http://instruct.uwo.ca/chemistry/2211a/2211a-VitB12-L26-L27.pdf
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Vitamin B12Properties and MetabolismVitamin B12 is the largest and most complex of the vitamins (Illus. 10-1), and is synthesized only by microorganisms. A series of structurally related compounds possess vitamin B12 activity. In addition, there are structurally similar vitamin B12 antagonists, produced by bacteria such as Streptomyces griseus, E. Coli and Propionibacterium shermanii. All these compounds contain a cobalt atom stabilized within a corrin ring structure that is similar to the porphyrin ring of hemoglobin and the cytochromes (Ellenbogen and Cooper, 1991). Cyanocobalamin is the common term used to refer to the most active and stable supplemental form of vitamin B12. Tissue metabolism converts cyanocobalamin to the two primary coenzyme forms, adenosylcobalamin and methylcobalamin (Ellenbogen and Cooper, 1991).
Illustration 10-1
Vitamin B12 is a dark-red, hygroscopic, needle-shaped crystal, freely soluble in water and alcohol but insoluble in acetone, chloroform and ether. Cyanocobalamin has a molecular weight of 1,355. 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 lower than 482°F (250°C). On average, 3% of dietary cobalt is converted to vitamin B12, and only 1% to 3% of the vitamin B12 synthesized in the rumen is absorbed from the small intestine (Girard, 1998a). Ruminal microflora also use dietary cobalt to produce different molecules, called analogs, closely related to cobalamin, but without biological activity for the host. Increasing dietary cobalt supply increases production of these analogs in the rumen at the expense of the biologically active forms of the vitamin. Most analogs are inactive for the host, but a few studies indicate that some analogs could have deleterious effects. In addition to cobalamin, the only biologically active molecule for the cow, seven analogs were identified in duodenal and ileal digesta (Girard et al., 2009). Although cobalamin was not the major form synthesized by ruminal microflora and, even if supplementary cyanocobalamin was extensively destroyed by ruminal microflora, based on calculations of apparent intestinal disappearance, cobalamin seems to be the major form absorbed in the small intestine. In ruminants, as in most species, the distal small intestine (ileum) is the primary absorptive site for vitamin B12. Substantial amounts of vitamin B12 are secreted into the duodenum and reabsorbed in the ileum, a process that is presumably subject to regulation based on the vitamin B12status of the animal. Absorption of vitamin B12 requires the presence of the intrinsic factor, a glycoprotein synthesized by the parietal cells of the gastric mucosa (McDowell, 2000). Pernicious anemia in humans is caused by a lack of the intrinsic factor. A second factor, transcobalamin II, has been identified in the intestinal villus (Quadros et al., 2000). Vitamin B12 absorption also occurs by diffusion, but only at very high concentrations (Ellenbogen and Cooper, 1991). In ruminants, vitamin B12 absorption requires the following: (a) adequate quantities of dietary cobalt, much of dietary vitamin B12 (e.g., cyanocobalamin) is destroyed by ruminal microflora (Girard et al., 2009); (b) a functional abomasum for digestion of food proteins and release of vitamin B12 and the production of intrinsic factor; (c) a functional pancreas (trypsin secretion) for protein digestion and release of bound vitamin B12; (d) a functional ileum. Intrinsic factor has been demonstrated in the cow but not the sheep. Factors that diminish vitamin B12 absorption include deficiencies of protein, iron and vitamin B6; thyroid removal/ hypothyroidism; and dietary tannic acid. Cobalamin analogs may compose up to 50% of the total plasma vitamin B12 in cattle, but are undetectable in sheep (Halpin et al., 1984).
Storage of vitamin B12 occurs 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 varies with the cobalt status of the animal. In cattle or sheep grazing cobalt-adequate pastures, most of the cobalt in the liver is in the form of vitamin B12. However, in cobalt deficiency only about 33% of liver cobalt is in the form of vitamin B12. This indicates that during cobalt deficiency vitamin B12 is depleted more rapidly than other forms of cobalt in the liver.
Unlike the other water-soluble vitamins, vitamin B12 is stored in significant amounts in the liver and other tissues, providing a reserve in times of cobalt depletion. Kominato (1971) reported a tissue half-life of 32 days for vitamin B12, indicating considerable tissue storage. Cattle and sheep with normal liver stores can tolerate a cobalt-deficient diet for several months without showing vitamin B12 deficiency signs.
In ruminants, both cobalt and vitamin B12 are excreted primarily in the feces, with smaller amounts excreted in urine (Smith and Marston, 1970). Lactating cows fed normal diets excrete 86% to 87.5% of all excreted cobalt in the feces (mainly associated with bile acids), 0.9% to 1.0% in the urine, and 11.5% to 12.5% in milk.
FunctionsVitamin 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. 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 include: (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). The primary role of vitamin B12 is as an essential cofactor for the enzymes methionine synthase and methylmalonyl-CoA mutase (McDowell, 2000). Methionine synthase effects the transfer of a methyl group from folic acid (N5-methyl-tetrahydrofolate) to homocysteine, forming methionine. Therefore, a vitamin B12 deficiency reduces methionine supply and metabolic recycling of methyl groups. The latter effect interrupts the normal metabolism of folic acid and blocks its utilization. Thus, a vitamin B12 deficiency produces a secondary deficiency of folic acid, and a characteristic anemia (McDowell, 2000).
Under intensive dairy systems, methionine may be limiting in certain diets. Under such circumstances, increasing methionine supply through feeding rumen-protected methionine has the potential to augment milk protein, fat concentrations and yields in high-producing dairy cows, probably through increased protein synthesis (NRC, 2001). Methionine is not only an amino acid involved in the structure of proteins per se but it also plays a unique role as the initiating amino acid for the synthesis of proteins (Brosnan et al., 2007). Supply of methionine, because of its role as donor of preformed labile methyl groups, affects the needs for methylneogenesis (Bailey and Gregory, 1999) and consequently, for folic acid and vitamin B12.
The nature of the ruminant digestive system imposes a huge dependence on gluconeogenesis because very little glucose is being absorbed (Reynolds, 2006). In ruminants, the rate of gluconeogenesis increases with feed intake, and one of the major substrates for gluconeogenesis is propionate. Metabolic utilization of propionate after its absorption from the gastrointestinal tract is dependent on the transformation of propionate into succinyl-coenzyme A, requiring the successive actions of biotin- and vitamin B12-dependent enzymes. Propionate is first transformed into propionyl-CoA, which is carboxylated to methylmalonyl-CoA by a biotin-dependent enzyme, propionyl-CoA carboxylase. Methylmalonyl-CoA is then isomerized in succinyl-CoA under the action of the vitamin B12-dependent enzyme, methylmalonyl-CoA mutase. Succinyl-CoA finally enters into the Krebs cycle, from where it can be directed toward gluconeogenesis (Le Grusse and Watier, 1993; McDowell, 2000). Flavin and Ochoa (1957) established that for succinate production the following steps are involved:
Propionate + ATP + CoA→Propionyl-CoA
Propionyl-CoA + Coz + 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 above).
Methylmalonyl-CoA also arises from the metabolism of odd-chained fatty acids and certain amino acids (valine, isoleucine, methionine and threonine) (Girard, 1998a). The propionic acid pathway is of particular importance in ruminants because of their dependence on propionic acid as the primary glucose precursor, and the production of propionic acid in the rumen fermentation of dietary carbohydrates. As a result of the blockage in utilization of propionic acid, a vitamin B12 or cobalt deficiency in ruminants results in the accumulation of succinic acid in the rumen and methylmalonic acid in blood and urine (Kennedy et al., 1995).
The availability of vitamin B12 for methyl group transfer affects the supply and transfer rate of methyl groups in intermediary metabolism. Thus, vitamin B12 status affects the metabolism of choline and its derivatives, and the synthesis of the purines and pyrimidines, components of DNA and RNA. As a result, rapidly dividing cells, in particular the red blood cells, are sensitive to vitamin B12 status. The nervous system is also quite sensitive to the availability of vitamin B12, possibly due to its high rate of metabolism. Reduction in the rate of protein synthesis is thought to be a principal cause of the growth depression frequently observed in vitamin B12-deficient animals (Friesecke, 1980).
Vitamin B12 is 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).
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). Cobalt deficient lambs had reduced concentrations of serum immunoglobulin G (IgG) and total protein (Fisher, 1991).
RequirementsThe 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; McDowell and Arthington, 2005). Vitamin B12 requirements are exceedingly small, measured in units of micrograms per kg of feed. The vitamin B12 requirement increases with higher rates of animal production and metabolism. The requirement is also affected by the relative intake of other nutrients. Excess dietary protein increases the vitamin B12 requirement. Interrelationships exist with dietary cobalt, choline, methionine and folic acid. Vitamin B12also appears to be affected by ascorbic acid metabolism (Scott et al., 1982).
Dietary cobalt and subsequent ruminal synthesis of vitamin B12 normally meet the requirement for vitamin B12 in ruminants. Under typical conditions, rumen synthesis of vitamin B12 would be functional by six to eight weeks of age, depending on intake of dry feed. Pre-ruminant calves, lambs and kids require supplemental vitamin B12. Vitamin B12requirements of the dairy calf are estimated between 0.34 and 0.68 µg per kg (0.15 to 0.31 µg per lb) body weight (NRC, 1989). On a dietary basis, the requirement of young dairy calves ranges from 20 to 40 µg per kg (9.1 to 18.2 µg per lb) of dry matter (Radostits and Bell, 1970).
The established dietary cobalt requirements for ruminants are 0.1 to 0.2 mg per kg (0.05 to 0.09 mg per lb) of diet. Precise estimates of minimum cobalt requirements are difficult because of the influence of variables such as seasonal changes in herbage cobalt concentrations, selective grazing and soil properties. Under grazing conditions, lambs are the most sensitive to cobalt deficiency, followed by mature sheep, calves, goats and mature cattle (Andrews, 1956).
Cobalt content of the diet is the primary factor affecting the synthesis of vitamin B12 by ruminal microflora. However, studies indicate that synthesis of vitamin B12 can be affected by other factors. High-concentrate diets can reduce the synthesis of vitamin B12 and also increase the production of vitamin B12 analogs, certain of which are antagonists of vitamin B12 (Sutton and Elliot, 1972). These natural analogs have little or no vitamin B12 activity.
Tiffany et al. (2006) recorded increased synthesis of vitamin B12 as the cobalt concentration of the diet increased from 0.10 to 1.0 mg per kg (0.045 to 0.455 mg per lb). Based upon performance and blood measures, the cobalt requirement for fattening cattle may be between 0.15 and 0.25 mg per kg (0.068 to 0.114 mg per lb) (Stangl et al., 2000a, b; Tiffany et al., 2003). However, ruminal synthesis of vitamin B12 was increased with dietary levels of 1.0 mg per kg (0.455 mg per lb) of cobalt (Tiffany et al., 2002).
Ruminants have higher vitamin B12 requirements per unit of body weight or diet than nonruminants, presumably because of the involvement of B12 in the metabolism of propionic acid. Experiments with sheep suggest an oral requirement for growing lambs of about 200 µg per day, about 10 times the reported oral requirement of other species per unit of food intake (Marston, 1970). The comparatively high cobalt requirement of ruminants arises partly from the low efficiency of production of vitamin B12 from cobalt by the rumen microflora and partly from the low efficiency of absorption of vitamin B12 (McDowell, 2000).
Grasslands containing 0.1 ppm cobalt will prevent deficiency symptoms, while levels of 0.05 to 0.07 ppm cobalt are deficient. Further evidence that 0.1 ppm cobalt in the diet will meet both the cobalt and the vitamin B12 requirements of sheep was provided by Mohammed (1983), who reported that ruminal concentrations of both vitamin B12 and propionic acid were maximized at 0.1 ppm dietary cobalt. However, cobalt concentration of 0.10 to 0.15 mg per kg (0.45 to 0.068 mg per lb) of dietary dry matter resulted in adequate vitamin B12 production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermenters (Tiffany et al., 2006).
