Essential to Every Cell: Cobalamin in Health and Disease

Cobalamin, commonly known as vitamin B₁₂, is a water-soluble vitamin used by every cell in the body. Cobalamin is required for maintenance of normal physiologic functions, including nucleic acid synthesis, amino acid metabolism, intestinal epithelial function, central nervous system maintenance, and hematopoiesis.^1,2 Given cobalamin’s essential role in health, it follows that cobalamin-deficient states, or hypocobalaminemia, can contribute to disease pathophysiology.

Physiology of Hypocobalaminemia

Ingestion and Absorption of Cobalamin

Dogs and cats cannot synthesize cobalamin; consequently, they rely on ingestion and intestinal absorption of dietary cobalamin. Most commercial pet foods, including vegetarian diets, are supplemented with cobalamin. Understanding cobalamin metabolism and absorption is important in evaluating patients for causes of cobalamin deficiency and in determining how to approach cobalamin-deficient states therapeutically.

Once ingested, cobalamin is liberated from dietary protein in the stomach, where it is immediately bound to the glycoprotein haptocorrin (R protein, transcobalamin I). Haptocorrin protects cobalamin from bacterial utilization within the proximal gastrointestinal tract. When it reaches the duodenum, pancreatic proteases digest haptocorrin, freeing cobalamin once again. Intrinsic factor (IF) takes over the role of protecting cobalamin from bacterial utilization, and the cobalamin-IF complex makes its way to the ileum for uptake.¹ In dogs, most IF comes from the exocrine pancreas; however, a small amount is produced from the gastric mucosa (the main site in humans).³ Cats produce IF entirely from their exocrine pancreas.⁴

In the ileum, the cobalamin-IF complex is absorbed by cubam receptors. Although most dietary cobalamin is absorbed via receptor-mediated endocytosis, approximately 1% is absorbed by passive diffusion across the intestinal mucosal epithelium. This process is thought to occur along the entire length of the gastrointestinal tract.¹ The ability of cobalamin to passively diffuse along the gastrointestinal tract likely explains why dogs and cats with severe ileal disease can still have a positive response to oral cobalamin supplementation.

Within enterocytes, lysosomes separate cobalamin from IF and the cubam receptor. Cobalamin is then transported in the bloodstream throughout the body.

Intracellular Role of Cobalamin

All eukaryotic cells use cobalamin as an essential cofactor for the intracellular enzymes methionine synthase and methylmalonyl-CoA mutase,¹ which play vital roles in nucleic acid synthesis and the citric acid cycle, respectively. Lack of cobalamin and the subsequent decreased activity of these enzymes cause blood levels of homocysteine and methylmalonic acid (MMA) to increase. MMA can inhibit the urea cycle, resulting in increases in plasma ammonia concentrations. This is thought to contribute to the neurologic disorders that can occasionally be seen with hypocobalaminemia.⁵

Clinical Signs of Hypocobalaminemia

Clinical signs associated with hypocobalaminemia are listed in BOX 1. Gastrointestinal signs, including dysrexia and weight loss, are the most common clinical signs in dogs and cats with cobalamin deficiency.¹⁴ Other clinical signs of hypocobalaminemia include failure to thrive, immunodeficiency, and neuropathies.^1,2 Given cobalamin’s central role in cellular metabolism, cobalamin deficiency should be considered as a differential diagnosis for young dogs being presented for suspicion of a portosystemic shunt or metabolic disorders such as hypoglycemia and hyperammonemia.¹

Cobalamin and Disease

There are several causes of hypocobalaminemia in dogs and cats, including inherited selective cobalamin malabsorption, exocrine pancreatic insufficiency (EPI), chronic enteropathy, alimentary lymphoma, and small intestinal dysbiosis.

Inherited Selective Cobalamin Malabsorption

Selective cobalamin malabsorption, known as Imerslund-Gräsbeck syndrome (IGS) in humans, has been reported in several breeds of dogs, including the giant schnauzer, border collie, Chinese Shar-Pei, Australian shepherd, beagle, and komondor.^1,5-8,14,15 This inherited disorder involves a lack of cubam receptor expression on the apical brush border of the ileum. Dogs with selective cobalamin malabsorption typically manifest clinical signs such as dysrexia, diarrhea, and failure to thrive within their first year of life (BOX 2).

