Friesecke, 1980). Gluconeogenesis and hemopoiesis are critically affected by cobalt deficiency; and carbohydrate, lipid, and nucleic acid metabolism are all dependent on adequate B12 and folic acid metabolism. An additional function of vitamin B12 relates to immune function. In mice, vitamin B12 deficiency 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 colo-rectal cancer because one-carbon metabolism has critical functions in biological methylation reactions and 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 + Co→Apropionyl-CoA


Propionyl-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).

 

Requirements

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 B12synthesis 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 poultry intestinal synthesis would be insignificant due to the short intestinal tract and rapid rate of feed movement through the gastrointestinal tract. Vitamin B12requirements are exceedingly small; an adequate allowance is only a few µg per kg of feed, making it the most potent of vitamins. Poultry species requirements vary from 3 to 10 µg per kg (1.4 to 4.5 µg per lb) of feed (NRC, 1994). Squires and Naber (1992) supplemented a corn-soybean diet for laying hens at control (no supplementation) or one, two or four times the NRC requirement for vitamin B12. Egg production was reduced after 12 weeks on the diets when hens were fed the two lowest vitamin B12 intakes. As vitamin B12 intake increased, shell thickness decreased and egg weight, hen weight, and hatchability increased. Maximum egg production, egg weight, hen weight, and hatchability were obtained when the diet contained 8 µg per kg (3.64 µg per lb) of vitamin B12.The vitamin B12 requirements of various species depend on the levels of several other nutrients in the diet. Excess protein increases the need for B12 as does performance level. The B12 requirement seems to depend on the levels of choline, methionine and folic acid in the diet, and B12 is also interrelated with ascorbic acid metabolism (Scott et al., 1982). Sewell et al. (1952) showed that B12 has a sparing effect on the methionine needs of the pig. A reciprocal relationship occurs between B12 and pantothenic acid in chick nutrition, with pantothenic acid sparing the 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). Propionic acid is often used as a feed preservative, and propionic acid is known to increase the need for vitamin B12.

Dietary need depends on intestinal synthesis and tissue reserves at hatching. Intestinal synthesis probably explains frequent failures to produce a B12 deficiency in pigs and rats on diets designed to be B12-free. The deficiency can be readily produced in rats, however, when coprophagy is prevented completely (Barnes and Fiala, 1958). Coprophagous animals, including poultry on deep litter, receive excellent sources of B12 from microbial fermentation. Litter would be a less valuable source of vitamin B12 under cold conditions, where bacterial numbers are greatly reduced. Poultry obtain some vitamin B12 by direct absorption of the vitamin produced by bacterial synthesis in the intestine (NRC, 1994), but the amount derived from this source is small and not reliable.

 

Sources

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. Vitamin B12 presence in tissues of animals is due to the ingestion of vitamin B12 in animal feeds or from intestinal ruminal synthesis. Among the richest sources are fermentation residues, are activated sewage sludge and manure.

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. The 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 of 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 elevation blood parameters compared to B12 from fish sources. Vitamin B12 was found to be more available in pasteurized milk (1.91 pmol per ml), and dried milk (1.27 pmol per ml), indicating that heat treatment probably affects 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 foods and feeds.

To supply the ruminant 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 the grazing ruminant in many parts of the world (McDowell, 1997; 1992). Concentration of cobalt in crops and forages is dependent on soil factors, plant species, maturity at storage, 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 nodules of legumes.

 

Deficiency

In growing chicks, turkey poults and quail, vitamin B12 deficiency reduces body weight gain, feed intake, and feed conversion. Vitamin B12 deficiency in growing chicks and turkeys may result in a nervous disorder and defective feathering. It has also been related to leg weakness and perosis; however, this appears to be a secondary effect. Perosis may occur in vitamin B12-deficient chicks or poults when the diet lacks choline, methionine, or betaine as a source of methyl groups. Addition of B12 may prevent perosis under these conditions because of its effect on synthesis of methyl groups. Additional clinical signs in B12 deficiency include anemia, gizzard erosion, and fatty deposits in the heart, liver, and kidneys. Poor feathering and mortality are the most obvious signs of a vitamin B12deficiency, and gizzard erosions may also appear (NRC, 1994). In hens, body weight and egg production are maintained despite a deficiency, but B12 has an important influence on egg size (Scott et al., 1982). However, Squires and Naber (1992) reported that both egg production and hen weight increased with vitamin B12 supplementation, as did hatchability and egg weight. Hatchability of incubated eggs may be severely reduced if the breeder diet contains inadequate vitamin B12 (Squires and Naber, 1992; Zhang et al., 1994). Changes that manifest themselves in vitamin B12-deficient chick embryos (Olcese et al., 1950) may be summarized as: (1) general hemorrhagic condition; (2) fatty liver in varying degrees; (3) heart often enlarged and irregular in shape; (4) kidneys pale or yellow, sometimes hemorrhagic; (5) incidence of perosis; (6) myoatrophy of the leg; (7) fewer myelinated fibers in the spinal cord; and (8) high incidence of embryonic malpositions. Hypertrophy of the thyroid gland has also been repeatedly observed (Ferguson and Couch, 1954). The most obvious change in B12-deficient embryos is myoatrophy of the leg, a condition characterized by atrophy of thigh muscles (Olceseet al., 1950). Two to five months may be needed to deplete hens of vitamin B12 stores to such an extent that progeny will hatch with low vitamin B12 reserves. The rate of depletion is most rapid when hens are fed high-protein diets (Scottet al., 1982). Chicks that do hatch without adequate carryover of vitamin B12 from the dam have a high rate of mortality. Vitamin B12-deficient embryos die at about day 17.

 

Fortification Considerations

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). Frye (1994) reported that storage time through six months had virtually no effect on B12 retention, whereas high heat exposure (45°C, 113°F) at three weeks decreased B12 to 34%. Verbeeck (1975) reported vitamin B12 to have good stability in premixes with or without minerals regardless of mineral source. 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 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 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 B12requirements.In a survey of 16 commercial egg layer and breeding flocks, Naber and Squires (1993a) reported considerable variation in vitamin B12. Some flocks sampled were of marginal vitamin B12 status to support hatchability and maximum egg size. For those flocks at one or two times the dietary requirement, vitamin transfer efficiency from diet to eggs was 43% for vitamin B12.Vitamin B12 is normally added to diets of all poultry species. Poultry raised in confinement, in management systems where there is limited access to feces for coprophagy, should have a greater dietary requirement for the vitamin. The vitamin B12 fortification of the ration should be adjusted to assure the margin of safety important to prevent deficiency and allow optimum performance in poultry.

 

Vitamin Safety

Addition of vitamin B12 to feeds 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 most species (NRC, 1987). Vitamin B12 is reported to be toxic with diets of around 5 mg per kg (2.3 mg per lb). Signs of toxicity are unclear, especially with many older reports, since results are likely confounded with toxic effects of fermentation residues, inadvertently included with B12 during manufacture (Leeson and Summers, 2001).