Tiffany and Spears (2005) found that ruminal vitamin B12 concentrations were greater and ruminal succinate concentrations were lower, in steers fed high-concentrate diets containing 0.19 mg of cobalt per kg (0.09 mg per lb) compared with those fed diets containing 0.04 or 0.09 mg cobalt per kg (0.018 or 0.041 mg per lb) of diet. Increasing dietary supply of cobalt from 0.19 to 0.93 mg per kg (0.086 to 0.423 mg per lb) of dry matter from parturition to 120 days of lactation had no effect on plasma concentrations of vitamin B12 or on milk production and milk component yields (Kincaid and Socha, 2007).
Sources Feedstuffs of animal origin are generally good sources of vitamin B12 (Driskell et al., 2001). Liver and kidney are especially rich sources. Fermentation products sometimes contain vitamin B12. Among the richest sources are fermentation residues including activated sewage sludge and manure. However, these materials are also a source of anti-vitamin B12.
The origin of vitamin B12 in nature is bacterial synthesis. Yeast and fungi do not appear to synthesize vitamin B12. Unlike other B-complex vitamins, vitamin B12 (cobalamin) is synthesized almost exclusively by bacteria and is therefore present only in foods that have been bacterially fermented or are derived from animals that have obtained this vitamin from their gastrointestinal microflora or their diet. There is no convincing evidence that the vitamin is produced in the tissues of of animals. Microbial synthesis of vitamin B12 in the alimentary tract is of considerable importance to ruminants and species that practice coprophagy.
Evidence suggests that the amount and type of roughage in the diet affects the rumen synthesis of vitamin B12 even when cobalt is adequate. Restricted roughage rations have been reported to increase vitamin B12 levels in rumen fluid and serum, reduce milk and liver concentrations and increase urinary excretion compared to cows fed roughage ad libitum (Walker and Elliot, 1972). Vitamin B12 levels in the rumen of dairy heifers fed corn silage is 1.4 to 2.7 times higher than when fed chopped or pelleted hay (Dryden and Hartman, 1970).
Plant products are practically devoid of vitamin B12. Minute quantities of vitamin B12 may be synthesized by soil microorganisms, released into the soil, and subsequently absorbed by plants. Root nodules of certain legumes contain small quantities of vitamin B12. Certain species of seaweed and other algae contain appreciable quantities of vitamin B12, up to 1 µg per gram of solids, synthesized by adherent bacteria (Scott et al., 1982). Commercial sources of vitamin B12 are produced by fermentation and are available as cyanocobalamin.
Cobalt is the primary dietary precursor of vitamin B12 in the ruminant. Although dietary cobalt of confined livestock is usually adequate, cobalt and secondary vitamin B12 deficiency is a significant problem for grazing livestock in many areas of the world (McDowell, 2000; McDowell and Arthington, 2005). Concentration of cobalt in forages is affected by soil properties, plant species, stage of maturity, yield, pasture management and climate. Soil levels of less than 2 ppm cobalt is generally considered deficient for ruminants (Correa, 1957). Liming of soils increases the pH, reduces the uptake of cobalt by plants and may increase the severity of a deficiency. Plants grown on soil containing 15 ppm cobalt with neutral or slightly acid pH can contain more cobalt than plants grown with 40 ppm cobalt and an alkaline pH (Latteur, 1962). Heavy rainfall tends to leach cobalt from the topsoil. This problem is often aggravated by rapid growth of forage crops during the rainy season, further diluting the cobalt content of the diet. Plants have varying degrees of affinity for cobalt, some being able to concentrate the element much more than others. Legumes, for example, generally have a greater ability to concentrate cobalt than do grasses (Underwood, 1977). The nitrogen-fixing bacteria in the root nodules of legumes require cobalt. High dietary potassium levels were reported to induce a transient, six-week reduction in plasma vitamin B12 concentrations (Smit et al., 1999).
Vitamin B12 is produced by fermentation and is available commercially as cyanocobalamin. Vitamin B12 is only slightly sensitive to heat, oxygen, moisture and pH (Gadient, 1986). Vitamin B12 has good stability in premixes with or without minerals, regardless of the source of the minerals, and is little affected by pelleting (Scott, 1966; Verbeeck, 1975). 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.
DeficiencyLassiter et al. (1953) demonstrated vitamin B12 deficiency in calves less than six weeks old that received no dietary animal protein. Clinical signs characterizing the deficiency included poor appetite and growth, lacrimation, muscular weakness, demyelination of peripheral nerves and emaciation. Young lambs (up to two months of age), require vitamin B12 supplementation, especially with early-weaning programs (NRC, 1985). In vitamin B12-deficient lambs, there is a sharp decrease of vitamin B12concentrations in blood and liver prior to the appearance of gross deficiency signs such as anorexia, weight loss and a decrease in blood hemoglobin concentration. At necropsy, the body of a severely deficient animal is extremely emaciated, often with a total absence of body fat. Fatty liver, hemosiderized spleen and hypoplasia of the erythrogenic tissue of bone marrow are also characteristic (Filmer, 1933). The anemia in lambs in normocytic and normochromic, but the anemia is mild and is not responsible for the primary deficiency signs of cobalt deficiency. Anorexia and marasmus invariably precede any considerable degree of anemia. The first discernible response to feeding cobalt or parenteral vitamin B12is a rapid improvement in appetite and body weight. Metabolically, vitamin B12 deficiency is characterized by decreases in the activity of methylmalonyl-CoA mutase, with a resultant increase in plasma and urinary methylmalonic acid (MMA), and by decreased activity of methionine synthase with a concomitant rise in plasma homocysteine (Kennedy et al., 1992, 1995). The reduction in methionine synthase activity in turn reduces the availability of methylated compounds, including the methylated phospholipids (Kennedy et al., 1992). The latter effect may be a cause of nerve demyelination. A recent study has associated high blood concentrations of methylmalonic acid and homocysteine with symptoms of acute and chronic hepatitis in vitamin B12-deficient twin-lambs (Vellema et al., 1999). Brain lesions were also observed in vitamin B12 deficient lambs in that study (Vellema et al., 1999). In ruminating cattle and sheep, vitamin B12 deficiency is most often caused by a cobalt deficiency. The syndrome is also known as “white-liver disease,” and the signs mimic those of pre-ruminant vitamin B12 deficiency and are reversible by either cobalt or vitamin B12 supplementation (Kennedy et al., 1997; Ulvund and Pestalozzi, 1990). Vitamin B12-deficient lambs exhibit a reduced lymphocyte response to Mycobacterium paratuberculosis vaccination and higher fecal egg counts from nematode infection compared to lambs supplemented with adequate cobalt (Vellema et al., 1996). Likewise, Paterson and MacPherson (1990) reported that cattle fed a cobalt-deficient diet for 10 weeks or longer had a significant reduction in neutrophil killing of Candida albicans yeast and greater weight loss when infected experimentally with brown stomach worm (Ostertagia ostertagi). Cobalt deficiency in beef cattle resulted in reduced appetite and weight gain, reduced vitamin B12 status, decreased triiodothyronine in serum, reduced folate levels in liver and a significant increase in liver accumulation of iron and nickel (Stangl et al., 1999).
Liver vitamin B12 concentrations of 0.10 µg or less per gram wet weight are “clearly diagnostic of cobalt deficiency disease” (Underwood, 1979). Liver cobalt concentrations in the range of 0.05 to 0.07 mg per kg (ppm; dry basis) or below are critical levels indicating deficiency (McDowell, 1985). Normal concentrations of serum MMA are tentatively suggested as being less than 2 µmol per liter, while subclinical cobalt deficiency is indicated by serum MMA concentrations of 2 to 4 µmol per liter and outright cobalt deficiency by more than 4 µmol per liter of serum (Paterson and MacPherson, 1990). In sheep, Marca et al. (1996) reported blood vitamin B12 concentrations greater than 0.2 µg/ml, in agreement with previous deficiency studies. Plasma MMA concentrations remained below 2 µmol/liter, considered normal for sheep. Milk vitamin B12 concentrations were 10 µg/ml in colostrum and early milk and declined thereafter to 50% or less the original value by 21 days of lactation (Marca et al., 1996).
Other histological and biochemical signs of cobalt/vitamin B12 deficiency in ruminants include: cardiovascular lesions resembling arteriosclerosis in sheep (Mohammed and Lamand, 1986); accumulation of odd-number, branched-chain fatty acids in tissues (Kennedy et al., 1994); and accumulation of succinate in rumen fluid due to the blockage in propionic acid synthesis (Kennedy et al., 1996).
Vitamin B12 deficiency induced by exposure to nitrous oxide resulted in almost complete inhibition of methionine synthase activity in liver, heart and brain tissue of sheep (Xue et al., 1986). The results led the authors to conclude that methyl group transfer via methionine synthase is required for adequate supply of methionine and methylated compounds. Amino acid concentrations are altered by cobalt deficiency in cattle as described by Stangl et al. (1998). In that study, feeding a cobalt-deficient diet reduced appetite, growth and vitamin B12 status and significantly reduced plasma methionine (by 53%) as well as the concentrations of valine, leucine, isoleucine, threonine, arginine, tyrosine and taurine. Conversely, plasma serine and homocysteine were significantly increased 2.7 and 4.8 times, respectively. Liver amino acid content was not affected.
Girard and coworkers have begun to explore the effects of vitamin B12, folic acid and methionine metabolism in lactating dairy cows (Girard et al., 1998; Girard and Matte, 2005; Preynat et al., 2009a, b; 2010; Girard et al., 2009). After observing an inverse relationship between serum vitamin B12 and folate concentrations during the course of lactation, Girard (1998a) conducted an experiment in which primiparous heifers were fed a diet supplemented with both folic acid and methionine and injected with either vitamin B12 or a placebo. In this preliminary trial, vitamin B12supplementation tended to increase milk production (8.8%), milk solids production (12%) and milk fat yield (16%), suggesting that vitamin B12 status may affect the response of lactating cows to folic acid and methionine supplementation.
Separate previously findings (Girard and Matte, 1998; 2005; Girard et al., 2005) showed that, in early lactation, a positive response of milk production and milk component yields to supplementary folic acid was observed in cows with higher plasma concentrations of vitamin B12. It appears that when folic acid was given in combination with vitamin B12 metabolic efficiency was improved, as suggested by similar lactational performance and dry matter intake over cows fed folic acid supplements alone. The effects of folic acid and vitamin B12 on lactation performance were not mainly explained by methionine economy because of a more efficient methylneogenesis but were rather related to increased glucose availability and changes in methionine metabolism (Preynat et al., 2009, a, b; 2010). These findings support the hypothesis that, in early lactation, supply of vitamin B12 was not optimal and limited the lactation performance of the cows.
Cobalt deficiency has been reported to reduce lamb survival and increase susceptibility to parasitic infection in cattle and sheep (Ferguson et al., 1988; Suttle and Jones, 1989). Cobalt deficiency has been associated with photosensitization of lambs characterized by a swollen head (Hesselink and Vellema, 1990). The condition responded to two injections of vitamin B12 three weeks apart. Cobalt deficiency in pregnant ewes reduced lamb numbers and increased stillbirths and neonatal mortality (Fisher, 1991). Lambs born from cobalt-deficient ewes were slower to nurse, had reduced concentrations of serum immunoglobulin G (IgG) and total protein, lower serum vitamin B12 and elevated serum MMA concentrations compared to normal lambs (Fisher, 1991). In African goats, cobalt deficiency reduced serum vitamin B12 and erythrocyte counts, although growth rate was not significantly affected, suggesting that goats, like cattle, are more resistant to cobalt/vitamin B12 deficiency than sheep (Mburu et al., 1993). Goats depleted of cobalt and thus vitamin B12 exhibited irregular estrus cycles, macrocytic anemia, decreased plasma progesterone and luteinizing hormone, and elevated plasma corticosteroids compared to normal animals (Mgongo et al., 1984).
With the exception of phosphorus and copper, cobalt is the most common mineral deficiency for grazing livestock in tropical countries (McDowell et al., 1984; McDowell, 1992). Cobalt deficiency signs are nonspecific, and it is often difficult to distinguish between a cobalt deficiency and basic malnutrition or parasitization. Acute clinical signs of cobalt deficiency mimic those of vitamin B12 deficiency (Illus. 10-2 and 10-3).
