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 cobalamin is used for compounds whose 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. Most of the metabolic studies utilize cyanocobalamin.
Vitamin B12 is a dark-red crystalline hygroscopic substance, freely soluble in water and alcohol but insoluble in acetone, chloroform and ether. Cyanocobalamin, which has a molecular weight of 1,355, 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 B12during cooking are usually not excessive because it is stable at temperatures lower than 250°C. Passage of vitamin B12 through the intestinal wall is a complex procedure that requires intervention of certain carrier compounds able to bind the vitamin molecule (McDowell, 2000; Green and Miller, 2007). For most species, vitamin B12 absorption requires: (1) adequate quantities of dietary vitamin B12, (2) normal stomach for breakdown of food proteins for release of vitamin B12, (3) normal stomach for production of intrinsic factor for absorption of vitamin B12 through the ileum, (4) normal pancreas (trypsin) required for release of bound vitamin B12 prior to combining the vitamin with the intrinsic factor and (5) normal 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). Additional factors that diminish vitamin B12absorption include deficiencies of protein, iron, 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 B12binding 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 one animal’s stomach do not in all cases increase B12 absorption in other species of animal or in man. There are structural differences in the vitamin B12 intrinsic factor among species. Species differences exist likewise for vitamin B12 transport proteins (Polak et al., 1979). Intrinsic factor has been demonstrated in man, monkey, pig, rat, cow, ferret, rabbit, hamster, fox, lion, tiger and leopard. It has not, at present, been detected in dog, guinea pig, horse, sheep, chicken and a number of minor species.
Illustration 10-2: Vitamin B12 Deficiency
Storage of vitamin B12 is found principally in liver; other sources are kidney, heart, spleen and brain. Even though vitamin B12 is water soluble, it has 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 (McDowell, 2000). The main excretion routes for absorbed vitamin B12 are urinary, biliary and fecal. Urinary excretion of the intact vitamin B12 by kidney glomerular filtration is minimal. Biliary excretion via feces is the major excretory route. The majority of cobalamin excreted in bile is reabsorbed; at least 65% to 75% is reabsorbed in the ileum by means of the intrinsic factor mechanism.
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, for instance methyl groups. Vitamin B12 is metabolically related to other essential nutrients, such as choline, methionine and folic acid (Stabler, 2006). Though 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 is as follows: (1) purine and pyrimidine synthesis, (2) transfer of methyl groups, (3) formation of proteins from amino acids and (4) carbohydrate and fat metabolism. A general function of vitamin B12 is 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 in ruminants are critically affected by cobalt deficiency. Carbohydrate, lipid, and nucleic acid metabolism are all dependent on adequate vitamin 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 that 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 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., 1990).
Deficiency of vitamin B12 induces a folic acid deficiency by blocking utilization of folic acid derivatives. A vitamin B12-containing enzyme removes the methyl group from methylfolate, thereby regenerating tetrahydrofolate (THF), from which is made the 5,20-methylene THF required for thymidylate synthesis. It has been suggested that vitamin B12status may affect systemic metabolism of folic acid during early pregnancy (Guay et al., 2002).
Wagle et al. (1958) demonstrated that rats and baby pigs deprived of vitamin B12 were less able to incorporate serine, methionine, phenylalanine and glucose into liver proteins. There is good reason to believe that impairment of protein synthesis is the principal reason for the growth depression that is frequently observed in animals deficient in vitamin B12 (Friesecke, 1980).
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). Vitamin B12 requirements are exceedingly small; an adequate allowance is only a few mg per kg of feed. Swine requirements vary from 5 to 20 µg per kg (2.3 to 9.1 µg per lb) of feed (NRC, 1998), with young pigs and breeding animals having the highest requirement. Early on, Anderson and Hogan (1950) suggested inclusion of orally administered vitamin B12 at the rate of 0.26 µg daily per kg (0.12 µg per lb) of live weight, or not over 1.5 µg per 100 g (6.8 µg/lb) of feed. Johnson et al. (1949) estimated the oral vitamin B12 requirement of the baby pig to be approximately 20 µg per kg (9 µg/lb) of dry matter consumed, or 0.6 µg B12 per kg (0.27 µg per lb) body weight daily when provided by injection. After monitoring growth performance in pigs supplied with 0, 17, 34, 51, or 68 µg of a vitamin B12 concentrate per kg dry matter, Neumann et al. (1950) estimated the vitamin B12requirement to be approximately 50 µg (22.7 µg per lb) of activity per kg of diet. An indication of inadequate folic acid and/or vitamin B12is a high plasma level of homocysteine, which is a detrimental intermediate metabolite of the vitamins. Plasma homocysteine in suckling piglets decreased with increasing concentrations of cyanocobalamin given to sows in gestation. The concentrations of dietary cyanocobalamin that maximized plasma vitamin B12 and minimized plasma homocysteine of sows during gestation were estimated to be 164 and 93 µg per kg (75.5 and 42.3 µg per lb) respectively (Simard et al. 2007). These researchers also reported that the maximal residual responses in sows and piglets during lactation were observed with treatments of 100 or 200 µg of cyanocobalamin per kg. (45.5 or 90.7 µg/lb).
