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Review of cobalamin status and disorders of cobalamin metabolism in dogs

Abstract Disorders of cobalamin (vitamin B12) metabolism are increasingly recognized in small animal medicine and have a variety of causes ranging from chronic gastrointestinal disease to hereditary defects in cobalamin metabolism. Measurement of serum cobalamin concentration, often in combination with serum folate concentration, is routinely performed as a diagnostic test in clinical practice. While the detection of hypocobalaminemia has therapeutic implications, interpretation of cobalamin status in dogs can be challenging. The aim of this review is to define hypocobalaminemia and cobalamin deficiency, normocobalaminemia, and hypercobalaminemia in dogs, describe known cobalamin deficiency states, breed predispositions in dogs, discuss the different biomarkers of importance for evaluating cobalamin status in dogs, and discuss the management of dogs with hypocobalaminemia.

The aim of this review is to critically evaluate the veterinary literature to define serum cobalamin status (normocobalaminemia, hypocobalaminemia and cobalamin deficiency, and hypercobalaminemia), describe causes of cobalamin deficiency states in dogs including breed predispositions, summarize breed-specific changes in cobalamin metabolism, discuss biomarkers of importance for evaluating cobalamin status in dogs, and discuss the treatment options for dogs in which a suboptimal or deficient cobalamin status has been diagnosed.