Illustration 10-2: Cobalt (Vitamin B12) Deficiencyin Cattle.
Wasting Disease
Illustration 10-3: Cobalt (Vitamin B12) in Cattle.
Wasting Disease
Courtesy of L.R. McDowell, University of Florida
The incidence of cobalt deficiency can vary greatly from year to year, from an undetectable mild deficiency to an acute stage. Lee (1963) illustrated this variation in a 14-year experiment with sheep in southern Australia. Half the ewes, replacements and progeny received supplemental cobalt and remained healthy (in no particular order by years). The undosed half had the following performance for the 14 years: in 2 years, lambs were unthrifty, but there were no deaths; in 3 years, growth rate of the lambs was slightly retarded; in 4 years, 30% to 100% of the lamb crop was lost; in 5 years, the performance of the remaining stock was as good as that of dosed animals. The conclusion would be that if there was no benefit from cobalt supplementation in a particular year, there was no guarantee that in the following year that a moderate to severe cobalt deficiency would not occur (McDowell, 2000). McDowell and Arthington (2003) have presented an extensive review of cobalt nutrition.
Fortification ConsiderationsRumen bacteria synthesize approximately 2 to 3 mg vitamin B12 per day when provided with adequate dietary cobalt. Synthesis of competitive analogs of vitamin B12 has been reported with higher levels of grain in the diet (Walker and Elliot, 1972). Serum vitamin B12 is lower during late pregnancy and early lactation than during mid-lactation, although correlations with milk yield, grain consumption or blood glucose concentration were poor (Elliot et al., 1965). Peters and Elliot (1983) reported that vitamin B12 injections increased milk protein production, but not milk yield, solids or fat production in vitamin B12-deficient ewes during early lactation. Liver capacity for gluconeogenesis from labeled propionic acid was increased in vitamin B12-treated ewes. Croom et al. (1981) did not detect any effect of supplementary vitamin B12 on milk fat synthesis in dairy cows. The authors had hypothesized that accumulation of MMA inhibits milk fat synthesis by reducing activity of lipogenic enzymes. Daugherty et al. (1986) found no beneficial effect of vitamin B12injections on growth performance or propionate-metabolizing enzymes in the livers of feedlot lambs fed 80% concentrate diets with an ionophore. Girard et al. (1998) in a preliminary trial reported that primiparous dairy cows fed diets supplemented with both folic acid and methionine responded to vitamin B12 injections with increased milk production between 25 and 125 days of lactation. In further studies, supplemental vitamin B12 and folic acid were found to be beneficial for milk production and milk component yields for high producing dairy cows in early lactation (Girard and Matte, 1998; 2005; Girard et al., 2005; 2009; Preynat et al., 2009 a, b; 2010). Weekly intramuscular injections of vitamin B12 and folic acid increased milk concentration of vitamin B12 by 68% in commercial dairy herds (Duplessis et al., 2011). A glass (250 ml) of milk from supplemented cows provided 54% of the recommended daily allowance (2.4 µg) for adults. Primiparous and multiparous cows differed in their milk yield response to dietary cobalt supplementation (Kincaid et al., 2003). Primiparous cows secreted colostrum and milk with higher cobalt concentrations than did multiparous cows. Likewise, serum B12levels were higher in primiparous than multiparous cows.
Young ruminants require supplemental vitamin B12 prior to full rumen development. Milk is a good source of vitamin B12. The NRC (2001) suggests that milk replacer for dairy calves contain 0.11 mg of cobalt per kg (0.05 mg per lb).
Although dietary supplementation of cobalt is the normal means of meeting the vitamin B12 requirement of ruminants, parenteral administration is used to treat animals with apparent deficiency symptoms or the general appearance of malnutrition or poor health. Vitamin B12 is sometimes administered parenterally to incoming feedlot cattle as a prophylactic measure.
Intramuscular injection of vitamin B12, at the rate of 100 µg per week, or 150 µg every other week, produced a rapid remission of all signs of deficiency in lambs, and was equivalent to cobalt administered orally at the rate of 7 mg per week (Andrews and Anderson, 1954). For treatment of cobalt deficiency in cattle, intramuscular administration of vitamin B12 at 500 to 3,000 µg per head is recommended, which may be repeated weekly (Graham, 1991). Administration of intramuscular vitamin B12 to cobalt-deficient animals produces overnight improvement in appetite, whereas oral dosing with cobalt requires seven to 10 days to produce similar effects (MacPherson, 1982).
Many forages and concentrate feeds do not supply adequate (0.10-0.20 ppm) cobalt, and thus supplementation is required. Growth responses to supplemental cobalt have been demonstrated in steers fed finishing diets based on barley grain (Raun et al., 1968), and sorghum grain and silage (Morris and Gartner, 1967). More recently, Tiffany and Spears (2005) used fattening cattle to show an effect of grain source on ruminal synthesis of vitamin B12 with greater response to cobalt supplementation when corn was the grain source compared with barley.
Vitamin SafetyAnimals can tolerate large excesses of vitamin B12. Dietary levels of at least several hundred times the requirement are suggested as safe for the mouse (NRC, 1987). Vitamin B12 is reported to be toxic for some animal species 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 vitamin B12 during manufacture (Leeson and Summers, 2001). Cobalt also has a low toxicity, with 10 mg per kg (4.5 mg per lb) the maximum dietary tolerable level for the common livestock species (NRC, 1980). Becker and Smith (1951) concluded that 150 mg per kg (68.2 mg per lb) on a dry diet basis, or 1,000 times normal levels, can be tolerated by sheep for many weeks without visible toxic effects. Characteristic signs of chronic cobalt toxicity in most species are reduced feed intake and body weight, emaciation, anemia, hyperchromenia, and increased liver cobalt (NRC, 1980). Cobalt toxicity in cattle is characterized by mild polycythemia; excessive urination, defecation and salivation; shortness of breath; and increased hemoglobin, red cell count and packed cell volume (NRC, 2000).
Illustration 10-1
Vitamin B12 is a dark-red, hygroscopic, needle-shaped crystal, freely soluble in water and alcohol but insoluble in acetone, chloroform and ether. Cyanocobalamin has a molecular weight of 1,355. 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 lower than 482°F (250°C). On average, 3% of dietary cobalt is converted to vitamin B12, and only 1% to 3% of the vitamin B12 synthesized in the rumen is absorbed from the small intestine (Girard, 1998a). Ruminal microflora also use dietary cobalt to produce different molecules, called analogs, closely related to cobalamin, but without biological activity for the host. Increasing dietary cobalt supply increases production of these analogs in the rumen at the expense of the biologically active forms of the vitamin. Most analogs are inactive for the host, but a few studies indicate that some analogs could have deleterious effects. In addition to cobalamin, the only biologically active molecule for the cow, seven analogs were identified in duodenal and ileal digesta (Girard et al., 2009). Although cobalamin was not the major form synthesized by ruminal microflora and, even if supplementary cyanocobalamin was extensively destroyed by ruminal microflora, based on calculations of apparent intestinal disappearance, cobalamin seems to be the major form absorbed in the small intestine. In ruminants, as in most species, the distal small intestine (ileum) is the primary absorptive site for vitamin B12. Substantial amounts of vitamin B12 are secreted into the duodenum and reabsorbed in the ileum, a process that is presumably subject to regulation based on the vitamin B12status of the animal. Absorption of vitamin B12 requires the presence of the intrinsic factor, a glycoprotein synthesized by the parietal cells of the gastric mucosa (McDowell, 2000). Pernicious anemia in humans is caused by a lack of the intrinsic factor. A second factor, transcobalamin II, has been identified in the intestinal villus (Quadros et al., 2000). Vitamin B12 absorption also occurs by diffusion, but only at very high concentrations (Ellenbogen and Cooper, 1991). In ruminants, vitamin B12 absorption requires the following: (a) adequate quantities of dietary cobalt, much of dietary vitamin B12 (e.g., cyanocobalamin) is destroyed by ruminal microflora (Girard et al., 2009); (b) a functional abomasum for digestion of food proteins and release of vitamin B12 and the production of intrinsic factor; (c) a functional pancreas (trypsin secretion) for protein digestion and release of bound vitamin B12; (d) a functional ileum. Intrinsic factor has been demonstrated in the cow but not the sheep. Factors that diminish vitamin B12 absorption include deficiencies of protein, iron and vitamin B6; thyroid removal/ hypothyroidism; and dietary tannic acid. Cobalamin analogs may compose up to 50% of the total plasma vitamin B12 in cattle, but are undetectable in sheep (Halpin et al., 1984).
Storage of vitamin B12 occurs 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 varies with the cobalt status of the animal. In cattle or sheep grazing cobalt-adequate pastures, most of the cobalt in the liver is in the form of vitamin B12. However, in cobalt deficiency only about 33% of liver cobalt is in the form of vitamin B12. This indicates that during cobalt deficiency vitamin B12 is depleted more rapidly than other forms of cobalt in the liver.
Unlike the other water-soluble vitamins, vitamin B12 is stored in significant amounts in the liver and other tissues, providing a reserve in times of cobalt depletion. Kominato (1971) reported a tissue half-life of 32 days for vitamin B12, indicating considerable tissue storage. Cattle and sheep with normal liver stores can tolerate a cobalt-deficient diet for several months without showing vitamin B12 deficiency signs.
In ruminants, both cobalt and vitamin B12 are excreted primarily in the feces, with smaller amounts excreted in urine (Smith and Marston, 1970). Lactating cows fed normal diets excrete 86% to 87.5% of all excreted cobalt in the feces (mainly associated with bile acids), 0.9% to 1.0% in the urine, and 11.5% to 12.5% in milk.
FunctionsVitamin 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. 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 include: (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). The primary role of vitamin B12 is as an essential cofactor for the enzymes methionine synthase and methylmalonyl-CoA mutase (McDowell, 2000). Methionine synthase effects the transfer of a methyl group from folic acid (N5-methyl-tetrahydrofolate) to homocysteine, forming methionine. Therefore, a vitamin B12 deficiency reduces methionine supply and metabolic recycling of methyl groups. The latter effect interrupts the normal metabolism of folic acid and blocks its utilization. Thus, a vitamin B12 deficiency produces a secondary deficiency of folic acid, and a characteristic anemia (McDowell, 2000).
Under intensive dairy systems, methionine may be limiting in certain diets. Under such circumstances, increasing methionine supply through feeding rumen-protected methionine has the potential to augment milk protein, fat concentrations and yields in high-producing dairy cows, probably through increased protein synthesis (NRC, 2001). Methionine is not only an amino acid involved in the structure of proteins per se but it also plays a unique role as the initiating amino acid for the synthesis of proteins (Brosnan et al., 2007). Supply of methionine, because of its role as donor of preformed labile methyl groups, affects the needs for methylneogenesis (Bailey and Gregory, 1999) and consequently, for folic acid and vitamin B12.
The nature of the ruminant digestive system imposes a huge dependence on gluconeogenesis because very little glucose is being absorbed (Reynolds, 2006). In ruminants, the rate of gluconeogenesis increases with feed intake, and one of the major substrates for gluconeogenesis is propionate. Metabolic utilization of propionate after its absorption from the gastrointestinal tract is dependent on the transformation of propionate into succinyl-coenzyme A, requiring the successive actions of biotin- and vitamin B12-dependent enzymes. Propionate is first transformed into propionyl-CoA, which is carboxylated to methylmalonyl-CoA by a biotin-dependent enzyme, propionyl-CoA carboxylase. Methylmalonyl-CoA is then isomerized in succinyl-CoA under the action of the vitamin B12-dependent enzyme, methylmalonyl-CoA mutase. Succinyl-CoA finally enters into the Krebs cycle, from where it can be directed toward gluconeogenesis (Le Grusse and Watier, 1993; McDowell, 2000). Flavin and Ochoa (1957) established that for succinate production the following steps are involved:
Propionate + ATP + CoA→Propionyl-CoA
Propionyl-CoA + Coz + 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 above).