Although the discovery of vitamin B₁₂ followed investigation of pernicious anemia in humans, the dyshematopoetic effects of hypocobalaminemia are less common in dogs and cats. Nonregenerative anemia has been reported in some dogs; however, unlike humans, in whom the anemia is characterized by megaloblastosis, in dogs it is typically normocytic and normochromic.^6-9,14 Dogs may also have neutropenia characterized by hypersegmented neutrophils.^6,14 Hepatic encephalopathic changes such as seizures may be seen as a consequence of increased ammonia concentrations in the blood.^1,2,5

Selective cobalamin deficiency can be treated with oral or parenteral administration of cobalamin.¹⁰ Following initiation of supplementation, appetite and demeanor often return to normal within 1 to 2 days. Weight gain follows over days to weeks, with hematologic abnormalities taking up to 14 days to resolve.⁹ If cobalamin supplementation is not instituted and maintained, dogs with selective cobalamin malabsorption die as a consequence of severe metabolic derangements and immunodeficiency.¹¹

Gastrointestinal Diseases

Exocrine Pancreatic Insufficiency

EPI is a disease in which inadequate production of digestive enzymes from pancreatic acinar cells results in clinical signs such as steatorrhea and weight loss in the face of polyphagia.¹² EPI predominantly affects young dogs (1 to 4 years of age; BOX 3), with German shepherds representing 60% of cases.¹³ Although less frequent in cats, EPI is most often seen with concurrent diseases and can affect cats of any age.¹⁶ In cats, diarrhea is not consistently present and weight loss or vomiting can be the lone clinical sign.

The exocrine pancreas is responsible for most IF production in the dog and all IF production in the cat. Consequently, many dogs and cats with EPI are cobalamin-deficient.^12,13 In addition to supplementing pancreatic enzymes, cobalamin supplementation is indicated in most dogs and cats with EPI. Supplementation is routinely parenteral; however, in a recent study, oral supplementation was effective in normalizing blood cobalamin concentrations in dogs with EPI.¹⁷ The prognosis for dogs with EPI is typically favorable; however, cobalamin concentrations <350 ng/L have been shown to be a negative prognostic indicator.¹³

Chronic Enteropathies

Chronic enteropathy is defined as persistent or recurrent clinical signs of gastrointestinal disease such as vomiting, diarrhea, weight loss, or dysrexia, or any combination of these, that has been present for longer than 3 weeks.¹⁸ The term chronic enteropathy does not imply which treatment will be needed to control clinical signs. Various treatment trials, including diet and antibiotic trials, are necessary to identify the underlying cause. Consequently, based on response to various therapies, chronic enteropathy is often further classified as food-responsive, antibiotic-responsive, immunosuppressant-responsive, or nonresponsive.

Hypocobalaminemia in patients with chronic enteropathy is thought to be secondary to chronic ileal mucosal disease (BOX 4). One study found a statistically significant correlation between the presence of hypocobalaminemia and an increased number of ileal intraepithelial lymphocytes.¹⁹ Additionally, the half-life of cobalamin in circulation has been shown to be reduced with severe intestinal disease.²⁰ The reported prevalence of hypocobalaminemia with chronic enteropathy is 30% and 61% in dogs and cats, respectively.^20,21

Hypocobalaminemia has been shown to be a negative prognostic indicator in dogs with chronic enteropathy. In one study, severe mucosal lesions in the duodenum, hypoalbuminemia (<2 g/dL), and hypocobalaminemia (<200 ng/L) were correlated with a negative outcome.²²

Alimentary Lymphoma

Similar to chronic enteropathy, alimentary lymphoma is presumed to result in hypocobalaminemia due to neoplastic lymphocytes infiltrating the ileum and disrupting receptor-mediated uptake of cobalamin.²³ The prevalence of hypocobalaminemia in dogs with low-grade gastrointestinal lymphoma has been reported to be as high as 71%, which is much higher than the 16% seen in dogs with multicentric lymphoma.^23,24

Small Intestinal Dysbiosis

Bacteria within the gastrointestinal tract compete with host cells for essential nutrients such as cobalamin. Consequently, changes in the composition or richness of the intestinal microbiota can result in hypocobalaminemia. Many different bacteria are implicated; however, Bacteroides species are considered the key competitors given their ability to use cobalamin complexed to IF.²⁵ Should folate-producing bacteria localize to the proximal small intestine and compete with host cells for cobalamin, hyperfolatemia may be seen concurrently with hypocobalaminemia. Historically, this has been referred to as small intestinal dysbiosis, and therapies to alter the microbiota (e.g., diet, probiotics, tylosin) are often recommended.

