Methylmalonic Aciduria Secondary to Selective Cobalamin Malabsorption in a Yorkshire Terrier

Introduction

Methylmalonic acidurias in people are genetically heterogenous and rare disorders of methylmalonate, homocysteine, and vitamin B₁₂ metabolism. Methylmalonic aciduria due to cobalamin malabsorption has previously been reported in giant schnauzers and a variety of other breeds, including border collies, Chinese shar peis, beagles, Labrador retrievers, as well as cats.^1–7 In giant schnauzers and border collies, it appears to be inherited as an autosomal recessive trait.^1,8 Cobalamin deficiency is also overrepresented in Chinese shar peis.^9,10 Breed predispositions for cobalamin deficiency have been the subject of surveys. Yorkshire terriers were not overrepresented in one survey, but a second survey found an apparent predisposition in that breed.^10,11 Cobalamin deficiency in some Yorkshire terriers has been associated with a possible protein-losing enteropathy as indicated by a decrease in serum α₁-proteinase inhibitor concentrations.¹²

Methylmalonic aciduria can arise as a result of a defect in cobalamin uptake from the ileum. Once cobalamin enters the small intestine, it is bound to a protein known as intrinsic factor (IF). The IF-cobalamin complex is recognized by an IF receptor located in the apical brush border of ileal enterocytes and absorbed via receptor-mediated endocytosis. In giant schnauzers, the ligand binding activity of the IF receptor is reduced 30-fold relative to normal dogs, and it is possible that similar mechanisms are responsible for primary cobalamin deficiencies seen in other breeds.¹³ This canine disorder resembles selective intestinal cobalamin malabsorption (Imerslund-Grasbeck syndrome) in people.

Cobalamin is an essential cofactor for methylmalonyl-CoA mutase and methionine synthase.¹⁴ Reduced activities of those enzymes lead to the accumulation of methylmalonic acid (MMA) and homocysteine, respectively.^13,15 Excess MMA is excreted in the urine. As well as having a direct neurotoxic effect, MMA inhibits carbamoyl phosphate synthase-1, an enzyme involved in the hepatic urea cycle, which in turn leads to hyperammonemia.^12,13 Together, those compounds can cause clinical signs of encephalopathy.

Reduced methionine synthase activity indirectly leads to a deficiency of the active form of folate. Folate is necessary for DNA synthesis, and deficiency leads to S-phase cell cycle arrest. That arrest primarily affects rapidly dividing cells; therefore, the gastrointestinal tract and bone marrow are commonly affected.

Yorkshire terriers are known to be predisposed to congenital portosystemic shunts and portal venous hypoplasia, which can result in hyperammonemia and identical clinical signs to those seen in dogs with hypocobalaminemia and methylmalonic aciduria. The treatment of those two conditions is very different; therefore, documenting the existence of methylmalonic aciduria in Yorkshire terriers is important.

Case Report

An 8 wk old male Yorkshire terrier was referred with a 2 wk history of recurrent collapse, seizure activity, and hypoglycemia. The patient was quiet but responsive, and no abnormalities were detected on physical and neurological examinations. The owner reported no obvious polydipsia and a normal appetite (the patient was fed a standard amount of commercial puppy food).

Hematology showed a mild regenerative anemia (hematocrit, 29.2 %; reference range, 37–55%), mature neutrophilia (15, 000/μL; reference range, 1800–7800/μL) and thrombocytosis (650 × 103/μL; reference range, 150–350 × 103/μL). Serum biochemical analysis documented hypoglycemia (48mg/dL; reference range, 70–110 mg/dL), hypoalbuminemia (1.5 g/dL; reference range, 3.5–5g/dL), hypoglobulinemia (18 g/L; reference range, 28–42 g/L), a mild increase in urea (30.5 mg/dL; reference range, 8–23 mg/dL), and reduced creatinine (0.55 mg/dL; reference range, 0.6–1.2 mg/dL). Postprandial bile acids were increased (6.1 μg/mL; reference range, 0.3–2.3 μg/dL) and serum ammonia was also increased (212 μg/dL; reference range, 15–45 μg/dL). The blood glucose and ammonia were measured immediately using validated in-house analyzers.