The vitamin B12 requirements of various species depend on the levels of several other nutrients in the diet. Excessive protein increases the need for vitamin B12, as 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 B12requirement. However, Luecke et al. (1952) did not observe a sparing effect of vitamin B12 on the pantothenic acid requirement of the pig. Luecke et al. (1952) indicated that the addition of 55 µg per kg (25 µg per lb) of vitamin B12 in feed may not have been sufficient to demonstrate a relationship between vitamin B12 and pantothenic acid. 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).
Colby and Ensminger (1950) were unable to detect a benefit of supplemental vitamin B12 when fed to growing pigs. However, Colby and Ensminger (1950) suggested that the experimental ration may not have been entirely free of the vitamin; intestinal synthesis may have occurred; or sufficient storage by the pigs from previous sources may have been involved in the lack of detection of a growth response that was different from that of the control animals. Bryantet al. (1981) reported that supplementation of a corn-soybean meal diet with 22 µg per kg (10 µg per lb) vitamin B12increased daily gains of both barrows and gilts by 6%, but this increase was not statistically significant. Dietary need for vitamin B12 depends on intestinal synthesis in addition to tissue reserves at birth. In order to study the vitamin B12needs of the young pig without prior accumulation of the vitamin, Bauriedel et al. (1954) utilized purified diets and baby pigs that were not allowed to suckle. It was reported that baby pigs can be raised from birth on purified diets and that a marked depletion of vitamin B12 could occur within 8 weeks. Early research findings (Richardson et al., 1951) suggested that when the intestinal flora is controlled through antibiotics administration, the vitamin B12 requirement of the weanling pigs is 11 µg or less per kg (5 µg or less per lb) of ration. When antibiotics and vitamin B12 were added to a corn-soybean meal diet, growth and feed efficiency were increased.
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). Coprophagous animals on deep litter receive excellent sources of vitamin B12 from microbial fermentation. The pig’s inclination toward coprophagy will supply part of the vitamin B12requirement. Swine also obtain some vitamin B12 by direct absorption of the vitamin produced by bacterial synthesis in the intestine (NRC, 1998). However, the amount from this source is not reliable. Hendricks et al. (1964) investigated the absorption of vitamin B12 from the colon of young pigs. The authors concluded that 41.6% to 58% of vitamin B12labeled with cobalt-57 was absorbed from the colon. Hendricks et al. (1964) also reported that some vitamin B12appeared to be metabolized during its passage through the lower digestive tract.
The origin of vitamin B12 in nature appears to be microbial synthesis. There is no convincing evidence that the vitamin is produced in tissues of higher plants or animals. It is synthesized by many bacteria but apparently not by yeast or by most fungi. Synthesis of this vitamin in the alimentary tract is of considerable importance for animals if sufficient cobalt is available.
Foods of animal origin— meat, liver, kidney, milk, eggs and fish— are reasonably good sources. Kidney and liver are excellent sources. These organs are richer in vitamin B12 for ruminants than most nonruminants. Neumann and Johnson (1950) found that injecting an extract from a pernicious anemia liver or an equivalent dose of crystalline vitamin B12 produced similar responses, suggesting the presence of significant amounts of vitamin B12 in the liver. Johnson et al. (1950) reported that the compound in the liver extract was indeed vitamin B12. Animal protein by-products, especially fish by-products, contain significant but variable quantities of vitamin B12. Among the richest sources are fermentation residues. Potent vitamin B12 sources include activated sewage sludge and manure.