| Ingestion and absorption of cobalamin
Cobalamin is a water-soluble vitamin, also referred to as vitamin B 12 .
It is mainly ingested with food of animal origin with liver, kidney, meat, egg, milk products as well as fish having high cobalamin content, but smaller amounts can also be produced by the intestinal microbiota. 1 Plants and plant products contain virtually no cobalamin and, whereas ruminants and other herbivores can produce a sufficient amount of cobalamin in their intestines, omnivorous or carnivorous animals including dogs and cats are not able to produce cobalamin.
The intestinal microbiota of dogs and cats can produce cobalamin in the presence of cobalt, but because this site of cobalamin production is distal to the site of its absorption, this ability appears to be of little benefit to the animal. Most commercial pet foods, including vegan or vegetarian diets, are supplemented with cobalamin, 2,3 but the content of dietary cobalamin varies between diets (dry diets: 0.05-0.25 mg/kg dry matter basis; canned diets: 0.03-0.59 mg/kg dry matter basis). 4 The National Research Council (NRC 2006) recommendation for cobalamin fortification is 35 μg/kg dry matter for canine diets, regardless of the canine life stage. 5 The concentration of serum cobalamin and cobalamin-dependent metabolites is not related to the dogs being fed a biologically appropriate raw food diet or a commercial dog food. 6 Bound to dietary protein, cobalamin reaches the stomach where it is released by activated pepsinogen and gastric acid. 7 Free cobalamin is then bound to haptocorrin (R protein, transcobalamin I) to protect it from bacterial utilization in the proximal GI tract. 7 In the duodenum, pancreatic proteases separate cobalamin from haptocorrin, and free cobalamin is bound to intrinsic factor (IF). The major site of IF synthesis is the gastric mucosa in humans, whereas in dogs IF is produced primarily by the exocrine pancreas and to a lesser extent in the stomach. 8,9 The cobalamin-IF-complex is then absorbed by receptor-mediated endocytosis. The receptor, known as cubam, is localized at the brush border of the ileum ( Fig. 1A and 2). This receptor complex is comprised of 2 subunits, the proteins amnionless (AMN) and cubilin (CUBN) (Fig. 1A and 2). 10 In addition to receptormediated cobalamin uptake at the ileal brush border epithelium, approximately 1% of dietary cobalamin is absorbed via passive diffusion across the intestinal mucosal epithelium, assumed to occur along the entire length of the GI tract. 11 Within the lysosomes of the enterocytes, cobalamin is separated from IF and the receptor. Bound to another transport protein, transcobalamin, cobalamin is then transported within the bloodstream to its target tissues. In humans, 20%-30% of the cobalamin is bound to transcobalamin II, whereas most of the circulating cobalamin (70%-80%) is bound to transcobalamin I and thus unavailable for cellular uptake. 12,13 Although transcobalamin II appears to be much more abundant in dogs than in humans, transcobalamin I exists in dogs. 14 At the target tissue, cobalamin enters the cells through specific receptors and is then released from transcobalamin II (Fig. 1A). 12 Approximately 15 μg/day of recirculating cobalamin is extracted by hepatocytes and secreted in bile bound to haptocorrin for enterohepatic recirculation. 15 Within all eukaryotic cells, cobalamin acts as an essential cofactor for the intracellular enzymes methionine synthase and methylmalonyl-CoA mutase ( Fig. 3). 16 F I G U R E 1 Schematic of the absorption of cobalamin by enterocytes in the distal small intestine (ileum). A, In the duodenum cobalamin is bound to intrinsic factor (IF). The cobalamin-IF complex is then absorbed by receptor-mediated endocytosis. This cubam receptor is localized at the brush border of the distal small intestine (ileum). The receptor complex is comprised of 2 subunits, the proteins amnionless (AMN) and cubilin (CUBN F I G U R E 2 Immunofluorescent staining of the cobalamin receptor subunits in the canine ileum epithelium. The expression of the cobalamin receptor was detected immunohistochemically in a cross section of an ileal villus from a dog using antibodies for the subunits (A) amnionless (AMN, green) and (B) cubilin (CUBN, green). Both subunits are localized throughout the entire enterocyte (cell membrane and cytosol). Nuclei are stained in blue with diamidine phenylindole (DAPI). The insert shows the corresponding secondary antibody control. Images were acquired using equipment at the Laser Scanning Microscopy Core Facility (College of Veterinary Medicine, University of Leipzig, Germany) F I G U R E 3 Intracellular pathways of cobalamin metabolism. Methionine synthase catalyzes the regeneration of methionine from homocysteine. Disorders associated with a deficiency in cellular cobalamin availability thus can lead to functional folate deficiency and increased concentrations of homocysteine. Methylmalonic CoA mutase catalyzes the reaction from methylmalonyl CoA to succinyl CoA which is a key molecule in the tricarboxylic acid cycle. A lack of intracellular cobalamin leads to a reduced enzyme activity and an accumulation of methylmalonic acid (MMA). Excess MMA is excreted in the urine. MMA can also inhibit the activity of carbamoyl phosphate synthetase I, an enzyme of the urea cycle. Carbamoyl phosphate synthetase I normally metabolizes ammonia to carbamoyl phosphate. When this metabolic process is impaired, plasma ammonia concentration increases 2.2.2 | Methylmalonyl-CoA mutase Methylmalonyl-CoA mutase, a cobalamin-dependent enzyme, catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, which is produced by the catabolism of odd-chain fatty acids and amino acids (Fig. 3). Succinyl-CoA is a key molecule in the citric acid cycle. A lack of intracellular cobalamin leads to a reduced enzyme activity and an accumulation of methylmalonic acid (MMA) intracellularly and subsequently also systemically (methylmalonic acidemia). On the one hand, excess MMA undergoes urinary excretion (methylmalonic aciduria); on the other hand, MMA can also inhibit the activity of carbamoyl phosphate synthetase I, an enzyme of the urea cycle. Carbamoyl phosphate synthetase I normally metabolizes ammonia to carbamoyl phosphate. When this metabolic process is impaired, plasma ammonia concentrations typically increase. 1,[17][18][19][20] Neurological disorders can thus be a complication of cobalamin deficiency states in dogs due to the increase in systemic MMA concentrations. 1,17,18,21 In humans, neurological symptoms also occur in organic acidemias including methylmalonic acidemia. 22

