We review the role of cobalt and vitamin B12 in animals, especially in ruminants. Vitamin B12 is an essential part of the enzyme systems involved in multiple metabolic reactions and mainly in the formation of energy from ruminal fermentation. Signs of deficiency, as well as cobalt toxicity, in animals are described. The level of cobalt in ruminants can be assessed by measuring the blood or tissue concentrations of cobalt or vitamin B12, as well as the level of some metabolites such as malonate, homocysteine or transobolamine in blood or methylmalonic acid in urine. The requirement for cobalt (Co) is around 0.11 ppm (mg/kg) dry matter (DM) in the diet, although current recommendations advise supplementing the diet up to 0.20 mg Co/kg DM, which seems to increase animal production, especially in dairy cattle.
Abstract
Cobalt, as a trace element, is essential for rumen microorganisms for the formation of vitamin B12. In the metabolism of mammals, vitamin B12 is an essential part of two enzymatic systems involved in multiple metabolic reactions, such as in the metabolism of carbohydrates, lipids, some amino acids and DNA. Adenosylcobalamin and methylcobalamin are coenzymes of methylmalonyl coenzyme A (CoA) mutase and methionine synthetase and are essential for obtaining energy through ruminal metabolism. Signs of cobalt deficiency range from hyporexia, reduced growth and weight loss to liver steatosis, anemia, impaired immune function, impaired reproductive function and even death. Cobalt status in ruminant animals can be assessed by direct measurement of blood or tissue concentrations of cobalt or vitamin B12, as well as the level of methylmalonic acid, homocysteine or transcobalamin in blood; methylmalonic acid in urine; some variables hematological; food consumption or growth of animals. In general, it is assumed that the requirement for cobalt (Co) is expressed around 0.11 ppm (mg/kg) in the dry matter (DM) diet; current recommendations seem to advise increasing Co supplementation and placing it around 0.20 mg Co/kg DM. Although there is no unanimous criterion about milk production, fattening or reproductive rates in response to increased supplementation with Co, in some investigations, when the total Co of the diet was approximately 1 to 1.3 ppm (mg/kg), maximum responses were observed in the milk production.
Keywords: cobalt, vitamin B12, deficiency, ruminant, cattle
1. Introduction
Cobalt (Co) is a metallic element with an atomic weight of 58.9. It is considered an essential trace element, because it is required in the human diet and of some animal species in very small amounts, close to 100 mg per kg of dry matter [1,2,3]. As such, cobalt has no known nutritional function, except as a component of vitamin B12, so when we refer to the Co status, we are really referring to the vitamin B12 status [4,5].
It is known that cobalt (Co), in ruminants, is an essential component for the microbial synthesis of vitamin B12, a water-soluble vitamin belonging to group B, commonly known as cobalamin, cyanocobalamin or also called the pernicious antianemia factor [6]. Although, technically, vitamin B12 refers only to cyanocobalamin, actually, the term vitamin B12 is the generic name used to refer to a group of compounds that have B12 activity, such as cyano, hydroxy, methyl or deoxyadenosylcobalamin, and which are also known as complete corrinoids [7,8]. There are many different analogs and derivatives devoid of biological activities [8,9], and there are even different isoforms of cobalamin (CBL) [7].
All of them present very complex structures, being the chemically largest of all vitamins, with a molecular weight of 1355 and whose empirical formula is C63H88O14N14PCo. One of its main characteristics is to present a cobalt content between 4.4% and 5.8% [1,8,10,11,12]. The molecule is made up of four main parts: the corrin ring (similar to that found in heme, chlorophyll and cytochromes); the remaining nucleotides; the aminopropanol residue that binds the nucleotide to the corrin ring and the ligand B attached to the cobalt atom as the central nucleus of the corrin ring [12,13,14,15,16]. In cyanocobalamin (CBL), 5,6-dimethylbenzimidazole (5,6-DMB) is the base in the nucleotide fraction (Figure 1) [17].
Figure 1
Figure 1
Empirical formula of vitamin B12.