Serum cobalamin levels were below the detectable limit of the available assay (i.e., <150 ng/L). Serum folate was within the reference range (10.6 ng/mL; reference range, 3–13 ng/mL).

The patient was initially treated using IV fluid therapy^a with 20 mmol/L of potassium chloride^b administered at a rate of 4 mL/kg/hr, an oral lactulose solution^c [2 mL per os (PO) q 8 hr], and amoxicillin trihydrate/clavulanate potassium^d (22 mg/kg IV q 8 hr). A single subcutaneous injection of cobalamin^e was administered (20 μg/kg). Due to the small size of the patient, urine was collected over the next 12 hr until 10 mL was obtained, which was submitted to measure MMA excretion using a previously validated gas-chromatography/mass spectrometry technique. Results were available 21 days later and showed increased urinary excretion of MMA (425 mmol/mol creatinine; reference range, <2 mmol/mol).⁴ Over the first 24 hr of hospitalization, IV glucose^f was administered as required based on blood glucose assessment. No evidence of a hepatic vascular abnormality was found on abdominal ultrasound.

Following 24 hr of treatment, the patient was brighter, eating well, and maintaining normoglycemia. A repeat blood ammonia level was within normal limits (28 μg/dL). Over the following 48 hr, the patient showed continual improvement in clinical signs and was discharged after 96 hr with lactulose syrup (2 mL PO q 8 hr) and ampicillin^g (20 mg/kg PO q 8 hr). The owner was advised to feed a prescription hepatic diet.^h Appointments for repeat cobalamin injections were scheduled for 7, 14, 21, and 28 days postdischarge.

The dog remained bright and neurologically normal on subsequent rechecks. Cobalamin concentrations (944 ng/L; reference range, >200 ng/L) and MMA excretion (<2 mmol/mol creatinine) were both checked 21 days after starting therapy and were within normal limits (the MMA result being available at day 42). At that point, the lactulose and ampicillin were discontinued. The scheduled cobalamin injections were continued, and the diet was transitioned to a commercial puppy food. A bile acid stimulation test was within normal limits on day 28 (preprandial bile acid, 1.4 μg/dL; postprandial bile acid, 2.3 μg/dL). The dog was scheduled to receive lifelong parenteral cobalamin supplementations q 4 wk and remained clinically normal at the time this report was written.

Due to the suspected inherited mode of transmission, the siblings of the dog reported herein were contacted. Of the two littermates, only a female sibling was tested and her serum cobalamin levels and urinary MMA were within normal limits. The owners of the remaining littermate declined testing due to geographic constraints but reported the dog to be clinically normal. Methylmalonic aciduria has been reported in clinically healthy dogs; therefore, the condition could not be excluded based on the absence of clinical signs alone.⁴ Unfortunately, it was not possible to screen the parents for cobalamin deficiency.

Discussion

Inborn errors of metabolism resulting in methylmalonic aciduria and hyperammonemia have never been reported in Yorkshire terriers before. In the United Kingdom, there has been only one previous case of methylmalonic aciduria reported in a dog.³ Fyfe et al. (1991) reported cobalamin malabsorption in giant schnauzers with clinical signs starting at 6–12 wk of age.¹ All other reports of this condition have been in dogs >6 mo of age.^3–5 The patient reported herein began showing clinical signs at 6 wk of age, making it one of the youngest animals to be diagnosed with this condition.

Lutz et al. (2012) documented urinary MMA levels >1800 mmol/mol of creatinine in border collies with cobalamin deficiency, and marked elevations in serum MMA concentrations have also been seen in 25% of dogs that had hypocobalaminemia secondary to chronic gastrointestinal disease.^4,19 The relatively mild elevation in MMA in the case reported herein could be either due to the fact that parental cobalamin was administered prior to obtaining the urine or that the severity of the disease in border collies is greater.