Plant products are practically devoid of vitamin B12. The vitamin B12 reported in higher plants in small amounts may result from synthesis by soil microorganisms, excretion of the vitamin into soil and 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, 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 B12 from 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 affects the bioavailability (Fie et al., 1994). Commercial sources of vitamin B12 are produced from fermentation products, and vitamin B12 is available as cyanocobalamin.
The general signs of a vitamin B12 deficiency in the pig are comparable with those observed in other species, principally a loss of appetite, variable feed intake and dramatic growth suppression (Illus. 10-2). In addition, sometimes there is rough skin and hair coat, vomiting and diarrhea, voice failure, and slight anemia (Catron et al., 1952). Anderson and Hogan (1950) recorded many of these symptoms of vitamin B12 deficiency. Microcytic to normocytic anemia is typical, and many researchers, including Neumann and Johnson (1950), have reported high neutrophil and low lymphocyte counts. However, observations on anemia are not unanimous and are sometimes contradictory. Even observations within one study show a wide variety of effects with regard to hematologic manifestations of vitamin B12 deficiency, as sometimes anemia does not develop at all and at other times moderately severe anemia occurs (Cartwright et al., 1951). Nervous disorders that occur in the pig include increased excitability, unsteady gait (i.e., hind leg incoordination) and posterior incoordination. The thymus and spleen become atrophied, while liver and tongue may be enlarged as a result of proliferation of granulomatous tissue.
Illustration 10-2: Vitamin B12 Deficiency
Courtesy of the late D.V. Catran andIowa State University
In the reproducing animal, litter size and pig survival are reduced. Abortions, small litters and birth weights, some deformities and inability to rear young occur in breeding sows. Late estrus, fewer corpora lutea and fewer embryos are produced in vitamin B12-deficient animals. During reproduction and lactation, vitamin B12 supplementation has been shown to increase birth weights and survival of young pigs. Frederick and Brisson (1961) found that sows deficient in vitamin B12 had fewer pigs and these had lower viability than the pigs born from sows that were supplemented with vitamin B12. Successive litters from deficient sows become progressively weaker. In a study of sows supplemented with vitamin B12 at 80 to 100 µg daily during pregnancy, there were improved piglet and litter weights and a decreased percentage of stillbirths (Reinisch and Gebhardt, 1987). Under some conditions, reproductive performance of sows has been improved by inclusion of higher than recommended levels of dietary vitamin B12 (Cunha, 1977). The response is evidenced by an increase in litter size and birth weight of pigs.
Vitamin B12 is produced by fermentation and as cyanocobalamin is available commercially for addition to feed. Vitamin B12 is only slightly sensitive to heat, oxygen, moisture and light (Gadient, 1986). Verbeeck (1975) reported vitamin B12to have good stability in premixes with or without minerals regardless of 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 vitamin B12 might have been converted into vitamin B12analogues. 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, including 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 vitamin B12 requirements. Perhaps the manner in which vitamin B12 is provided may affect the response as well. Wilson et al. (1991) found that intramuscular injection of 2 mg of vitamin B12 to pigs at weaning increased their growth rate and especially the weight gain during the third week. Wilson et al. (1991) did not determine whether dietary vitamin B12 would have produced a similar response. Nesheim et al. (1950) compared the effects of injected with oral administration of vitamin B12. Nesheim et al. (1950) found significant improvements in average daily gains for the vitamin B12-supplemented pigs. Furthermore, the injectable vitamin B12 requirement was around 0.6 µg of vitamin B12 per kg (0.27 µg per lb) of body weight daily, while the oral requirement was approximately 20 µg per kg (9.1 µg per lb) of dry matter consumed. Thus, in this experiment, the injectable requirement was approximately half of the oral requirement for vitamin B12.Vitamin B12 is normally added to diets of all swine. Swine raised in confinement or in management systems in which there is limited access to feces should have a greater dietary requirement for the vitamin. Vitamin B12 supplementation may be warranted under certain conditions where stress, disease or parasites lower feed intake and (or) reduce intestinal absorption. In practice, vitamin B12 fortification of the ration should be adjusted to ensure the margin of safety important to prevent deficiency and allow optimal performance of swine.
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 safe for the mouse (NRC, 1998). No data are available pertaining to vitamin B12 safety in swine.