| Excretion of cobalamin
Body stores of cobalamin in dogs far exceed the amount of cobalamin that is normally lost through the intestinal tract. Cobalamin undergoes biliary excretion (bound to haptocorrin), and a large amount of cobalamin is conserved by enterohepatic recirculation. 1 Renal glomerular filtration of the transcobalamin II-cobalamin complex is followed by tubular reabsorption to minimize urinary losses of cobalamin. Megalin, an endocytic receptor in the proximal renal tubulus that has a high affinity for the transcobalamin II-cobalamin complex, mediates renal reabsorption and retention of cobalamin. Free cobalamin is excreted in the urine. 23,24 The cubam receptor is also present in the kidneys 25 and is involved in the renal reabsorption of several proteins (eg, albumin, transferrin, and vitamin D-binding protein).  (Table 1). An immunoassay is also available for measurement of serum cobalamin concentrations in dogs but yields different results than the chemiluminescent assay. 29 The main indication for measuring serum cobalamin is to identify a subnormal cobalamin status. 30 Because cobalamin-dependent metabolic reactions are localized to the intracellular compartment (ie, cytoplasm and mitochondria), the serum cobalamin concentration does not necessarily exactly reflect the whole-body cobalamin status in an individual dog. Intracellular storage of cobalamin is mostly as a cofactor bound to cobalamin-dependent enzymes that might or might not be saturated with cobalamin. Decreased liver and kidney cobalamin stores, and certain defects in cobalamin metabolism, such as malabsorption due a defect in the ileal receptor, can result in cobalamin deficiency. Therefore, other markers that more closely reflect the intracellular availability of cobalamin such as the serum concentration of HCY and MMA should ideally be included when assessing cobalamin status. 31 Cobalamin is stable in serum even if samples are not strictly protected from light. 32 Thus, in clinical practice, measurement of serum cobalamin can be conveniently performed in serum samples that are archived for up to 5 days. However, the long-term (>5 days) stability of cobalamin in serum samples has not been reported. 5.9-31.9 μmol/L. 33 In human medicine, other methods that are used for measuring HCY are a chemiluminescent microparticle immunoassay and an enzyme-cycling assay (Homocysteine Cobas C, Integra, Roche).

| Serum HCY concentration
Homocysteine is a sulfurated intermediate amino acid that is produced from dietary methionine and is then either remethylated to methionine or metabolized to cysteine. 34 Serum HCY concentrations can also be increased with renal insufficiency 40 or hypothyroidism. 41 In humans, HCY has direct toxic effects on neurons and endothelial cells, can induce DNA strand damage, oxidative stress, and apoptosis 36,42 and also induce hepatic degeneration and fibrosis. 43 Even small changes in serum HCY concentrations (≥5-10 μmol/L) increase the risk of cardiovascular diseases in people. 36,37 Increased serum HCY concentrations occur in dogs with cardiac or renal disease. 44 Circulating HCY is mostly bound to albumin. As a consequence, diseases associated with hypoalbuminemia, such as protein-losing enteropathy (PLE), might be associated with a lower degree of hyperhomocysteinemia than expected or even with normohomocysteinemia. 38,45 However, it is likely that breed-specific effects exist. For example in Greyhounds, 38  Because MMA production increases as a result of decreased intracellular availability of cobalamin, MMA is a useful marker of cobalamin deficiency at the cellular level. Such a deficiency can occur due to cobalamin malabsorption, deficient cobalamin transport (which is not reported in dogs), depletion of cobalamin stores in the liver and kidneys secondary to cobalamin malabsorption, or a combination of these, leading to insufficient amounts of cobalamin entering the cells.
Tissues or cells with a high turnover rate such as enterocytes or blood cells are primarily affected. Dogs with a serum cobalamin concentration below RI have significantly higher serum MMA concentrations compared to dogs with normocobalaminemia, but serum MMA concentrations are increased in some (12%) normocobalaminemic dogs. 26 In Of healthy normocobalaminemic Border Collies, 38% have increased urine MMA concentrations. 6 None of these dogs have increased serum HCY or decreased cobalamin concentrations compared to cobalamin-deficient dogs. These cobalamin-deficient Border Collies have a primary methylmalonic aciduria. However, bacterial contamination of urine also has to be considered as a cause for increased urinary MMA concentrations. 47 A similar presentation occurs in children with a defect in the methylmalonyl-CoA mutase. 37,51,52 Urine MMA concentrations are also measured by GC/MS 26 or LC/MS-MS. 47 The RI was determined as 0-4.2 mmol/mol of creatinine in 1 report. 6 Dogs normally excrete <10 mg MMA/g of creatinine (<9.6 mmol MMA/mol of creatinine). 1 Normalizing urine MMA to creatinine concentration minimizes the concentration or dilution effect of urine on MMA concentrations. An advantage of measuring urinary MMA concentration is easier determination because MMA concentrations in urine are up to 40-fold higher than in serum. Furthermore, MMA is stable in urine, and sample collection is less invasive compared to the collection of blood samples. 6