Although cobalt is known as an essential trace element for humans and animals, there are differences in the way it must be supplied in the diet of different species. While vegetables cannot synthesize this vitamin, humans and most monogastric animals (exclusive animals with cecotrophy or coprophagia) require cobalt in its active form (vitamin B12). However, in adult ruminants, vitamin B12 is produced during the microbial fermentation of food in the stomachs and, mainly, in the rumen [18]. The ruminal flora—that is, the microorganisms, bacteria and yeasts present in the rumen—can synthesize vitamin B12, provided that the cobalt concentration in the ruminal fluid is higher than 0.5 mg/mL, while if this level is not reached, the ruminal synthesis of vitamin B12 remains inhibited, reducing its contribution to blood and other tissue [6,9,19,20].
Even the ciliated protozoa present in the rumen need vitamin B12, which they obtain from ruminal bacteria that synthesize vitamin B12 [17]. In addition, these bacteria, present in the rumen, use dietary cobalt to produce vitamin B12 analogs, molecules chemically related to cyanocobalamin but devoid of biological activity [17,21]. The production of vitamin B12 by ruminal microflora, as with folates, is generally considered sufficient to avoid deficiency symptoms in ruminants [12,22], although, in steers, it has been shown that the ruminal microflora extensively destroys dietary folic acid and vitamin B12 [22,23].
As early as 1935, cobalt was shown to be an essential nutrient for ruminants when it was discovered that it corrects a disorder characterized by reduced appetite and weight loss [6]. A few years later, in 1948, it was found that cobalt was an essential component of vitamin B12 for sheep and cattle, and its lack caused conditions such as coastal disease (in sheep), wasting disease or enzootic marasmus in cattle [6]. Vitamin B12 deficiency is associated with conditions such as methylmalonic aciduria, megaloblastic anemia and pernicious anemia [6]. Its deficiency has been described in various regions around the world (Australia: Marston in 1935, New Zealand: McNaught in 1948, United States: Ammermann in 1969 and tropical regions: McDowell et al. in 1993). Animals with vitamin B12 deficiency show nonspecific clinical symptoms, such as reduced food intake, retarded growth, muscle wasting, rough coat and thickening of the skin. Reproductive disorders and decreased milk yield are often observed [4,6].
In young ruminants (preruminant lambs and calves) up to the ages of six to eight weeks, the rumen is not fully developed and not functional for the synthesis of this vitamin [18]. Therefore, they require a dietary source of vitamin B12, such as colostrum, milk or milk replacers [18,20]. In contrast, adult domestic ruminants do not necessarily depend on a dietary source of vitamin B12, because ruminal microorganisms are capable of synthesizing vitamin B12 from Co [24]. The efficiency at which Co is used by ruminal microorganisms that produce vitamin B12 is very low, and according to Smith (1987), the amount of Co in the diet converted into vitamin B12 in the rumen ranges between 3–13% of the intake [25,26]. The amount of cobalamin synthesized depends on multiple factors, including the composition of the ration (ratio of forage to concentrate; fiber content) and dry matter intake [20]. However, it has long been recognized that the most important factor for the production of vitamin B12 and analogs synthesized by ruminal bacteria is the concentration of cobalt in the diet [9,17,20]. Without cobalt in the diet, ruminal production of vitamin B12 decreases rapidly (within a few days). However, vitamin B12 stored in the liver of adult ruminants is usually sufficient to last for several months when placed on a cobalt-deficient diet [19].
Ruminants appear to be more sensitive to vitamin B12 deficiency than nonruminants, in large part because they are highly dependent on gluconeogenesis to meet their tissue glucose needs. A decompensation in propionate metabolism at the point where methylmalonyl coenzyme A (CoA) is converted to succinyl-CoA may be a primary problem arising from vitamin B12 deficiency [19].
The purpose of this contribution is to establish the existing nexus between a mineral element, cobalt, with the interpretation of its role in the synthesis of vitamin B12, as well as its participation in animal metabolism, especially in ruminants. First, we will analyze the effect of this element from a physiological point of view, and later, we will interpret the effect of both a possible deficit and an excessive cobalt ingestion. Finally, we want to provide possible diagnostic methods and some management protocols in the contribution of cobalt to the feeding of cattle and sheep.