Methylmalonyl-CoA also arises from the metabolism of odd-chained fatty acids and certain amino acids (valine, isoleucine, methionine and threonine) (Girard, 1998a). The propionic acid pathway is of particular importance in ruminants because of their dependence on propionic acid as the primary glucose precursor, and the production of propionic acid in the rumen fermentation of dietary carbohydrates. As a result of the blockage in utilization of propionic acid, a vitamin B12 or cobalt deficiency in ruminants results in the accumulation of succinic acid in the rumen and methylmalonic acid in blood and urine (Kennedy et al., 1995).
The availability of vitamin B12 for methyl group transfer affects the supply and transfer rate of methyl groups in intermediary metabolism. Thus, vitamin B12 status affects the metabolism of choline and its derivatives, and the synthesis of the purines and pyrimidines, components of DNA and RNA. As a result, rapidly dividing cells, in particular the red blood cells, are sensitive to vitamin B12 status. The nervous system is also quite sensitive to the availability of vitamin B12, possibly due to its high rate of metabolism. Reduction in the rate of protein synthesis is thought to be a principal cause of the growth depression frequently observed in vitamin B12-deficient animals (Friesecke, 1980).
Vitamin B12 is 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).
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). Cobalt deficient lambs had reduced concentrations of serum immunoglobulin G (IgG) and total protein (Fisher, 1991).
RequirementsThe 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; McDowell and Arthington, 2005). Vitamin B12 requirements are exceedingly small, measured in units of micrograms per kg of feed. The vitamin B12 requirement increases with higher rates of animal production and metabolism. The requirement is also affected by the relative intake of other nutrients. Excess dietary protein increases the vitamin B12 requirement. Interrelationships exist with dietary cobalt, choline, methionine and folic acid. Vitamin B12also appears to be affected by ascorbic acid metabolism (Scott et al., 1982).
Dietary cobalt and subsequent ruminal synthesis of vitamin B12 normally meet the requirement for vitamin B12 in ruminants. Under typical conditions, rumen synthesis of vitamin B12 would be functional by six to eight weeks of age, depending on intake of dry feed. Pre-ruminant calves, lambs and kids require supplemental vitamin B12. Vitamin B12requirements of the dairy calf are estimated between 0.34 and 0.68 µg per kg (0.15 to 0.31 µg per lb) body weight (NRC, 1989). On a dietary basis, the requirement of young dairy calves ranges from 20 to 40 µg per kg (9.1 to 18.2 µg per lb) of dry matter (Radostits and Bell, 1970).
The established dietary cobalt requirements for ruminants are 0.1 to 0.2 mg per kg (0.05 to 0.09 mg per lb) of diet. Precise estimates of minimum cobalt requirements are difficult because of the influence of variables such as seasonal changes in herbage cobalt concentrations, selective grazing and soil properties. Under grazing conditions, lambs are the most sensitive to cobalt deficiency, followed by mature sheep, calves, goats and mature cattle (Andrews, 1956).
Cobalt content of the diet is the primary factor affecting the synthesis of vitamin B12 by ruminal microflora. However, studies indicate that synthesis of vitamin B12 can be affected by other factors. High-concentrate diets can reduce the synthesis of vitamin B12 and also increase the production of vitamin B12 analogs, certain of which are antagonists of vitamin B12 (Sutton and Elliot, 1972). These natural analogs have little or no vitamin B12 activity.
Tiffany et al. (2006) recorded increased synthesis of vitamin B12 as the cobalt concentration of the diet increased from 0.10 to 1.0 mg per kg (0.045 to 0.455 mg per lb). Based upon performance and blood measures, the cobalt requirement for fattening cattle may be between 0.15 and 0.25 mg per kg (0.068 to 0.114 mg per lb) (Stangl et al., 2000a, b; Tiffany et al., 2003). However, ruminal synthesis of vitamin B12 was increased with dietary levels of 1.0 mg per kg (0.455 mg per lb) of cobalt (Tiffany et al., 2002).
Ruminants have higher vitamin B12 requirements per unit of body weight or diet than nonruminants, presumably because of the involvement of B12 in the metabolism of propionic acid. Experiments with sheep suggest an oral requirement for growing lambs of about 200 µg per day, about 10 times the reported oral requirement of other species per unit of food intake (Marston, 1970). The comparatively high cobalt requirement of ruminants arises partly from the low efficiency of production of vitamin B12 from cobalt by the rumen microflora and partly from the low efficiency of absorption of vitamin B12 (McDowell, 2000).
Grasslands containing 0.1 ppm cobalt will prevent deficiency symptoms, while levels of 0.05 to 0.07 ppm cobalt are deficient. Further evidence that 0.1 ppm cobalt in the diet will meet both the cobalt and the vitamin B12 requirements of sheep was provided by Mohammed (1983), who reported that ruminal concentrations of both vitamin B12 and propionic acid were maximized at 0.1 ppm dietary cobalt. However, cobalt concentration of 0.10 to 0.15 mg per kg (0.45 to 0.068 mg per lb) of dietary dry matter resulted in adequate vitamin B12 production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermenters (Tiffany et al., 2006).
Tiffany and Spears (2005) found that ruminal vitamin B12 concentrations were greater and ruminal succinate concentrations were lower, in steers fed high-concentrate diets containing 0.19 mg of cobalt per kg (0.09 mg per lb) compared with those fed diets containing 0.04 or 0.09 mg cobalt per kg (0.018 or 0.041 mg per lb) of diet. Increasing dietary supply of cobalt from 0.19 to 0.93 mg per kg (0.086 to 0.423 mg per lb) of dry matter from parturition to 120 days of lactation had no effect on plasma concentrations of vitamin B12 or on milk production and milk component yields (Kincaid and Socha, 2007).
Sources Feedstuffs of animal origin are generally good sources of vitamin B12 (Driskell et al., 2001). Liver and kidney are especially rich sources. Fermentation products sometimes contain vitamin B12. Among the richest sources are fermentation residues including activated sewage sludge and manure. However, these materials are also a source of anti-vitamin B12.
The origin of vitamin B12 in nature is bacterial synthesis. Yeast and fungi do not appear to synthesize vitamin B12. Unlike other B-complex vitamins, vitamin B12 (cobalamin) is synthesized almost exclusively by bacteria and is therefore present only in foods that have been bacterially fermented or are derived from animals that have obtained this vitamin from their gastrointestinal microflora or their diet. There is no convincing evidence that the vitamin is produced in the tissues of of animals. Microbial synthesis of vitamin B12 in the alimentary tract is of considerable importance to ruminants and species that practice coprophagy.
Evidence suggests that the amount and type of roughage in the diet affects the rumen synthesis of vitamin B12 even when cobalt is adequate. Restricted roughage rations have been reported to increase vitamin B12 levels in rumen fluid and serum, reduce milk and liver concentrations and increase urinary excretion compared to cows fed roughage ad libitum (Walker and Elliot, 1972). Vitamin B12 levels in the rumen of dairy heifers fed corn silage is 1.4 to 2.7 times higher than when fed chopped or pelleted hay (Dryden and Hartman, 1970).
Plant products are practically devoid of vitamin B12. Minute quantities of vitamin B12 may be synthesized by soil microorganisms, released into the soil, and subsequently absorbed by plants. Root nodules of certain legumes contain small quantities of vitamin B12. Certain species of seaweed and other algae contain appreciable quantities of vitamin B12, up to 1 µg per gram of solids, synthesized by adherent bacteria (Scott et al., 1982). Commercial sources of vitamin B12 are produced by fermentation and are available as cyanocobalamin.
Cobalt is the primary dietary precursor of vitamin B12 in the ruminant. Although dietary cobalt of confined livestock is usually adequate, cobalt and secondary vitamin B12 deficiency is a significant problem for grazing livestock in many areas of the world (McDowell, 2000; McDowell and Arthington, 2005). Concentration of cobalt in forages is affected by soil properties, plant species, stage of maturity, yield, pasture management and climate. Soil levels of less than 2 ppm cobalt is generally considered deficient for ruminants (Correa, 1957). Liming of soils increases the pH, reduces the uptake of cobalt by plants and may increase the severity of a deficiency. Plants grown on soil containing 15 ppm cobalt with neutral or slightly acid pH can contain more cobalt than plants grown with 40 ppm cobalt and an alkaline pH (Latteur, 1962). Heavy rainfall tends to leach cobalt from the topsoil. This problem is often aggravated by rapid growth of forage crops during the rainy season, further diluting the cobalt content of the diet. Plants have varying degrees of affinity for cobalt, some being able to concentrate the element much more than others. Legumes, for example, generally have a greater ability to concentrate cobalt than do grasses (Underwood, 1977). The nitrogen-fixing bacteria in the root nodules of legumes require cobalt. High dietary potassium levels were reported to induce a transient, six-week reduction in plasma vitamin B12 concentrations (Smit et al., 1999).
Vitamin B12 is produced by fermentation and is available commercially as cyanocobalamin. Vitamin B12 is only slightly sensitive to heat, oxygen, moisture and pH (Gadient, 1986). Vitamin B12 has good stability in premixes with or without minerals, regardless of the source of the minerals, and is little affected by pelleting (Scott, 1966; Verbeeck, 1975). 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.
DeficiencyLassiter et al. (1953) demonstrated vitamin B12 deficiency in calves less than six weeks old that received no dietary animal protein. Clinical signs characterizing the deficiency included poor appetite and growth, lacrimation, muscular weakness, demyelination of peripheral nerves and emaciation. Young lambs (up to two months of age), require vitamin B12 supplementation, especially with early-weaning programs (NRC, 1985). In vitamin B12-deficient lambs, there is a sharp decrease of vitamin B12concentrations in blood and liver prior to the appearance of gross deficiency signs such as anorexia, weight loss and a decrease in blood hemoglobin concentration. At necropsy, the body of a severely deficient animal is extremely emaciated, often with a total absence of body fat. Fatty liver, hemosiderized spleen and hypoplasia of the erythrogenic tissue of bone marrow are also characteristic (Filmer, 1933). The anemia in lambs in normocytic and normochromic, but the anemia is mild and is not responsible for the primary deficiency signs of cobalt deficiency. Anorexia and marasmus invariably precede any considerable degree of anemia. The first discernible response to feeding cobalt or parenteral vitamin B12is a rapid improvement in appetite and body weight. Metabolically, vitamin B12 deficiency is characterized by decreases in the activity of methylmalonyl-CoA mutase, with a resultant increase in plasma and urinary methylmalonic acid (MMA), and by decreased activity of methionine synthase with a concomitant rise in plasma homocysteine (Kennedy et al., 1992, 1995). The reduction in methionine synthase activity in turn reduces the availability of methylated compounds, including the methylated phospholipids (Kennedy et al., 1992). The latter effect may be a cause of nerve demyelination. A recent study has associated high blood concentrations of methylmalonic acid and homocysteine with symptoms of acute and chronic hepatitis in vitamin B12-deficient twin-lambs (Vellema et al., 1999). Brain lesions were also observed in vitamin B12 deficient lambs in that study (Vellema et al., 1999). In ruminating cattle and sheep, vitamin B12 deficiency is most often caused by a cobalt deficiency. The syndrome is also known as “white-liver disease,” and the signs mimic those of pre-ruminant vitamin B12 deficiency and are reversible by either cobalt or vitamin B12 supplementation (Kennedy et al., 1997; Ulvund and Pestalozzi, 1990). Vitamin B12-deficient lambs exhibit a reduced lymphocyte response to Mycobacterium paratuberculosis vaccination and higher fecal egg counts from nematode infection compared to lambs supplemented with adequate cobalt (Vellema et al., 1996). Likewise, Paterson and MacPherson (1990) reported that cattle fed a cobalt-deficient diet for 10 weeks or longer had a significant reduction in neutrophil killing of Candida albicans yeast and greater weight loss when infected experimentally with brown stomach worm (Ostertagia ostertagi). Cobalt deficiency in beef cattle resulted in reduced appetite and weight gain, reduced vitamin B12 status, decreased triiodothyronine in serum, reduced folate levels in liver and a significant increase in liver accumulation of iron and nickel (Stangl et al., 1999).