| Interpretation of cobalamin status
The currently recommended interpretation of the cobalamin status in dogs is summarized in  33 These data suggest that a subtle or mild subclinical intracellular cobalamin deficiency exists in these dogs. In humans with suboptimal cobalamin status, the response to cobalamin supplementation is a marked reduction in serum MMA concentration. 55 Dogs with serum cobalamin concentrations at the lower end of the RI have a normalization of serum MMA concentrations after cobalamin supplementation. 33 This could indicate that the discrepancy between an increased serum MMA and a normal cobalamin concentration reflects intracellular cobalamin deficiency despite that body stores of cobalamin are sufficient to maintain normocobalaminemia.
Such a "cobalamin resistance" occurs in geriatric, diabetic, and hemodialysis human patients. 53 Alternative explanations could be the shift to an increased pool of circulating cobalamin with hepatic disease 53 or a compromised translocation of cobalamin to the intracellular space (which is not reported in dogs).

| Hypocobalaminemia
Hypocobalaminemia is typically referred to in dogs with a serum cobalamin concentration between the lower limit of quantification (LoQ) of the assay and the lower reference limit (Fig. 4). However, careful interpretation is necessary because different laboratories use different LoQs (Table 1).
Thirty-one percent of hypocobalaminemic dogs (as defined above) have serum MMA concentrations above the RI, 26 and these dogs are assumed to be cobalamin-deficient on the cellular level. However, it is

F I G U R E 4 Interpretation of the cobalamin status in dogs.
Hypocobalaminemia is typically referred to as a serum cobalamin concentration between the lower limit of quantification (LoQ) of the assay and the lower reference limit. Dogs with cobalamin deficiency have an undetectable serum cobalamin concentration (ie, below LoQ) and a serum MMA concentration above RI. Cobalamin should be supplemented whenever serum cobalamin concentration is suboptimal (ie, less than approximately 400 ng/L) possible that other factors (such as acute secondary SI dysbiosis due to an acute flare of the underlying enteropathy, proton-pump inhibitor use, 56

| Cobalamin deficiency
The term cobalamin deficiency is not well-defined in the veterinary literature. Most current cobalamin assays have an LoQ between 45 and 150 ng/L (Table 1). We propose using the term cobalamin deficiency for dogs with an undetectable serum cobalamin concentration and a serum MMA concentration above RI (Table 2, Figure 4).

| Hypercobalaminemia
Hypercobalaminemia refers to a serum cobalamin concentration above the normal RI. Until recently, hypercobalaminemia was considered as a benign finding and was basically ignored in companion animals. However, recent data in people suggest that hypercobalaminemia is an underestimated finding that can reflect a number of serious underlying diseases such as solid neoplasms, hematological and other malignancies, and hepatic or renal diseases. [60][61][62] Abnormally high serum cobalamin concentrations occur in cats with hepatic and neoplastic diseases. 63 A retrospective analysis of the medical records and data from 592 dogs that were presented for further diagnostic workup at the University of Leipzig Small Animal Clinic showed that 2% (n = 11) of all dogs in which serum cobalamin status was determined and that did not receive any supplemental cobalamin (n=487 dogs) had a serum cobalamin concentration above RI, ranging from 1,100-3,561 ng/L (median: 1,334 ng/L) (unpublished data).
These hypercobalaminemic dogs had mostly (73%) signs of chronic GI disease, and a neoplastic condition could not be definitively excluded in 82% of the dogs. These findings suggest that hypercobalaminemia is less frequently observed in dogs than in cats, 63 in which a prevalence of 14% (28 of 202 cats) was observed in the study above  (Fig. 1B). 49,[64][65][66][67][68][69][70][71] Hypocobalaminemia also occurs in people with IBD (Crohn's disease and ulcerative colitis). There are no reported cases of cobalamin deficiency of dietary origin in dogs. 31

| Clinicopathological findings and clinical presentation
Blood cell count abnormalities, typically nonregenerative anemia with megaloblastosis, neutropenia, and hypersegmented neutrophils, can be early signs of derangements in cobalamin metabolism. 72 With progression, disorders of phospholipid and amino acid metabolism have been documented in other species and might also occur in dogs. 21,73 In addition to blood cell dyscrasias and organic acidemias associ-  Breed-specific changes in serum cobalamin concentrations and distinct clinical manifestations exist in the Border Collie, Beagle, and Chinese Shar-Pei breeds (Table S1).