2. Cobalt and Vitamin B12 Essential Functions
Vitamin B12 or cobalamin is an essential part of several enzyme systems that carry out a series of very basic metabolic functions. It is crucial for energy metabolism and for cell replication processes, since it behaves like a coenzyme, catalyzing intramolecular mutations and reactions of transfers of one-carbon groups [7]. Most cobalamins serve as a cofactor for important enzymes; thus, the metabolism of carbohydrates, lipids, amino acids and DNA involve reactions in which vitamin B12 is an essential cofactor [4,10].
2.1. Forms of Vitamin B12
Cyanocobalamin, the generally available form of vitamin B12, and hydroxycobalamin are nonphysiological forms of cobalamin, which are rapidly transformed in the body into methylcobalamin or deoxyadenosylcobalamin (or 5-deoxyadenosylcobalamin), the physiologically active forms or coenzymes of vitamin B12 (Figure 2) [1,9,24,25]. Cyanocobalamin on exposure to light and reducing agents rapidly changes to the form of hydroxycobalamin [10]. Hydroxycobalamin, methylcobalamin and deoxyadenosylcobalamin are chemically more labile than cyanocobalamin (Figure 1 and Figure 2) [1,19,25].
Figure 2
Figure 2
The forms of vitamin B12 and their role in mammalian metabolism. CoA: coenzyme A.
Most of vitamin B12 is found in cells, in mitochondria, in the form of 5′-deoxyadenosylcobalamin, whereas methylcobalamin is the main form of cobalamin in plasma, although small amounts of this coenzyme can be found in cells. Other Co-containing corrinoids, which are not cobalamins, have been detected in plasma and other organs, called cobalamin analogs due to their structural similarity to the vitamin, from which they differ due to alterations in the corrinic nucleus. The biological significance of these cobalamin analogs is not well-known, and although some may be inert, it is suspected that others may have coenzyme activity, be toxic or even inhibit the action of vitamin B12 [10].
2.2. Participation in Biochemical Reactions
At least 10 different vitamin B12-dependent metabolic reactions have been identified in microorganisms. However, in mammals, only two important enzyme systems are recognized in which three vitamin B12-dependent enzymes participate: methylmalonyl CoA mutase and leucine mutase, which require adenosylcobalamin as a coenzyme, and methionine synthetase, which is required by the coenzyme methylcobalamin [1,7,10,12,15,16,19,24,27,28,29].
(1) The first, methylmalonyl CoA mutase, is a mitochondrial enzyme, dependent on cobalamin, involved in the metabolism of propionate to succinate. Methylmalonyl-CoA mutase catalyzes the transformation of methylmalonyl-CoA into succinyl-CoA, where 5′adenosylcobalamin functions as a coenzyme of the mutase, allowing the transformation of methylmalonyl CoA, which, in turn, comes from the propionate formed well as a product of ruminal fermentation, of the degradation of odd-chain fatty acids or of some amino acids: valine, isoleucine, methionine and threonine [25,28]. In ruminants, methylmalonyl-CoA plays a unique regulatory role in gluconeogenesis and fatty acid oxidation [30].
In short, propionate is first transformed into propionyl-CoA; then, through the action of propionyl-CoA carboxylase, a biotin-dependent enzyme, it receives an additional carbon and is converted to methylmalonyl-CoA. Under the action of methylmalonyl-CoA mutase, a vitamin B12-dependent enzyme, the CO- succinyl-CoA (SCoA) group is transferred from one carbon to another in the molecule, forming succinyl-CoA, which can enter the Krebs cycle (Figure 3) [28]. Two molecules of adenosyl cobalamin are required to convert methylmalonyl CoA to succinyl-CoA. In the case of vitamin B12 deficiency, the activity of methylmalonyl CoA mutase is affected; methylmalonyl-CoA accumulates and degrades to methylmalonic acid [14,28,31]. This is a critical reaction for glucose homeostasis in ruminants, because propionic acid is their most important energy source and will be used as a gluconeogenic precursor (Figure 3) [27,28,32,33].
