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Cobalamin deficiency (Proceedings)
Cobalamin (vitamin B12) is a cyclic tetrapyrrol that contains a corrin ring with a cobalt atom in the center. Cobalamin is actually made up of a group of compounds and is exclusively derived from bacterial sources. The biologically active forms of this vitamin are methylcobalamin (required for methyl-group transfers) and adenosylcobalamin (required for adenosyl-group transfers), but there are other molecules that belong to this group of vitamins, such as hydroxocobalamin or cyanocobalamin.
Cobalamin (vitamin B12) is a cyclic tetrapyrrol that contains a corrin ring with a cobalt atom in the center. Cobalamin is actually made up of a group of compounds and is exclusively derived from bacterial sources. The biologically active forms of this vitamin are methylcobalamin (required for methyl-group transfers) and adenosylcobalamin (required for adenosyl-group transfers), but there are other molecules that belong to this group of vitamins, such as hydroxocobalamin or cyanocobalamin. Cyanocobalamin, does not occur naturally, but is manufactured by bacterial fermentation and cyanide incorporation for treatment of cobalamin deficiency. Cobalamin has important functions in amino acid metabolism and DNA synthesis.
Cobalamin is an essential cofactor for several enzyme systems in mammalian species. The first enzyme system, methylmalonyl-CoA mutase, is located in the mitochondria and plays a crucial role in the transformation of propionyl-CoA to succinyl-CoA. Thus, cobalamin plays a major role in the metabolism of several amino acids.
Cobalamin is also important in the transformation of the sulfur-containing amino acids methionine and cysteine. Homocysteine is an intermediary amino acid that is being formed from methionine and is not found in the diet. The transformation of homocysteine to methionine is linked to another metabolically crucial process, the generation of the biologically active tetrahydrofolate from N5-methyltetrahydrofolate. Simplistically, the cobalamin-dependant enzyme methionine synthase transfers a methyl group from N5-methyltetrahydrofolate to homocysteine, which results in tetrahydrofolate and methionine. Thus, this enzyme not only plays a role in the transformation of sulfur-containing amino acids, but may be even more important in the generation of the biologically active tetrahydrofolate, which is involved in the synthesis of both purines and pyrimidines.
Dietary cobalamin is tightly bound to dietary animal-derived protein. In the stomach, dietary protein is partially digested by pepsin and HCl and cobalamin is being released. However, cobalamin immediately binds to a transporter protein called haptocorrin or R-protein. Haptocorrin is mostly synthesized and secreted by the gastric mucosa. Haptocorrin in turn is digested by pancreatic proteases in the small intestine. Free cobalamin binds to intrinsic factor. In humans, intrinsic factor is mostly synthesized and secreted by the parietal cells of the gastric mucosa, but there is good evidence that in dogs and cats most of intrinsic factor is synthesized and secreted by pancreatic acinar cells. Cobalamin/intrinsic factor complexes are being absorbed by a complex receptor in the microvillus pits of the apical brush border membrane of the ileal enterocytes. Thus, the absorption of cobalamin is an extremely complex system that relies on a multitude of factors and processes. As cobalamin is being absorbed into the intestinal epithelial cells, it dissociates from intrinsic factor and free cobalamin is released into the circulation, where most of it binds to yet another protein, transcobalamin II. The main storage compartments for cobalamin in the body are the liver and the kidney, which maintain serum cobalamin concentrations by releasing cobalamin when needed.
Cobalamin deficiency is quite common in humans. Several causes of cobalamin deficiency have been described in humans, including hereditary causes that are due to mutations of the genes encoding carrier proteins, dietary insufficiency, postsurgical malabsorption after gastrectomy or ileal resections, food-cobalamin malabsorption, and idiopathic cobalamin deficiency. Dietary insufficiency is quite uncommon as a cause of cobalamin deficiency and mainly occurs in elderly people, who are already malnourished and consume an all vegetarian diet. Food-cobalamin malabsorption is a term that combines any cause of cobalamin deficiency that is not due to dietary deficiency, such as exocrine pancreatic insufficiency (EPI), intestinal lymphoma or tuberculosis, celiac disease or Crohn's disease.