Liver vitamin B12 concentrations of 0.10 µg or less per gram wet weight are “clearly diagnostic of cobalt deficiency disease” (Underwood, 1979). Liver cobalt concentrations in the range of 0.05 to 0.07 mg per kg (ppm; dry basis) or below are critical levels indicating deficiency (McDowell, 1985). Normal concentrations of serum MMA are tentatively suggested as being less than 2 µmol per liter, while subclinical cobalt deficiency is indicated by serum MMA concentrations of 2 to 4 µmol per liter and outright cobalt deficiency by more than 4 µmol per liter of serum (Paterson and MacPherson, 1990). In sheep, Marca et al. (1996) reported blood vitamin B12 concentrations greater than 0.2 µg/ml, in agreement with previous deficiency studies. Plasma MMA concentrations remained below 2 µmol/liter, considered normal for sheep. Milk vitamin B12 concentrations were 10 µg/ml in colostrum and early milk and declined thereafter to 50% or less the original value by 21 days of lactation (Marca et al., 1996).
Other histological and biochemical signs of cobalt/vitamin B12 deficiency in ruminants include: cardiovascular lesions resembling arteriosclerosis in sheep (Mohammed and Lamand, 1986); accumulation of odd-number, branched-chain fatty acids in tissues (Kennedy et al., 1994); and accumulation of succinate in rumen fluid due to the blockage in propionic acid synthesis (Kennedy et al., 1996).
Vitamin B12 deficiency induced by exposure to nitrous oxide resulted in almost complete inhibition of methionine synthase activity in liver, heart and brain tissue of sheep (Xue et al., 1986). The results led the authors to conclude that methyl group transfer via methionine synthase is required for adequate supply of methionine and methylated compounds. Amino acid concentrations are altered by cobalt deficiency in cattle as described by Stangl et al. (1998). In that study, feeding a cobalt-deficient diet reduced appetite, growth and vitamin B12 status and significantly reduced plasma methionine (by 53%) as well as the concentrations of valine, leucine, isoleucine, threonine, arginine, tyrosine and taurine. Conversely, plasma serine and homocysteine were significantly increased 2.7 and 4.8 times, respectively. Liver amino acid content was not affected.
Girard and coworkers have begun to explore the effects of vitamin B12, folic acid and methionine metabolism in lactating dairy cows (Girard et al., 1998; Girard and Matte, 2005; Preynat et al., 2009a, b; 2010; Girard et al., 2009). After observing an inverse relationship between serum vitamin B12 and folate concentrations during the course of lactation, Girard (1998a) conducted an experiment in which primiparous heifers were fed a diet supplemented with both folic acid and methionine and injected with either vitamin B12 or a placebo. In this preliminary trial, vitamin B12supplementation tended to increase milk production (8.8%), milk solids production (12%) and milk fat yield (16%), suggesting that vitamin B12 status may affect the response of lactating cows to folic acid and methionine supplementation.
Separate previously findings (Girard and Matte, 1998; 2005; Girard et al., 2005) showed that, in early lactation, a positive response of milk production and milk component yields to supplementary folic acid was observed in cows with higher plasma concentrations of vitamin B12. It appears that when folic acid was given in combination with vitamin B12 metabolic efficiency was improved, as suggested by similar lactational performance and dry matter intake over cows fed folic acid supplements alone. The effects of folic acid and vitamin B12 on lactation performance were not mainly explained by methionine economy because of a more efficient methylneogenesis but were rather related to increased glucose availability and changes in methionine metabolism (Preynat et al., 2009, a, b; 2010). These findings support the hypothesis that, in early lactation, supply of vitamin B12 was not optimal and limited the lactation performance of the cows.
Cobalt deficiency has been reported to reduce lamb survival and increase susceptibility to parasitic infection in cattle and sheep (Ferguson et al., 1988; Suttle and Jones, 1989). Cobalt deficiency has been associated with photosensitization of lambs characterized by a swollen head (Hesselink and Vellema, 1990). The condition responded to two injections of vitamin B12 three weeks apart. Cobalt deficiency in pregnant ewes reduced lamb numbers and increased stillbirths and neonatal mortality (Fisher, 1991). Lambs born from cobalt-deficient ewes were slower to nurse, had reduced concentrations of serum immunoglobulin G (IgG) and total protein, lower serum vitamin B12 and elevated serum MMA concentrations compared to normal lambs (Fisher, 1991). In African goats, cobalt deficiency reduced serum vitamin B12 and erythrocyte counts, although growth rate was not significantly affected, suggesting that goats, like cattle, are more resistant to cobalt/vitamin B12 deficiency than sheep (Mburu et al., 1993). Goats depleted of cobalt and thus vitamin B12 exhibited irregular estrus cycles, macrocytic anemia, decreased plasma progesterone and luteinizing hormone, and elevated plasma corticosteroids compared to normal animals (Mgongo et al., 1984).
With the exception of phosphorus and copper, cobalt is the most common mineral deficiency for grazing livestock in tropical countries (McDowell et al., 1984; McDowell, 1992). Cobalt deficiency signs are nonspecific, and it is often difficult to distinguish between a cobalt deficiency and basic malnutrition or parasitization. Acute clinical signs of cobalt deficiency mimic those of vitamin B12 deficiency (Illus. 10-2 and 10-3).
Illustration 10-2: Cobalt (Vitamin B12) Deficiencyin Cattle.
Wasting Disease
Illustration 10-3: Cobalt (Vitamin B12) in Cattle.
Wasting Disease
Courtesy of L.R. McDowell, University of Florida
The incidence of cobalt deficiency can vary greatly from year to year, from an undetectable mild deficiency to an acute stage. Lee (1963) illustrated this variation in a 14-year experiment with sheep in southern Australia. Half the ewes, replacements and progeny received supplemental cobalt and remained healthy (in no particular order by years). The undosed half had the following performance for the 14 years: in 2 years, lambs were unthrifty, but there were no deaths; in 3 years, growth rate of the lambs was slightly retarded; in 4 years, 30% to 100% of the lamb crop was lost; in 5 years, the performance of the remaining stock was as good as that of dosed animals. The conclusion would be that if there was no benefit from cobalt supplementation in a particular year, there was no guarantee that in the following year that a moderate to severe cobalt deficiency would not occur (McDowell, 2000). McDowell and Arthington (2003) have presented an extensive review of cobalt nutrition.
Fortification ConsiderationsRumen bacteria synthesize approximately 2 to 3 mg vitamin B12 per day when provided with adequate dietary cobalt. Synthesis of competitive analogs of vitamin B12 has been reported with higher levels of grain in the diet (Walker and Elliot, 1972). Serum vitamin B12 is lower during late pregnancy and early lactation than during mid-lactation, although correlations with milk yield, grain consumption or blood glucose concentration were poor (Elliot et al., 1965). Peters and Elliot (1983) reported that vitamin B12 injections increased milk protein production, but not milk yield, solids or fat production in vitamin B12-deficient ewes during early lactation. Liver capacity for gluconeogenesis from labeled propionic acid was increased in vitamin B12-treated ewes. Croom et al. (1981) did not detect any effect of supplementary vitamin B12 on milk fat synthesis in dairy cows. The authors had hypothesized that accumulation of MMA inhibits milk fat synthesis by reducing activity of lipogenic enzymes. Daugherty et al. (1986) found no beneficial effect of vitamin B12injections on growth performance or propionate-metabolizing enzymes in the livers of feedlot lambs fed 80% concentrate diets with an ionophore. Girard et al. (1998) in a preliminary trial reported that primiparous dairy cows fed diets supplemented with both folic acid and methionine responded to vitamin B12 injections with increased milk production between 25 and 125 days of lactation. In further studies, supplemental vitamin B12 and folic acid were found to be beneficial for milk production and milk component yields for high producing dairy cows in early lactation (Girard and Matte, 1998; 2005; Girard et al., 2005; 2009; Preynat et al., 2009 a, b; 2010). Weekly intramuscular injections of vitamin B12 and folic acid increased milk concentration of vitamin B12 by 68% in commercial dairy herds (Duplessis et al., 2011). A glass (250 ml) of milk from supplemented cows provided 54% of the recommended daily allowance (2.4 µg) for adults. Primiparous and multiparous cows differed in their milk yield response to dietary cobalt supplementation (Kincaid et al., 2003). Primiparous cows secreted colostrum and milk with higher cobalt concentrations than did multiparous cows. Likewise, serum B12levels were higher in primiparous than multiparous cows.
Young ruminants require supplemental vitamin B12 prior to full rumen development. Milk is a good source of vitamin B12. The NRC (2001) suggests that milk replacer for dairy calves contain 0.11 mg of cobalt per kg (0.05 mg per lb).
Although dietary supplementation of cobalt is the normal means of meeting the vitamin B12 requirement of ruminants, parenteral administration is used to treat animals with apparent deficiency symptoms or the general appearance of malnutrition or poor health. Vitamin B12 is sometimes administered parenterally to incoming feedlot cattle as a prophylactic measure.
Intramuscular injection of vitamin B12, at the rate of 100 µg per week, or 150 µg every other week, produced a rapid remission of all signs of deficiency in lambs, and was equivalent to cobalt administered orally at the rate of 7 mg per week (Andrews and Anderson, 1954). For treatment of cobalt deficiency in cattle, intramuscular administration of vitamin B12 at 500 to 3,000 µg per head is recommended, which may be repeated weekly (Graham, 1991). Administration of intramuscular vitamin B12 to cobalt-deficient animals produces overnight improvement in appetite, whereas oral dosing with cobalt requires seven to 10 days to produce similar effects (MacPherson, 1982).
Many forages and concentrate feeds do not supply adequate (0.10-0.20 ppm) cobalt, and thus supplementation is required. Growth responses to supplemental cobalt have been demonstrated in steers fed finishing diets based on barley grain (Raun et al., 1968), and sorghum grain and silage (Morris and Gartner, 1967). More recently, Tiffany and Spears (2005) used fattening cattle to show an effect of grain source on ruminal synthesis of vitamin B12 with greater response to cobalt supplementation when corn was the grain source compared with barley.
Vitamin SafetyAnimals can tolerate large excesses of vitamin B12. Dietary levels of at least several hundred times the requirement are suggested as safe for the mouse (NRC, 1987). Vitamin B12 is reported to be toxic for some animal species 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 vitamin B12 during manufacture (Leeson and Summers, 2001). Cobalt also has a low toxicity, with 10 mg per kg (4.5 mg per lb) the maximum dietary tolerable level for the common livestock species (NRC, 1980). Becker and Smith (1951) concluded that 150 mg per kg (68.2 mg per lb) on a dry diet basis, or 1,000 times normal levels, can be tolerated by sheep for many weeks without visible toxic effects. Characteristic signs of chronic cobalt toxicity in most species are reduced feed intake and body weight, emaciation, anemia, hyperchromenia, and increased liver cobalt (NRC, 1980). Cobalt toxicity in cattle is characterized by mild polycythemia; excessive urination, defecation and salivation; shortness of breath; and increased hemoglobin, red cell count and packed cell volume (NRC, 2000).