Figure 3
Figure 3
The participation of vitamin B12 in biochemical reactions. Enzymatice systems in which it participates as a coenzyme.
As previously indicated, methylmalonyl-CoA mutase is also an enzyme that participates in the degradation of odd-chain fatty acids and in the metabolic pathways that involve branched-chain amino acids and cholesterol. It catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA, which enters the Krebs cycle for catabolic utilization. The Krebs cycle accepts only two-carbon molecules, so odd-chain fatty acids could not be fully catabolized without this pathway involving the cofactor cobalamin. In the absence of adenosylcobalamin, an accumulation of methylmalonic acid (MMA) occurs as a by-product [7].
(2) Methionine synthetase (or 5-methyl-tetrahydrofolate-homocysteine methyl transferase) is an intracellular, cytosolic enzyme that requires methylcobalamin as a cofactor and that catalyzes the conversion of the amino acid homocysteine to methionine. This reaction links the metabolism of cobalamin and folate (Figure 3) [28,32]. It plays an essential role in the transfer of methyl groups between methyltetrahydrofolate (the methylated form of folic acid) and homocysteine to regenerate methionine and tetrahydrofolate, two essential compounds for the synthesis of S-adenosylmethionine and nucleic acids [14,16,25,27,28,32]. Additionally, methionine synthetase is crucial for nucleic acid synthesis (tetrahydrofolate is a precursor of purine and pyrimidine synthesis) [7].
(3) Another enzyme, leucine mutase (or L-leucine-2,3-aminomutase), participates in the isomerization of L-α-leucine to L-β-leucine. The metabolic importance of this enzyme has not been characterized but has been identified in rat liver and kidneys, sheep and monkey liver and human leukocytes [28].
So far, in dairy cows, the roles of only two vitamin B12-dependent enzymes have been described [28,31]. On the one hand is methionine synthase, described above, which plays an essential role in the transfer of one-carbon units from the methylated form of folic acid to homocysteine [28]. On the other hand is the methylmalonyl CoA mutase enzyme involved in neoglucogenesis, in the passage from propionate to succinate, facilitating its entry into the Krebs cycle (Figure 2 and Figure 3) [28]. In the opinion of Girard and Matte [28], it is likely that this metabolic pathway plays an important role in the energy metabolism of dairy cows.
Cobalt deficiency has been shown to negatively affect the immune function of ewes and calves, leading to increased susceptibility to infection in ewes, with particularly serious consequences for the viability of newborn lambs [9,34]. Even the increase in the synthesis of vitamin B12 by ruminal microbes restored neutrophil function [34,35] and was able to reduce stress [36].
3. Cobalt Metabolism: Absorption, Storage and Excretion
3.1. Factors that Modify the Production of Vitamin B12
The metabolism of cobalt (and its radioisotopes) has been studied in humans, in laboratory animals, in companion animals and in pets and, especially, as a component of vitamin B12. It is highly probable that the efficacy of the bacterial use of cobalt for the synthesis of cobalamin is influenced by multiple factors. These include the adequate contribution of cobalt [11,15,37], the ingredients and composition of the diet and their effects on fermentation and the composition of the ruminal microbiome [9,15,17,37].
The composition of the diet, and especially the forage-concentrate ratio, play a fundamental role. The synthesis of vitamin B12 is positively associated with the dietary concentrations of neutral detergent fiber (NDF) and acid detergent fiber (ADF) and is negatively correlated with the concentration of starch in the diet [38,39]. Thus, the synthesis of vitamin B12 in the rumen is three times higher in cows that receive a diet high in fiber than in cows that receive a diet high in starch [39].
Other factors, such as initial body reserves, the genetic selection of animals [40,41], the supplementation of different vitamins (alone or in combination) and even the adequacy or deficiency of other nutrients, play an important role in the production and use of vitamin B12, causing variable responses [12,17,22,30,37,40,42].
