Vitamin B12 deficiency is a potential long-term hazard in patients with short bowel syndrome (SBS). Time to onset of deficiency may be delayed for several years (1), and predicting those at risk may be hampered by unavailable information about postsurgical anatomy. Measuring B12 alone is inadequate for assessing status because functional deficiency may precede subnormal serum levels and hematologic abnormalities (2). Inadequate B12 impairs the function of B12-dependent metabolic pathways, resulting in increased production of alternative byproducts (eg, methylmalonic acid [MMA], homocysteine). Therefore, measuring plasma levels of MMA is a helpful ancillary test for detecting metabolic deficiency of B12 before onset of decreased serum levels (2,3). We present an adolescent male with SBS (intestinal resection for neonatal necrotizing enterocolitis), small bacterial overgrowth (SBBO), and history of vitamin B12 deficiency who developed elevated plasma levels of MMA despite being replenished with vitamin B12. Plasma MMA decreased after therapy with metronidazole. Bacterial overgrowth should be considered a potential cause of persistently elevated plasma MMA despite B12 therapy in patients with SBS.
A 17-year-old boy with SBS, chronic SBBO, and history of vitamin B12 deficiency was found to have elevated plasma levels of MMA during routine surveillance laboratory tests. The SBS came from surgical resections of neonatal necrotizing enterocolitis that extensively involved the ileum (ileocecal valve included), cecum, and right colon. He required parenteral nutrition therapy for 2 years and supplemental gastrostomy feeds for 5 years. The chronic SBBO was diagnosed based on recurring symptoms of gaseous abdominal discomfort and diarrhea confirmed by abnormal hydrogen breath test. He responded to periodic antibiotics (metronidazole) and decreasing consumption of simple sugars. At age 14 years he was diagnosed with vitamin B12 deficiency based on low serum B12, 191 pg/mL (normal 200–1000 pg/mL) in association with increased plasma MMA, 1.96 μmol/L (normal <0.4 μmol/L) (4). He was asymptomatic, had normal hemoglobin and red blood cell indices, and had no signs of neurological disease or peripheral neuropathy. He was treated with vitamin B12 (cyanocobalamin) 1000 μg (administered as intramuscular injections of 100 μg for 10 days), following which the serum B12 increased to 519 pg/mL and plasma MMA normalized, thus indicating a good biochemical response to therapy. He was thereafter placed on maintenance therapy with monthly injections of cyanocobalamin 100 μg. After 2 years of relative stability, normal growth, and compliance with monthly cyanocobalamin, his laboratory tests showed increased plasma levels of MMA; however, with normal serum vitamin B12, complete blood count, red blood cell indices, and acid/balance (Table 1). He remained asymptomatic and there was no history of disordered amino acid metabolism. We hypothesized that the increased plasma MMA resulted from excessive intestinal production of propionate (and the MMA derived from it) that overwhelmed his capacity to completely metabolize it, and that this could be curtailed by therapy with an appropriate antibiotic. Therefore, he was treated with metronidazole for 14 days. Four weeks after the antibiotic course, repeat laboratory tests showed significantly decreased plasma MMA, whereas the B12 levels remained unchanged (Table 1).
The derivatives of vitamin B12 involved in intermediary metabolism are methylcobalamin and deoxyadenosylcobalamin. They are required as cofactors for 2 essential metabolic processes in humans: Methylcobalamin is a cofactor in the remethylation reaction of homocysteine to methionine, catalyzed by methionine synthase. Deoxyadenosylcobalamin (coenzyme form of vitamin B12) is required for the isomerization of methylmalonyl coenzyme A (CoA) to succinyl CoA, catalyzed by methylmalonyl Co mutase (Fig. 1). Deficiency of vitamin B12 leads to functional deficiency of methionine synthase and methylmalonyl CoA mutase enzymes, resulting in accumulation of homocysteine and methylmalonyl CoA, respectively (1–3,5). The metabolic fate of methylmalonyl CoA is either isomerization to succinyl CoA then oxidation in the tricarboxylic acid cycle (Fig. 1) or hydrolysis to MMA and excretion in the urine. The risk factors for developing vitamin B12 deficiency in patients with SBS are malabsorption secondary to loss of the terminal ileum, gastric acid suppression therapy that interferes with digestion and release of cobalamin from dietary proteins (6), and bacterial competition for B12 despite its linkage to intrinsic factor (7,8). The neurological sequel of vitamin B12 deficiency may be irreversible, thus underscoring the importance of early detection and initiation of replacement therapy. Decreased or low normal serum B12 (200–400 pg/mL) in association with increased plasma MMA suggests impaired function of cobalamin-dependent enzymes, consistent with metabolic deficiency of B12. Many assays discriminate poorly between serum B12 concentrations that fall between 100 and 400 pg/mL (1). Therefore, additionally measuring the plasma concentrations of MMA is recommended as a confirmatory diagnostic test for vitamin B12 deficiency (1–3,5). The subject in this report developed elevated plasma levels of MMA despite normal serum levels of B12 and monthly replacement therapy. Bacterially derived inactive analogues also may lead to normal serum levels of B12 in patients with clinical deficiency (9,10). The specificity of B12 assays to exclude bacterially derived analogues in our patient was unknown. Nonetheless, lack of assay specificity was unlikely because the MMA levels declined following antibiotic therapy, whereas serum B12 remained unchanged, thus indicating the source of MMA had no effect on the measured levels of B12.