In dogs and cats the most common causes of cobalamin deficiency are chronic and severe small intestinal disease and EPI. In addition, hereditary cobalamin deficiency has been described in various dog breeds. A recent study has shown that 82% of dogs with EPI were cobalamin deficient. Similar studies in cats have shown that most, if not all, cats with EPI are cobalamin deficient. While cobalamin deficiency can occur in humans with EPI it is certainly not as prominent of a feature as it is in dogs and cats, which can be explained by the differences in cobalamin absorption between species. As discussed above, intrinsic factor in humans is mostly supplied through the gastric mucosa, while in dogs and cats it is mostly supplied by pancreatic acinar cells. The lack of pancreatic proteases and the alteration of the small intestinal microbiota may also play a role, but appear to be less important than the lack of intrinsic factor.
Dogs and cats with severe and long-standing small intestinal disease involving the ileum may also show cobalamin deficiency. In one study, 49 of 80 cats (61%) with chronic signs of gastrointestinal disease had cobalamin deficiency, as evidenced by a subnormal serum cobalamin concentration. It is interesting to note that there is one study from the UK that would suggest that cobalamin deficiency is much less common in cats in the UK than in the USA. However, there are other reports from the UK that would suggest that cobalamin deficiency does occur frequently in cats with gastrointestinal disease in the UK. These differences are interesting as they point to difficulties of measuring serum cobalamin concentrations in dogs and cats. As in humans, there appear to be considerable cobalamin stores. It is unclear where most of the cobalamin stores are located in dogs or cats, but as in humans it takes a considerable amount of time for these body stores to be depleted if an insufficient amount of cobalamin is being absorbed. In one study the half-life for exogenously administered cobalamin in healthy cats was estimated to be between 11 and 14 days, while the half-life in cats with gastrointestinal disease was being estimated at 4.5 to 5.5 days in the same study. However, it should be noted that serum concentrations may not accurately reflect the true half life of cobalamin in cobalamin-deficient patients as body stores may get replenished with the exogenously administered cobalamin. Regardless, this study would suggest that similarly to humans, cobalamin stores are large and cobalamin malabsorption has to be long-standing before cobalamin deficiency occurs in dogs and cats.
Hereditary cobalamin deficiency has been recorded in a few dog breeds, including the Giant Schnauzer, Beagle, Border Collie, Australian Shepherd, and Chinese Shar Pei. Recently, a region of chromosome 13 has been identified that cosegregates with cobalamin deficiency in the Chinese Shar Pei, but the actual gene causing the disease has not yet been identified.
Clinical signs or complications of cobalamin deficiency have been reported in humans and veterinary species. The most important clinical signs observed in humans with cobalamin deficiency are hematological (i.e., macrocytosis, neutrophil hypersegmentation, non-regenerative macrocytic anemia, thrombocytopenia, neutropenia, pancytopenia) and neuro-psychiatric (i.e., polyneuritis, dementia, others). In addition, digestive abnormalities (i.e., villous blunting, inflammatory infiltration of the GI mucosa, cobalamin malabsorption, malabsorption of other nutrients), immunodeficiencies, and other signs (i.e., tiredness, loss of appetite) have been reported, but are still under debate.
Clinical signs or complications of cobalamin deficiency have been much less well described in dogs and cats. Most dogs and cats with cobalamin deficiency only show clinical signs of gastrointestinal disease, which could either be a cause or the effect of cobalamin deficiency. In a recent case report a Border Collie with selective cobalamin deficiency was described. The dog presented with hyperammonemic encephalopathy and fully responded to cobalamin supplementation. In another case report a juvenile Beagle presented with failure to gain weight, lethargy, intermittent vomiting, seizures, anemia, and leucopenia. This dog also fully responded to treatment with cobalamin supplementation. In a separate case report a 4-year old cat presented with severe encephalopathy and was diagnosed with an organic acidemia and cobalamin deficiency. Interestingly, in contrast to the Border Collie mentioned above, this cat had a normal plasma ammonia concentration.