At the time of initial presentation, there was a documented mild elevation in postprandial bile acids that suggested hepatic dysfunction. As a breed, Yorkshire terriers are predisposed to hepatic vascular abnormalities, which is why the patient was treated for presumed hepatic encephalopathy. No vascular abnormality was identified on abdominal ultrasound, and a bile acid stimulation test was subsequently within normal limits. A computed tomographic angiogram was considered but deemed unnecessary on the basis of a normal bile acid stimulation test and continual resolution of clinical signs after treatment to control hepatic encephalopathy was discontinued. A liver biopsy to exclude portal venous hypoplasia was also deemed unnecessary for the same reasons. The mild elevation in postprandial bile acids at presentation could have been due to laboratory error, transient cholestasis, or unidentified underlying gastrointestinal disease.

The case described here improved within 24 hr of hospitalization. During that time, the patient was treated for hepatic encephalopathy as well as cobalamin deficiency. It is therefore impossible to determine how much the hyperammonemia contributed to the clinical signs and how much they were due to the methylmalonic aciduria, which has been associated with neurological abnormalities in people.^3,18 Given that ammonia returned to normal during the first 24 hr it would seem probable that this was a significant contributory factor.

The patient was continued on medications aimed at addressing an underlying liver disease (i.e., lactulose and ampicillin) as well as a prescription diet until the scheduled 21 day recheck when the results of the urinary MMA taken at presentation were available. The authors felt that until the abnormal urinary MMA excretion was confirmed and given the initially increased bile acid assessment, it was prudent to continue the above medications until that time. A repeat urine sample obtained on day 21 confirmed normalization of the urinary MMA following the cobalamin administration (that result was returned 21 days after submission). Unfortunately, a repeat urinary MMA assessment was not performed once the patient was transitioned onto weekly cobalamin injections for 4 wk.

Hematologic abnormalities characteristically seen in dogs with cobalamin deficiency include neutropenia with hypersegmented neutrophils and nonregenerative anemia with occasional erythroblasts.^1,2 The case reported herein had a mildly regenerative anemia, a moderate neutrophilia, and thrombocytosis. The combination of those abnormalities may have been due to underlying gastrointestinal hemorrhage; however, the absence of the characteristic hematological anomalies seen in cases of selective cobalamin deficiency cannot be fully explained.

The ability to maintain blood glucose following correction of the hyperammonemia supports the assumption that the low blood glucose was likely secondary to the encephalopathy-induced anorexia and seizures.

Hypocobalaminemia has been reported secondary to dogs with pancreatitis/pancreatic insufficiency and chronic gastrointestinal disease (including small intestinal bacterial overgrowth).¹⁹ Although there was no reported history consistent with gastrointestinal disease, it is impossible to fully exclude that the reduced absorption of cobalamin was secondary to small intestinal bacterial overgrowth because this case was treated with antibiotics. Pancreatic insufficiency cannot be fully excluded either because trypsin-like immunoreactivity was not assessed; however, the signalment, clinical signs, normal ultrasonographic appearance of the pancreas, and response to therapy make pancreatic insufficiency unlikely. It is possible that this dog had a transient hypocobalaminemia secondary to either gastrointestinal or pancreatic disease rather than a primary defect in cobalamin metabolism, but the sustained and complete response to cobalamin supplementation without any other long-term therapy would make that unlikely.

Conclusion

This report documents the first recorded incidence of hyperammonemia secondary to selective cobalamin deficiency in Yorkshire terriers. Cobalamin deficiency should therefore be added to the list of differential diagnoses for this breed presenting with neurological abnormalities and increased blood ammonia levels. Cobalamin concentrations should be measured in Yorkshire terriers with hyperammonemia and no other evidence of hepatic dysfunction.