Propionate is a short-chain fatty acid produced by fermentation of complex carbohydrates by enteric organisms: bacteroides, peptostreptococcus, succinomonas, and succinovibrio (11). Enterically produced propionate is absorbed into the portal circulation and may account for up to 25% of the plasma levels in patients with propionic acidemia (12). Catabolism of amino acids and odd chain fatty acids contributes to approximately 75% of the propionate load in humans (12). In the cell, propionate is acetylated to propionyl CoA then carboxylated to methylmalonyl CoA (Fig. 1). The association between intestinally derived propionate and increased plasma levels of MMA has been described in patients with methylmalonic and propionic acidurias. In these patients, therapy with metronidazole effectively lowers increased plasma levels of MMA and propionic acid (13–15). The significant decline in elevated plasma concentrations of MMA in our patient following therapy with metronidazole was evidence that bacteria played a role in the pathogenesis. The improvement was attributed to eradication of bacteroides and other metronidazole-sensitive propionate-producing intestinal flora (16). SBBO-induced elevated plasma MMA depends on the nature of the intestinal flora (ie, whether they produce propionate). Propionate and methylmalonate exist as CoA esters within the cell and as their respective acids in plasma. We speculate that SBBO produced a large propionate load that overwhelmed our patient's capacity to rapidly metabolize it to succinyl CoA (Fig. 1), resulting in increased plasma levels of MMA. Interestingly, our patient remained asymptomatic and did not develop metabolic acidosis. The clinical implication of increased plasma levels of MMA in the absence of metabolic acidosis, B12 deficiency, and clinical symptoms is unknown. Thus, other than to differentiate whether increased plasma MMA is of enteric origin versus B12 deficiency, the benefit of using antibiotics solely to decrease MMA levels in patients with SBS is unknown.
The importance of high folate status in patients at risk for B12 deficiency is that it may mask hematologic abnormalities, delay diagnosis, and thus increase risk for neurological complications (17). Our subject had high serum levels of folic acid (Table 1) while consuming a regular diet and not taking supplements of a multivitamin or folic acid. The foods naturally rich in folate include leafy green vegetables, spinach, turnip greens, citrus fruits, dried beans, and peas (http://www.nal.usda.gov/fnic/cgi-bin/nut_search.pl). Furthermore, in 1996 the Food and Drug Administration, in an initiative to reduce the incidence of congenital neural tube defects, published regulations requiring fortification of several foods with folate (18). This practice has been credited for the recent US population-wide increase in serum folate levels (19). Patients with atrophic gastritis, a disorder predisposing to SBBO, also have elevated serum levels of folate (20). Bacterially derived folate and related metabolites are readily assimilated from the upper gastrointestinal tract and may be detected in urine (21). Thus, SBBO can be credited for synthesis of folic acid that is bioavailable to the human host. Our subject had no hematologic abnormalities at the time that his B12 deficiency was diagnosed, but serum folate was not measured. It is possible that hematologic changes of B12 deficiency were masked by a high folate status from increased dietary intake and SBBO. The frequency of masked hematologic abnormalities of B12 deficiency in this era of increased dietary intake and serum levels of folate is unknown. This underscores the importance of measuring MMA and not relying on hematologic indices when evaluating B12 status in patients with SBS.
Elevated MMA is a helpful confirmatory diagnostic test in patients with decreased or low normal serum levels of vitamin B12(2). We have presented a clinical scenario highlighting other potential sources of elevated MMA that need to be considered during assessment of vitamin B12 status. Intestinal flora should be considered among the causes of persistently elevated MMA despite adequate B12 therapy in patients with SBS.
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