Diagnosis of cobalamin deficiency
A definitive diagnosis of cobalamin deficiency can be challenging. Clinical signs are ultimately caused by cobalamin deficiency on a cellular level. However, the cellular cobalamin status is difficult to assess. Serum cobalamin concentration has been traditionally measured to help assess cobalamin status, but some patients with cobalamin deficiency on a cellular level do not always have severely decreased serum cobalamin concentrations. Thus, in order to avoid missing patients with cobalamin deficiency, cobalamin supplementation should be considered even when serum cobalamin concentration is low normal. Several assays for the measurement of serum concentrations of cobalamin in humans are available. In order to be used in dogs and cats, these assays designed for use in humans must be validated for use in dogs and cats. The GI Lab at Texas A&M University has recently analytically validated an automated chemiluminescence assay designed for the measurement of cobalamin concentration in humans for use in dogs and cats. A reference range for serum cobalamin concentration in dogs and cats was established using this assay (http://www.cvm.tamu.edu/gilab/assays/b12folate.shtml). It should be noted that reference ranges are not transferrable between labs and each lab should have their own reference range established.
Serum or urine methylmalonic acid (MMA) concentration can also be used as an indicator of cobalamin status. Cobalamin deficiency leads to accumulation of MMA and thus concentrations of MMA are often dramatically increased in the serum or urine of patients with cobalamin deficiency. Serum MMA concentrations have been shown to be increased in cats with cobalamin deficiency and have been shown to decrease with cobalamin supplementation. Also, recently dogs with severely decreased serum cobalamin concentrations were shown to have increased serum MMA concentrations. Interestingly, several dogs with low-normal serum cobalamin concentrations were also shown to have increased serum MMA concentrations, demonstrating that a severely decreased serum cobalamin concentration is not optimally sensitive for the diagnosis of cobalamin deficiency on a cellular level and that a cut-off value for cobalamin supplementation should be chosen that is in the low-normal reference range. This is especially true if one considers that cobalamin supplementation is minimally invasive, safe, and relatively cheap. As suggested by these data, measurement of serum MMA concentration may be a better diagnostic test for cobalamin deficiency than serum cobalamin concentration. However, measurement of MMA concentration in serum or urine is technically involved and expensive. Thus, MMA is currently not routinely assessed in patients evaluated for cobalamin deficiency.
Another way to assess cobalamin status is indirectly by use of a Schilling test. The Schilling test involves the oral administration of radioactively marked cobalamin that has been bound to an animal protein. However, to the author's knowledge the Schilling test has never been systematically evaluated in dogs or cats and is not practical for routine clinical practice.
Thus, the only routinely available diagnostic tool to assess cobalamin status in dogs and cats is serum cobalamin concentration, which should be evaluated in every dog and cat with chronic signs of gastrointestinal disease or with clinical signs compatible with cobalamin deficiency that cannot be attributed to other conditions (i.e., unexplained immunodeficiencies, anemias, neuropathies).
Patients with severe cobalamin deficiency often do not respond to therapy of the underlying gastrointestinal disorder until cobalamin is supplemented. Unfortunately, only empirical suggestions are available concerning protocols for cobalamin supplementation in dogs or cats. However, there is no indication that over-supplementation of cobalamin leads to clinical disease. In humans, the standard route of cobalamin application is by parenteral administration. This is because cobalamin deficiency has been shown to lead to cobalamin malabsorption in the ileum. However, there are recent data that would suggest that with certain forms of cobalamin deficiency, oral or nasal supplementation may be efficacious. There are no such data for dogs or cats and the data in humans is controversial. Thus, currently, the recommendation is to supplement cobalamin in veterinary patients by subcutaneous injection. The most common form of cobalamin used for supplementation is cyanocobalamin, but hydroxocobalamin can also be used. The author empirically uses the following dosing schedule: 150-250 μg per injection in cats, 250-1500 μg per injection in dogs; SC q 7 days for 6 weeks, then q 14 days for 6 weeks, then q 30 days for one injection, then reevaluate serum cobalamin concentration one month later. If the underlying disease process has resolved and cobalamin body stores have been replenished, serum cobalamin concentration should be supranormal at the time of re-evaluation. However, if serum cobalamin concentration is in the normal range, treatment should be continued at least monthly and the owner should be warned that clinical signs may recur sometime in the future. Finally, if serum cobalamin concentration at the time of recheck is subnormal, further work-up is required to definitively diagnose the underlying disease process and cobalamin supplementation should be continued weekly or bi-weekly. In a recent study cats with chronic gastrointestinal disease that had failed therapy and were shown to have cobalamin deficiency quickly gained weight after initiation of cobalamin supplementation.