Vitamin B12, their types, clinical manifestations and aetiology of B12 deficiency, its types and normal absorption mechanism Vitamin B12, also called cobalamin, is a water-soluble vitamin involved in the optimal functioning of the hemopoetic, neuro-cognitive and vascular systems. It is involved in DNA synthesis, fatty acid metabolism and energy production (Yamada 2013). Vitamin B12 exerts its physiological effects by facilitating the methylation of homocysteine to methionine which is later activated into S-adenosyl methionine that donates its methyl group to methyl acceptors (Bottiglieri et al 2000). Similarly, vitamin B12 mediates the conversion of methyl malonyl coenzyme A (coA) to succinyl coA, a process when hindered, results in accumulation of serum methylmalonic acid (MMA) thereby causing defective fatty acid synthesis of the neuronal membranes (Malouf and Areosa 2003). Vitamin B12 (cobalamin) plays an important role in DNA synthesis and neurological function. Deficiency can lead to a wide spectrum of hematologic and neuropsychiatric “The detection and correction of vitamin B12 deficiency prevents megaloblastic anemia and potentially irreversible neuropathy and neuropsychiatric changes”. -Harrington 2016 . 63 disorders that can often be reversed by early diagnosis and prompt treatment (Robert and Brown 2003). Adequate serum levels are necessary for nervous system maintenance and the development of normal red blood cells (Butler et al 2006). Vitamin B12 cannot be synthesised in the body and must therefore be obtained from the diet. B12 deficiency can cause several forms of anemia, most notably pernicious anemia. The earlier treatments of pernicious anemia have a rather fascinating story. Up until the late 1920’s pernicious anemia was untreatable and fatal. Three American physiologists (William Murphy, George Minot and George Whipple) devised the concept that food could be used to treat pernicious anemia. The diet they constructed containing liver “in such quantities [that] seemed very outrageous” had dramatic beneficial effects on the once untreatable pernicious anemia (Minot and Murphy 1926). In 1934, the three colleagues were awarded the Nobel Prize in physiology and medicine. Despite the proven efficacy of the liver therapy, a more satisfactory method of treatment than the daily consumption of a half pound of liver was needed. An American doctor named William Bosworth Castle devised an idea that enabled him to investigate the pathophysiology of this disease. Liver seemed to be necessary for the patients’ bone marrow to function properly and Castle questioned why normal people did not have to eat such large amounts of liver to stave off pernicious anemia. Castle proposed a theory that an “intrinsic factor” is secreted by the stomach of normal healthy individuals which is required for the formation of an “anti-pernicious anemia complex [from an] extrinsic factor”, present in beef and liver (Castle 1929). The B12 molecule was first isolated in its cyano-form in 1948 and was then identified as the active component of the “extrinsic factor” proposed by Castle (Smith 1948). The chemical structure of the B12 molecule was later confirmed, using X-ray crystallography by Dorothy Hodgkin in 1956. She was subsequently awarded the Nobel Prize in chemistry for her significant findings (Smith 2008). a) Coenzyme forms/Types of Vitamin B12 Vitamin B12 is a rather large molecule. One part of its structure is a “corrin” nucleus, which resembles the “haem” of haemoglobin. While the haem moiety in haemoglobin holds an atom of iron, in B12 the corrin group holds an atom of cobalt. In order to be true B12, cobalamin must have one of a number of attachments to the corrin group: . 64 depending on the attachment, cobalamin can be cyanocobalamin, hydroxycobalamin, aquacobalamin, nitritocobalamin, methylcobalamin, or adenosylcobalamin. Only two of these cobalamins are active as co-enzymes in the human body: methylcobalamin and adenosylcobalamin. Most supplemental B12 is supplied as cyanocobalamin, in which form it is stable; cyanocobalamin must be converted to methyl- or adenosylcobalamin before it is biologically active (Harrington 2012). Vitamin B12 is synthesised by micro-organisms and enters the diet with food of animal origin. Plants do not require B12 for any function, and therefore have no mechanisms to produce or store B12. The biosynthesis of this nutrient never seems to have made the transition to the higher, eukaryotic forms of life. In humans, the vitamin is required in trace amounts (approximately 1 µg/day) to act as a co-enzyme to two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase (Ehrenpreis 2002; Schjonsby 1989). B12 is the only co-enzyme for methylmalonylCoA mutase, which catalyses the conversion of methylmalonyl-CoA to succinyl-CoA. When adequate B12 is not available, methylmalonyl-CoA production increases. Because it is toxic, methylmalonyl CoA is then rapidly converted to methylmalonic acid (MMA), which accumulates in the blood and urine. Since this reaction only requires B12 as a co-enzyme, MMA levels are a good indicator of B12 status (Harrington 2012). Vitamin B12 is a complex compound that is converted into several coenzymes. It is used for shifting of hydrogen between carbon atoms, usually in conjunction with a shift of some other group (e.g., NH2, or CH3); Vitamin B12 can also act as a methyl group carrier, accepting the carbon from tetrahydrofolate derivatives. In humans, vitamin B12 has only two known functions: 1) synthesis of methionine from homocysteine and 2) the rearrangement of methylmalonyl-CoA (from odd chain fatty acid metabolism and some amino acids) to succinyl-CoA. The structures below include the structure of the actual vitamin and of the major coenzyme forms found in humans. (The cyanide group in cyanocobalamin is not necessarily present, and is typically an artifact of purification.) 5´-Deoxyadenosyl cobalamin is the coenzyme required by methylmalonyl-CoA mutase, while methylcobalamin acts as the methylgroup acceptor and donor during the methionine synthase reaction (Brandt 2011). . 65 Vitamin B12 is a collective term for a group of cobalt-containing compounds known as corrinoids which when assembled with 5th and 6th position ligands are known as cobalamins. Vitamin B12 (cyanocobalamin, CNCbl) was discovered in the first half of the 20th century and identified as the anti-pernicious anemia factor. The two coenzyme forms, 5 - deoxy-5 -adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl), were discovered later on (Hodgkin et al 1956; Lenhert and Hodgkin 1961). Cobalamin (Cbl) is necessary for synthesis of DNA bases, transfer of methyl group [i.e. regeneration of S-adenosylmethionine (SAM)], as well as metabolism of branched chain amino acids and fatty acids with an odd number of carbon atoms. There are a variety of Cbl forms, which share the core structure of Cbl but contain different upper ligands. Also the physiological forms of cobalamin (hydoxycobalamin, adenosylcobalamin and methylcobalamin) are available as supplements with different routes of administrations (Obeid et al 2015) as: Yang et al 2015 *The corrin ring of vitamin B12 is similar but not identical to the porphyrin ring found in heme-containing proteins. Vitamin B12 is not used as a source of heme. Figure 2.7.1: Chemical structures of vitamin B12 forms 1. Cyanocobalamin (CNCbl) This synthetic version of vitamin B12 is created in a lab, which makes it the cheapest supplement option. It offers the most stable form of B12, although it does so through the presence of a cyanide molecule. While the amount of cyanide is not dangerous, it does require the body to expend energy to convert and remove it. CNCbl is a stable and inexpensive synthetic form commonly used for food fortification and oral or parenteral supplements. . 66 2. Methylcobalamin (MeCbl) This is the most active form in the human body. It converts homocysteine into methionine, which helps protect the cardiovascular system. Methylcobalamin also offers overall protection to the nervous system. This B12 form can also cross the blood-brain barrier–without assistance–to protect brain cells. It contributes essential methyl groups needed for detoxification and to start the body’s biochemical reactions. In mammalian cells, MeCbl is a cofactor for the cytosolic methionine synthase. 3. Hydroxocobalamin (HOCbl) Hydroxycobalamin is an abundant and physiologically relevant intermediate form. Exposure of aerated aqueous solutions of MeCbl and AdoCbl to ambient light causes formation of HOCbl in vitro. Bacteria naturally create this form of vitamin B12, making it the main type found in most foods. It easily converts into methylcobalamin in the body. Hydroxocobalamin is commonly used via injection as a treatment for B12 deficiency as well as a treatment for cyanide poisoning. 4. Adenosylcobalamin (AdoCbl) AdoCbl is a cofactor for the mitochondrial methylmalonyl-CoA (MM-CoA) mutase. The energy formation that occurs during the Citric Acid cycle requires this form of B12. Although naturally occurring, it is the least stable of the four types of B12 outside the human body and does not translate well into a tablet-based supplement. It can be difficult to find this one in supplement form. When supplemented, CNCbl needs to be converted into MeCbl and AdoCbl in order to exert its anticipated biological effect on the cell. The concept of replacing CNCbl/HOCbl with the coenzyme forms as ready-to-use sources of the cofactors has recently emerged. Supplementation of MeCbl and AdoCbl is postulated to be more effective than that of CNCbl/HOCbl (Thakkar and Billa 2015; Zhang and Ning 2008). Clinical Manifestations: Vitamin B12 deficiency is associated with hematologic, neurologic, and psychiatric manifestations (Table 2.7.1). It is a common cause of macrocytic (megaloblastic) anemia and, in advanced cases, pancytopenia. Neurologic sequelae from vitamin B12 . 67 deficiency include paresthesias, peripheral neuropathy, and demyelination of the corticospinal tract and dorsal columns (subacute combined systems disease). Vitamin B12 deficiency also has been linked to psychiatric disorders, inclu . 68 Normal absorption mechanism of vitamin B12 in action The main dietary sources of B12 are dairy products, meat (especially liver) and eggs. When humans eat animal foods, the B12 they consume is protein bound. The acidic environment of the stomach enables the release of B12 that is bound to food (Robert and Brown 2003). The free B12 is then rapidly bound by intrinsic factor (IF), a muco-polysaccharide secreted by the gastric parietal cells that line the stomach. The binding of B12 to IF occurs in the duodenum causing the formation of the IF-B12 complex (Butler et al 2006). This complex is resistant to digestion by gastric juices. Upon reaching the terminal ileum, it binds to and is absorbed by the intestinal microvilli. Approximately 1 percent of a large oral dose of vitamin B12 is absorbed by this second mechanism (Elia 1998). This pathway is important in relation to oral replacement. In the plasma, about 20% of the absorbed B12 binds to the serum protein holotranscobalamin (Holo-TC) for transport (Hin et al 2006). Holo-TC is the protein that delivers bound B12 to all cells in the body. The majority of B12 (80%) circulating in the blood binds to the serum protein haptocorrin and is biologically unavailable for most cells (Hvas and Nexo 2006). The function of haptocorrin remains unknown. The interruption of one or any combination of these steps places a person at risk of developing deficiency (Figure 2.7.2). . 69 Robert and Brown 2003 Figure 2.7.2: Vitamin B12 absorption and transport. . 70 Methylmalonic Acid and Homocysteine In humans, only two enzymatic reactions are known to be dependent on vitamin B12. In the first reaction, methylmalonic acid is converted to succinyl-CoA (a necessary component of the citric acid cycle) using vitamin B12 as a cofactor (Figure 2.7.3). Vitamin B12 deficiency, therefore, can lead to increased levels of serum methylmalonic acid. In the second reaction, homocysteine is converted to methionine by using vitamin B12 and folic acid as cofactors. The latter reaction is accompanied by the conversion of methyltetrahydrofolate to tetrahydrofolate, which is necessary for efficient DNA synthesis (Hvas and Nexo 2006). Therefore, a deficiency in B12 can impair DNA synthesis and may lead to increased homocysteine levels (Robert and Brown 2003; Martin 1998). Stabler 1995 Figure 2.7.3: Vitamin B12 deficiency leads to a serum build-up of methylmalonic acid. Deficiency of vitamin B12 or folic acid can lead to increased homocysteine levels. It has been hypothesized that a pathway of oxidation of homocysteine to homocysteic acid is the potential explanation of the dangerous effect of homocysteine. Elevated levels of homocysteine in the blood predispose to arteriosclerosis and stroke (Lipton . 71 et al 1997). Indeed it has been recently estimated that as many as 47% of patients with arterial occlusions manifest modest elevations in plasma homocysteine. Included among the many causes are genetic alterations in enzymes such as cystathionine betasynthase, a defect found in 1-2% of the general population, and deficiencies in vitamins B6, B12, and folate whose intake is suboptimal in perhaps 40% of the population (Perry et al 1995). Aetiology of Vitamin B12 deficiency- The causes of vitamin B12 deficiency can be divided into three classes: i) nutritional deficiency, ii) malabsorption syndromes and iii) other gastrointestinal (GI) causes (Snow 1999). i). Nutritional deficiency: Dietary sources of vitamin B12 are primarily meats and dairy products. Normally, humans maintain a large vitamin B12 reserve, which can last two to five years even in the presence of severe malabsorption. Nevertheless, nutritional deficiency of B12 can occur in specific populations. Elderly patients with “tea and toast” diets and chronic alcoholics are at especially high risk due to the dietary deficits of B12 frequently found within these groups. The dietary limitations of strict vegans make them another, less common at-risk population. Table 2.7.2: Etiologies of Vitamin B12 Deficiency Nutritional deficiency Inadequate intake (e.g., alcoholics, elderly, vegans) Malabsorption syndromes Food-bound B12 malabsorption Prolonged use of proton pump inhibitors Prolonged use of histamine H2 receptor blockers Lack of intrinsic factor or parietal cells Pernicious anemia Atrophic gastritis Postgastrectomy Other gastrointestinal causes Ileal malabsorption Enteritis (Crohn’s disease) Ileal resection Biologic competition Bacterial overgrowth Tapeworm infestation Defective transport Transcobalamin II deficiency Snow 1999 . 