Border Collie
Several case reports and case series have described a selective cobalamin malabsorption in the Border Collie breed. 18 66 Pancreatic acinar atrophy is also an inherited disorder in the Eurasian breed. 96 Exocrine pancreatic insufficiency is characterized by an inadequate production of digestive enzymes from pancreatic acinar cells, leading to the typical clinical signs of weight loss in the face of polyphagia and increased fecal volume. 97,98 Failure to absorb cobalamin in dogs with EPI might be caused by 4 postulated or hypothetical mechanisms: pancreatic secretion of IF is reduced or absent, digestive enzymes are lacking causing an impaired release of cobalamin from haptocorrin and thus no binding of cobalamin to IF, secondary SI dysbiosis compromising the endogenous production of cobalamin, and the intestinal mucosa might also be compromised by the presence of toxic metabolites due to SI dysbiosis. 9,99-101 However, in people cobalamin assimilation does not improve with antibiotic treatment, 102 and there is no data that demonstrate a benefit of probiotics (eg, Enterococcus faecium SF68 103 ) or prebiotics.
Because the exocrine pancreas is the major site of IF synthesis in dogs, cobalamin supplementation is often necessary in these dogs.
However, cobalamin deficiency in some cases is not corrected by enzyme replacement alone likely because IF is not consistently a component of the exogenous enzyme mixture. 99 Treatment with bovine pancreatic enzyme extract is also not sufficient to restore cobalamin absorption in dogs with EPI, because IF appears to be species-specific.
The fact that serum cobalamin concentrations <100 ng/L are a negative prognostic factor 66 underlines the importance to adequately supplement dogs with EPI, but only 4 of 135 (3%) hypocobalaminemic dogs with EPI were given supplemental cobalamin in that study. 66 Another recent study revealed a prevalence of hypocobalaminemia (serum cobalamin concentration <350 ng/L) of 55% in dogs with EPI. 104 Hypocobalaminemia was also shown to be a negative prognostic factor in that study, and 112 of 116 dogs (82%) received cobalamin supplementation. 104  In addition, cobalamin has been hypothesized to be decreased in dogs with CIE because of secondary SI dysbiosis. Once the body stores of cobalamin are depleted, cobalamin deficiency can occur. 105 The half-life of cobalamin in healthy dogs is approximately 6-16 weeks. 106 It is assumed that this half-life is decreased in dogs with CIE as a result of a reduced cobalamin absorption in the face of normal biliary excretion and enterohepatic circulation. 49 Reported prevalences of hypocobalaminemia in dogs with CIE ranged from 19% to 38%, 49,65,[107][108][109][110] and similar findings have been reported in cats. 111 Dogs with suboptimal serum cobalamin status might not respond to treatment of the primary disease process unless given supplemental cobalamin, because cobalamin is essential for many cell functions and mucosal regeneration, and a deficiency can contribute to mucosal inflammatory infiltration and villous atrophy. 72,105 Correlation of hypocobalaminemia with histopathologic findings Hypocobalaminemia correlates with an increased number of intraepithelial lymphocytes in the ileal mucosa, 114 presenting evidence of an association between ileal mucosal inflammatory changes and hypocobalaminemia in dogs with CIE. However, the expression of the cobalamin-receptor in dogs with CIE has not been evaluated.