Diagnosis of Hypocobalaminemia

Hypocobalaminemia is diagnosed based on measurement of serum cobalamin concentrations. Normal serum cobalamin levels depend on the reference laboratory used. The Texas A&M University Gastrointestinal Laboratory reports a reference interval of 251 to 908 ng/L for dogs and 290 to 1500 ng/L for cats. However, it is important to remember that cobalamin functions intracellularly. Consequently, patients that have low to normal serum cobalamin levels (250 to 350 ng/L) often have intracellular cobalamin deficiency. Additional biomarkers, including MMA and homocysteine, may provide more insight in these cases, as both of these increase with tissue cobalamin deficiency in dogs. Although measurement of canine MMA concentrations is commercially available through Texas A&M University, homocysteine measurement is not currently commercially available.

Treatment & Monitoring for Hypocobalaminemia

Indications for Hypocobalaminemia

Although hypocobalaminemia is defined by a serum cobalamin concentration that falls below the reference range (250 ng/L), 12% of normocobalaminemic dogs with chronic enteropathy have been reported to have increased serum MMA concentrations, suggesting intracellular deficiency.²⁵ Given these findings, the current recommendation is to supplement whenever cobalamin falls below normal or within the low to normal range (<400 ng/L) in both dogs and cats.

Route, Formulation, and Dose

Parenteral cyanocobalamin administered once weekly has traditionally been used for supplementation, as it is inexpensive and widely available. In humans with cobalamin deficiency, hydroxycobalamin is the mainstay of treatment due to its superior absorption and tissue retention rates when compared to cyanocobalamin. A recent study in cats demonstrated that hydroxycobalamin administered intramuscularly every 2 weeks is effective. Furthermore, in comparison to previous studies, hydroxycobalamin injections every other week were superior to once weekly cyanocobalamin injections in lowering serum MMA levels in cats.²⁶

Once-daily oral cobalamin supplementation (250 to 1000 µg in dogs, 250 µg in cats) is an effective alternative route in dogs with inherited selective cobalamin malabsorption and in dogs and cats with chronic enteropathies.^17,27,28 Once-daily oral supplementation of cobalamin may also be effective in dogs with EPI.¹⁶ Further information regarding dose, frequency, and administration of cobalamin can be found on the Texas A&M Gastrointestinal Laboratory website (vetmed.tamu.edu/gilab/research/cobalamin-information).

Reevaluation

Dogs and cats with EPI or selective cobalamin deficiency require lifelong supplementation. However, in patients with underlying diseases that can go into remission (e.g., food-responsive enteropathy), the current recommendation is that serum cobalamin concentrations be assessed 1 month after the seventh injection of parenteral cyanocobalamin or 1 week after completing 12 weeks of oral cobalamin therapy. In these cases, long-term supplementation is often not necessary, unless disease relapse occurs.

Given that the half-life of cobalamin in healthy dogs is reported to range from 6 to 16 weeks, recheck serum cobalamin levels should be supranormal.²⁹ This indicates that cobalamin body stores have been replenished. However, recent data in humans and cats have suggested that hypercobalaminemia can reflect a serious underlying neoplastic, hepatic, or renal disease.^30,31 Consequently, the recommendations for serum cobalamin monitoring and supplementation may change. Currently, if the serum cobalamin concentration is within the normal range at reevalution, it is recommended that cobalamin supplementation be continued and the underlying cause readdressed, as it is likely not being optimally treated.

Prognosis for Hypocolbalaminemia

Hypocobalaminemia has been associated with a shorter survival time in animals with diseases such as EPI, chronic enteropathy, and multicentric lymphoma.^12,22,23 That being said, the prognosis largely depends on the underlying disease resulting in hypocobalaminemia. In diseases with the potential for remission, the outcome is favorable.