72 ii.) Malabsorption syndromes: The primary example of a malabsorption syndrome is pernicious anaemia. This condition is the result of an autoimmune disease in which antibodies attack the parietal cells of the stomach, almost completely blocking the release of IF as a result. This hindered IF release prevents the formation of the IF-B12 complex, subsequently impairing B12 absorption. Researchers now believe there is an age-associated decline in the intestinal absorption of B12 (Hin et al 2006). Therefore, it comes as no surprise that B12 deficiency has been reported in about 15% of adults older than 65 years. Laboratory evidence of parietal cell antibodies is approximately 85 to 90 percent sensitive for the diagnosis of pernicious anemia. However, the presence of parietal cell antibodies is nonspecific and occurs in other autoimmune states. Intrinsic factor antibody is only 50 percent sensitive, but it is far more specific for the diagnosis of pernicious anemia (Robert and Brown 2003). The phenomenon of food-bound malabsorption occurs when vitamin B12 bound to protein in foods cannot be cleaved and released. Any process that interferes with gastric acid production can lead to this impairment. Atrophic gastritis, with resulting hypochlorhydria, is a major cause, especially in the elderly (Stabler 1995). Subtotal gastrectomy, once common before the availability of effective medical therapy for peptic ulcer disease, also can lead to vitamin B12 deficiency by this mechanism. The widespread and prolonged use of histamine H2-receptor blockers and proton pump inhibitors for ulcer disease also may cause impaired breakdown of vitamin B12 from food, causing malabsorption and eventual depletion of B12 stores. Studies in the past have confirmed that long-term use of omeprazole can lead to lower serum vitamin B12 levels (Marcuard et al 1994; Termanini et al 1998). While more studies are needed to identify the incidence and prevalence of vitamin B12 deficiency in this subset of patients, screening for subclinical B12 deficiency should be a consideration in patients who have received long-term acid-suppression therapy (Bradford and Taylor 1999). iii.) Other GI causes: Although quite rare, certain GI conditions can also cause B12 deficiency. If a patient has an intestinal parasite infestation such as Diphyllobothrium latum (fish tapeworm) this may compete with the intestinal microvilli for the absorption of B12 (Snow 1999). Similarly, other etiologies of vitamin B12 deficiency, although less common, deserve mention. Patients with evidence of vitamin B12 . 73 deficiency and chronic gastrointestinal symptoms such as dyspepsia, recurrent peptic ulcer disease, or diarrhea may warrant evaluation for such entities as Whipple’s disease (a rare bacterial infection that impairs absorption), Zollinger-Ellison syndrome (gastrinoma causing peptic ulcer and diarrhea), or Crohn’s disease. Patients with a history of intestinal surgery, strictures, or blind loops may have bacterial overgrowth that can compete for dietary vitamin B12 in the small bowel, as can infestation with tapeworms or other intestinal parasites. Congenital transport-protein deficiencies, including transcobalamin II deficiency, are another rare cause of vitamin B12 deficiency (Robert and Brown 2003). Vitamin B12 deficiency is common in the elderly, affecting as much as 10% to 15% people over the age of 60 as a consequence of inadequate intake or malabsorption (Baik and Russell 1999). The incidence, however, appears to increase with age (Robert and Brown 2003).Vitamin B12 deficiency causes a classical neurological and hematological syndrome (Langan and Zawistoski 2011; Savage and Lindenbaum 1995) and has long been associated with cognitive and psychiatric disturbances (Strachan and Henderson 1965). Vitamin B12 deficiency has been widely implicated in the milder forms of cognitive decline as observed in a consecutive series of patients attending a memory clinic, 3.3% of the MCI patients had low serum vitamin B12 values (Pereira et al 2006). In another studies, non-demented cognitively impaired elderly patients with vitamin B12 deficiency had lower verbal fluency scores as compared to those with normal values of vitamin B12 and poorer performances in spatial copying test (Eastley et al 2000; Riggs et al 1996). In a community-based sample of non-demented elderly subjects, both normal and cognitively impaired, the values of methylmalonic acid (MMA), which inversely reflects the level of vitamin B12, were associated with worse performances in language and praxis tests (McCracken et al 2006). Similarly, in a sample of non-demented subjects older than 75 years, low levels of vitamin B12 were associated with decreased performance in a modified block design test, which evaluates abstraction and visuospatial abilities (Robins et al 2001). Thus, vitamin B12 profoundly affects the various cognitive domains in the non-demented elderly. . 74 Mode of Vitamin B12 therapy In a cohort study, all (n=15) vitamin B12 deficient MCI patients were started on vitamin B12 therapy (1 mg orally or 1 mg by intramuscular administration every month) and the normalization of serum levels was achieved (Silva et al 2013). B12 supplementation is now widely used for the treatment of B12 deficiency. Mostly the B12 deficient individuals are treated with an intramuscular B12 injection (Butler et al 2006). Treatment schedules for intramuscular administration vary widely but usually consist of initial loading doses followed by monthly maintenance injections. One regimen consists of daily injections of 1,000 mcg for one to two weeks, then a maintenance dose of 1,000 mcg every one to three months (Robert and Brown 2003). Table 2.7.3: Schedule for Vitamin B12 Therapy Route of administration Initial dosage Maintenance dosage Oral 1,000 mcg per day for one to two weeks 1,000 to 2,000 mcg per day for life Intramuscular 100 to 1,000 mcg every day or every other day for one to two weeks 100 to 1,000 mcg every one to three months Robert and Brown 2003 Beneficial impact of Vitamin B12 Experiments carried out largely have highlighted that B vitamins are important for the methylation and assembly of phospholipids (da Silva et al 2014; Akesson et al 1982). Vogiatzoglou et al (2008) in their study confirmed that the atrophy rate of the brain is faster at low plasma vitamin B-12 concentrations. In a brain aging study involving 49 cognitively normal individuals (age 25–72 years, 69% women) with dietary information, 11C-PiB (11C-Pittsburgh Compound-B- PiB: a measure of amyloid-β (Aβ) load) and 18F-FDG PET (18F-fluorodeoxyglucose –FDG: a proxy of neuronal activity) scan examinations illustrated that the higher intake of vitamin B12 from food sources was associated with lower Aβ load in cognitively normal individuals (Mosconi et al 2014). . 75 Role of Vitamin B and homocysteine Some studies have shown that higher intakes of vitamin B12 and folate are related to better cognitive functioning or lower AD risk in the elderly (Bryan et al 2004; Corrada et al 2005; La et al 1997; Luchsinger et al 2007; Morris et al 2005; Durga et al 2007; Morris et al 2006), possibly due to their ability to reduce homocysteine levels, although results are not conclusive (Morris et al 2006). The central nervous system requires a constant supply of glucose, and adequate brain function and maintenance depend on almost all the essential nutrients. For those B vitamins that participate in one-carbon metabolism (i.e. folate, vitamin B12, and vitamin B6) deficiency of the enzymes involved in these pathways is associated with severe impairment of brain function. As shown in figure 2.7.4 vitamin B12 participate in methylation process. The de novo synthesis of methionine requires vitamin B12, which is involved directly in the transfer of the methyl group to homocysteine (Wang 2001). Wang 2001 Figure 2.7.4: Metabolic relationship between folate, Vitamin B12 and homocysteine In case of folate or vitamin B12 deficiency, the methionine synthetase reaction is severely impaired. In particular, vitamin B12 is the necessary coenzyme, adequate for . 76 the correct functioning of the methyl donation from 5 methyltethrahydrofolate in tetrhahydrofolate, necessary for methionine synthetase. The folic acid “obliges” the entire vitamin B12 to subserve as coenzyme, and therefore enforces the otherwise limited damage caused by the vitamin B12 defect (Moretti 2001). Diagnosis of vitamin B12 deficiency Serum vitamin B12 (cobalamin) testing has long been – and continues to be – recommended as part of the routine screening of patients with dementia (American Psychiatric Association 2007). The diagnosis of vitamin B12 deficiency has traditionally been based on low serum vitamin B12 levels, usually less than 200 pg per mL (150 pmol per L), along with clinical evidence of disease. However, studies indicate that older patients tend to present with neuropsychiatric disease in the absence of hematologic findings (Lee 1999; Lindenbaum et al 1988). The APA recommendation reflects the widespread assumption that B12 deficiency is a reversible cause of dementia, or at least is commonly associated with cognitive impairment that may be partially correctable. A full diagnostic work-up is frequently not undertaken until a dementing illness is well established; and when B12 deficiency is diagnosed in a demented patient, it is usually not the only (or even primary) etiological process involved. Furthermore, B12 deficiency is often incidentally detected during the course of a dementia of other etiology (e.g. Alzheimer’s disease). In such circumstances there is little prognostic data available to guide clinicians regarding the likely response to B12 replacement therapy (Dyck et al 2009). Further diagnostic challenges are posed by patients who present with subtle B12 deficiency (Carmel et al 1987) or B12-related neuropsychiatric syndromes without anemia or macrocytosis (Lindenbaum et al 1988). These diagnostic uncertainties prompted the quest for biochemical markers of tissue B12 deficiency and the development of assays for two metabolites that accumulate if B12 is lacking: methylmalonic acid (MMA) and homocysteine (Hcys) (Stabler et al 1986; 1988). The measurement of serum MMA and Hcys in combination with serum B12 levels has enlarged the understanding of B12 deficiency considerably beyond the classically described megaloblastic anemia and subacute combined systems disease (Dyck et al 2009). The MMA and Hcys metabolite measurements have been shown to be more sensitive in . 77 the diagnosis of vitamin B12 deficiency than measurement of serum B12 levels alone (Stabler 1995; Savage et al 1994; Sumner et al 1996; Frenkel and Yardley 2000; Lindenbaum et al 1990 ; Snow 1999). This finding suggests that MMA and Hcys levels can be early markers for tissue vitamin B12 deficiency, even before hematologic manifestations occur. Vitamin B12 or folic acid deficiency can cause the Hcys level to rise. Also, MMA levels can be elevated in patients with renal disease (the result of decreased urinary excretion); thus, elevated levels must be interpreted with caution (Savage et al 1994). In the cross-sectional Banbury B12 study carried by Dr. Harold Hin and his colleagues (2006) on 1,000 community-dwelling individuals more than 75 years, the associations of cognitive impairment, depression and neuropathy with blood measurements of B12 in elderly were examined. 13% (125 free-living) participants having the serum B12 concentration of less that 133 pmol L¯¹ were deemed to be B12 deficient. Low B12 concentrations correlated with cognitive impairment. A further finding was that participants with B12 levels in the bottom quartile had a two-fold risk of cognitive impairment, when compared to those in the top quartile. Low B12 levels were also associated with peripheral neuropathy, based on the findings that the B12 deficient participants were observed to have missing knee and ankle jerk reflexes. Vitamin B12 and neuropsychiatric symptoms Data obtained from the literature state that vitamin B12 is somehow bound to cognition and to the implementation of active strategies to coordinate and do well in active problem solving (Savage et al 1994). Psychiatric symptoms attributable to vitamin B12 deficiency have been described for decades. These symptoms seem to fall into several clinically separate categories: slow cerebration, confusion, memory changes, delirium with or without hallucinations and or delusions, depression, acute psychotic states, and more rarely, reversible manic and schizophreniform states (Nillson 1998. A higher prevalence of lower serum vitamin B12 levels have been found in subjects with AD, other dementias and in people with different cognitive impairments, as compared with controls (Bell et al 1998). In the prevalence of low vitamin B12 serum . 