Correlation of hypocobalaminemia with outcome in dogs
Several factors have been evaluated for their potential to predict outcome in dogs with CIE. 65 In addition to the presence of severe mucosal lesions in the duodenum and hypoalbuminemia (serum albumin concentration of <20 g/L), hypocobalaminemia (defined as a serum cobalamin concentration of <200 ng/L in this study) at the time of diagnosis might predict refractoriness to treatment and was strongly correlated with a negative outcome despite supplementation of cobalamin for 6 weeks. 65 However, despite that hypocobalaminemia is a negative prognostic factor, adequate supplementation of cobalamin appears to be essential for the success of treatment. Thus, further studies need to investigate if early initiation of cobalamin supplementation is associated with a better outcome in dogs with CIE.
Hypocobalaminemia also correlates with hypoalbuminemia in dogs with CIE. 65,108,109 Protein-losing enteropathy Protein-losing enteropathy is a syndrome associated with an abnormal loss of albumin into the intestinal lumen.  69 Prevalences of hypocobalaminemia range from 43%-75% in dogs with non-neoplastic, noninfectious causes of PLE. 65,107,124 Hypocobalaminemia was also associated with decreased serum alpha 1 -proteinase inhibitor concentrations (presumably due to PLE) in Yorkshire Terriers. 125 Serum cobalamin concentrations should be evaluated in all dogs with PLE and, if needed, supplemental cobalamin be started early. 69

| Alimentary lymphoma
Sixteen percent of dogs with multicentric lymphoma have hypocobalaminemia, and this finding is associated with a poor outcome. 68 A higher rate of hypocobalaminemia (40% and 71%) is seen in dogs with low-grade (lymphocytic) GI lymphoma. 126,127 It is presumed that the hypocobalaminemia in dogs with lymphoma is a consequence of the ileal infiltration with neoplastic lymphocytes hypothetically resulting in a disruption of the receptor-mediated GI uptake of cobalamin. 68

| Small intestinal dysbiosis
Small intestinal (SI) dysbiosis is defined as an altered composition, richness, or both of the intestinal microbiota. 50 Competition between bacteria and host cells for essential nutrients such as cobalamin can lead to malnutrition. 67

| TREATMENT
Cobalamin should be supplemented whenever serum cobalamin concentration is subnormal. There is a 12% probability that CIE dogs with low-normal serum cobalamin concentration (ie, less than approximately 400 ng/L) might also benefit from supplementation. 26,33 Cyanocobalamin has traditionally been used for supplementation, as it is widely available and inexpensive.  Table S3) over a period of 12 weeks, 33 followed by a recheck of serum cobalamin concentration after discontinuation of supplementation. Dogs were rechecked the day after the last PO cobalamin dose in this study, 33 but the authors typically recheck serum cobalamin concentration 1 week after the last cobalamin tablet.

| Monitoring
Several studies in human patients with hypocobalaminemia suggest that supplementation with oral cobalamin might be as effective as parenteral administration. [138][139][140] A study that included 51 dogs showed that serum cobalamin concentrations increased significantly and normocobalaminemia was achieved in all hypocobalaminemic dogs with chronic enteropathy after daily oral cobalamin supplementation for at least 3 weeks (20-202 days). 141 However, similar to this study, another investigation by the same research group showed that supranormal serum cobalamin concentrations were achieved in only about half of the dogs. The dogs in this study reached normal serum cobalamin concentrations within 4 weeks of receiving PO cobalamin, but a hypercobalaminemic state was reached after 12 weeks of PO cobalamin supplementation in about two thirds of these dogs. 28 In addition, other recent studies suggest that daily PO cobalamin supplementation is effective to normalize the serum cobalamin status in dogs with CIE and also to maintain normal cobalamin status in dogs with hereditary cobalamin malabsorption that had previously been supplemented with parenteral hydroxocobalamin. 28,47 Thus, oral cobalamin supplementation is a simple, noninvasive, and pain-free alternative to weekly SC injections of either cyanocobalamin or hydroxocobalamin. 28,33 An alternative pathway of intestinal cobalamin absorption beyond receptor-mediated transport in the ileum is a possible explanation why oral cobalamin supplementation might be effective in spite of a defective receptor-mediated uptake into enterocytes.
This has also been reported in people. 11 However, expression levels of the ileal cobalamin-IF receptor in dogs with chronic enteropathies have not been investigated.

ACKNOWLEDGMENTS
Parts of this manuscript will be reproduced in Stefanie Kather's doctoral thesis, which is generously supported by a research stipend con-

CONFLICT OF INTEREST DECLARATION
Romy Heilmann has received speaker honoraria from Dechra company for presentations at continuing education meetings. Dechra has an oral cyanocobalamin formulation commercially available.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
Authors declare no IACUC or other approval was needed.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.