78 levels is consistent with that found in community-dwelling elderly persons in general but is associated with greater overall cognitive impairment. (Whyte et al 2002). Furthermore, some intervention studies have shown the effectiveness of vitamin B12 supplementation in improving cognition in demented or cognitively impaired subjects. Chronic dementia responds poorly but should nevertheless be treated if there is a metabolic deficiency (Nillson etal 1998) However, a treatment effect was demonstrated among the patients presenting with cognitive impairment, improving when compared to matched patients on the verbal fluency test. A prospective investigation by Cunha et al (1995) on a total of 181 consecutive, demented (DSMIII or DSMIIIR criteria and score below 24 on the MMSE) elderly outpatients demonstrated beneficial effects on cognitive function of demented and cobalamin deficient patients after cobalamin replacement. The frequency of vitamin B12 deficiency (less than 200 pg/mliter) was 25% (46 patients) and the treatment outcome obtained in 19 patients (19 of 46) showed improvement (MMSE returned to normal values) with all having the mild dementia history of less than 2 years. The implications from this study suggest that screening for B12 deficiency should be considered in patients with recent onset of mild mental status changes. The extensive investigations conducted in the Homocysteine and B Vitamins in Cognitive Impairment (VITACOG), randomized clinical trial with homocysteinelowering B vitamins in older people with MCI, showed that treatment with high doses of B vitamins markedly reduced the global brain atrophy rate, as well as atrophy rates in those gray matter regions most commonly associated with AD (Smith et al 2010, Douaud et al 2013). In 2012, Blasko and co-workers in a prospective cohort with a retrospective vitamin intake evaluation of older adults (N=81) with MCI from the Vienna Transdanube aging study reported that users of vitamin B12 or folate, independent of time and pattern of use, had lower grades of peri-ventricular hyperintensities and deep white matter lesions associated with a lower conversion rate to dementia as compared to nonusers. . 79 Reports of patients with cognitive deficits showed notable improvement, namely in language and frontal lobe functions, after the vitamin B12 supplementation (Eastley et al 2000). Several cross-sectional and longitudinal studies performed in both healthy and cognitively impaired older subjects reported inverse associations between vitamin B12 levels and the degree of cognitive impairment (Engelborghs et al 2004; Elias et al 2006; Hin et al 2006; Tangney et al 2009). On the contrary, some studies found no association between the two (Ariogul et al 2005; Paulionis et al 2005; Kang et al 2006; Faux et al 2011). Moreover, a review also found that vitamin B12 deficiency is associated with cognitive impairment, but supplementation did not improve cognitive function in patients with previous deficits (Moore et al 2012). Similarly the findings from a retrospective monocentric study conducted on 125 cobalamin deficient elderly patients over 14 months having received oral (88.8%) and intramuscular (11.2%) cobalamin supplementation, depicted a significant increase of Vitamin B12 levels (p<0.001) but it proved to be less effective in patients with dementia (p=0.04) (Couderc et al 2015). Contrary to this, a review based on evidence from the limited studies stated that daily oral dose of 2000 mcg vitamin B12, an initial daily 1000 mcg doses, thereafter weekly and then monthly may be as effective as intramuscular administration for obtaining short term haematological and neurological responses in vitamin B12 deficient patients (Vidal-Alaball et al 2005). Wellmer et al (2006) suggested from their case study of successful oral vitamin B12 that a monitored oral substitution therapy should be used as the first line therapy for neurological disorders related to vitamin B12 deficiency. It could be concluded that that vitamin B12 treatment may improve frontal lobe and language function in patients with cognitive impairment, but rarely reverses dement
Biochemistry of B12-Cofactors in Human Metabolism (Krautler 2012)
Vitamin B12, the “antipernicious anaemia factor”, is a crystallisable cobalt-complex, which belongs to a group of unique “complete” corrinoids, named cobalamins (Cbl). In humans, instead of the “vitamin”, two organometallic B12-forms are coenzymes in two metabolically important enzymes: Methyl-cobalamin, the cofactor of methionine synthase, and coenzyme B12 (adenosyl-cobalamin), the cofactor of methylmalonyl-CoA mutase. The cytoplasmatic methionine synthase catalyzes the transfer of a methyl group from N-methyl-tetrahydrofolate to homocysteine to yield methionine and to liberate tetrahydrofolate. In the mitochondrial methylmalonyl-CoA mutase a radical process transforms methylmalonyl-CoA (a remains e.g. from uneven numbered fatty acids) into succinyl-CoA, for further metabolic use. In addition, in the human mitochondria an adenosyl-transferase incorporates the organometallic group of coenzyme B12. In all these enzymes, the bound B12-derivatives engage (or are formed) in exceptional organometallic enzymatic reactions. This chapter recapitulates the physiological chemistry of vitamin B12, relevant in the context of the metabolic transformation of B12-derivatives into the relevant coenzyme forms and their use in B12-dependent enzymes.
Vitamin B12, the “antipernicious anaemia factor”, is a crystallisable cobalt-complex, which belongs to a group of unique “complete” corrinoids, named cobalamins (Cbl). In humans, instead of the “vitamin”, two organometallic B12-forms are coenzymes in two metabolically important enzymes: Methyl-cobalamin, the cofactor of methionine synthase, and coenzyme B12 (adenosyl-cobalamin), the cofactor of methylmalonyl-CoA mutase. The cytoplasmatic methionine synthase catalyzes the transfer of a methyl group from N-methyl-tetrahydrofolate to homocysteine to yield methionine and to liberate tetrahydrofolate. In the mitochondrial methylmalonyl-CoA mutase a radical process transforms methylmalonyl-CoA (a remains e.g. from uneven numbered fatty acids) into succinyl-CoA, for further metabolic use. In addition, in the human mitochondria an adenosyl-transferase incorporates the organometallic group of coenzyme B12. In all these enzymes, the bound B12-derivatives engage (or are formed) in exceptional organometallic enzymatic reactions. This chapter recapitulates the physiological chemistry of vitamin B12, relevant in the context of the metabolic transformation of B12-derivatives into the relevant coenzyme forms and their use in B12-dependent enzymes.
COBALAMIN DEFICIENCY (Herrman 2012)
Cobalamin (Cbl, vitamin B12) consists of a corrinoid structure with cobalt in the centre of the molecule. Neither humans nor animals are able to synthesize this vitamin. Foods of animal source are the only natural source of cobalamin in human diet. There are only two enzymatic reactions in mammalian cells that require cobalamin as cofactor. Methylcobolamin is a cofactor for methionine synthase. The enzyme methylmalonyl-CoA-mutase requires adenosylcobalamin as a cofactor. Therefore, serum concentrations of homocysteine (tHcy) and methylmalonic acid (MMA) will increase in cobalamin deficiency. The cobalamin absorption from diet is a complex process that involves different proteins: haptocorrin, intrinsic factor and transcobalamin (TC). Cobalamin that is bound to TC is called holotranscobalamin (holoTC) which is the metabolically active vitamin B12 fraction. HoloTC consists 6 and 20% of total cobalamin whereas 80% of total serum cobalamin is bound to another binding protein, haptocorrin. Cobalamin deficiency is common worldwide. Cobalamin malabsorption is common in elderly subjects which might explain low vitamin status. Subjects who ingest low amount of cobalamin like vegetarians develop vitamin deficiency. No single parameter can be used to diagnose cobalamin deficiency. Total serum cobalamin is neither sensitive nor it is specific for cobalamin deficiency. This might explain why many deficient subjects would be overlooked by utilizing total cobalamin as status marker. Concentration of holotranscobalamin (holoTC) in serum is an earlier marker that becomes decreased before total serum cobalamin. Concentrations of MMA and tHcy increase in blood of cobalamin deficient subjects. Despite limitations of these markers in patients with renal dysfunction, concentrations of MMA and tHcy are useful functional markers of cobalamin status. The combined use of holoTC and MMA assays may better indicate cobalamin status than either of them. Because Cbl deficiency is a risk factor for neurodegenerative diseases an early diagnosis of a low B12 status is required which should be followed by an effective treatment in order to prevent irreversible damages.
Cobalamin (Cbl, vitamin B12) consists of a corrinoid structure with cobalt in the centre of the molecule. Neither humans nor animals are able to synthesize this vitamin. Foods of animal source are the only natural source of cobalamin in human diet. There are only two enzymatic reactions in mammalian cells that require cobalamin as cofactor. Methylcobolamin is a cofactor for methionine synthase. The enzyme methylmalonyl-CoA-mutase requires adenosylcobalamin as a cofactor. Therefore, serum concentrations of homocysteine (tHcy) and methylmalonic acid (MMA) will increase in cobalamin deficiency. The cobalamin absorption from diet is a complex process that involves different proteins: haptocorrin, intrinsic factor and transcobalamin (TC). Cobalamin that is bound to TC is called holotranscobalamin (holoTC) which is the metabolically active vitamin B12 fraction. HoloTC consists 6 and 20% of total cobalamin whereas 80% of total serum cobalamin is bound to another binding protein, haptocorrin. Cobalamin deficiency is common worldwide. Cobalamin malabsorption is common in elderly subjects which might explain low vitamin status. Subjects who ingest low amount of cobalamin like vegetarians develop vitamin deficiency. No single parameter can be used to diagnose cobalamin deficiency. Total serum cobalamin is neither sensitive nor it is specific for cobalamin deficiency. This might explain why many deficient subjects would be overlooked by utilizing total cobalamin as status marker. Concentration of holotranscobalamin (holoTC) in serum is an earlier marker that becomes decreased before total serum cobalamin. Concentrations of MMA and tHcy increase in blood of cobalamin deficient subjects. Despite limitations of these markers in patients with renal dysfunction, concentrations of MMA and tHcy are useful functional markers of cobalamin status. The combined use of holoTC and MMA assays may better indicate cobalamin status than either of them. Because Cbl deficiency is a risk factor for neurodegenerative diseases an early diagnosis of a low B12 status is required which should be followed by an effective treatment in order to prevent irreversible damages.
Physiological and Molecular Aspects of Cobalamin Transport (Fedosev 2012)
Minute doses of a complex cofactor cobalamin (Cbl, vitamin B12) are essential for metabolism. The nutritional chain for humans includes: (1) production of Cbl by bacteria in the intestinal tract of herbivores; (2) accumulation of the absorbed Cbl in animal tissues; (3) consumption of food of animal origin. Most biological sources contain both Cbl and its analogues, i.e. Cbl-resembling compounds physiologically inactive in animal cells. Selective assimilation of the true vitamin requires an interplay between three transporting proteins – haptocorrin (HC), intrinsic factor (IF), transcobalamin (TC) – and several receptors. HC is present in many biological fluids, including gastric juice, where it assists in disposal of analogues. Gastric IF selectively binds dietary Cbl and enters the intestinal cells via receptor-mediated endocytosis. Absorbed Cbl is transmitted to TC and delivered to the tissues with blood flow. The complex transport system guarantees a very efficient uptake of the vitamin, but failure at any link causes Cbl-deficiency. Early detection of a negative B12 balance is highly desirable to prevent irreversible neurological damages, anaemia and death in aggravated cases. The review focuses on the molecular mechanisms of cobalamin transport with emphasis on interaction of corrinoids with the specific proteins and protein-receptor recognition. The last section briefly describes practical aspects of recent basic research concerning early detection of B12-related disorders, medical application of Cbl-conjugates, and purification of corrinoids from biological samples.
Minute doses of a complex cofactor cobalamin (Cbl, vitamin B12) are essential for metabolism. The nutritional chain for humans includes: (1) production of Cbl by bacteria in the intestinal tract of herbivores; (2) accumulation of the absorbed Cbl in animal tissues; (3) consumption of food of animal origin. Most biological sources contain both Cbl and its analogues, i.e. Cbl-resembling compounds physiologically inactive in animal cells. Selective assimilation of the true vitamin requires an interplay between three transporting proteins – haptocorrin (HC), intrinsic factor (IF), transcobalamin (TC) – and several receptors. HC is present in many biological fluids, including gastric juice, where it assists in disposal of analogues. Gastric IF selectively binds dietary Cbl and enters the intestinal cells via receptor-mediated endocytosis. Absorbed Cbl is transmitted to TC and delivered to the tissues with blood flow. The complex transport system guarantees a very efficient uptake of the vitamin, but failure at any link causes Cbl-deficiency. Early detection of a negative B12 balance is highly desirable to prevent irreversible neurological damages, anaemia and death in aggravated cases. The review focuses on the molecular mechanisms of cobalamin transport with emphasis on interaction of corrinoids with the specific proteins and protein-receptor recognition. The last section briefly describes practical aspects of recent basic research concerning early detection of B12-related disorders, medical application of Cbl-conjugates, and purification of corrinoids from biological samples.