Cobalamin (vitamin B12) is a water-soluble vitamin involved in neuronal function, hematopoiesis, DNA and fatty acid synthesis, and energy production.[1-3] CBL absorption requires binding proteins and specific receptors along various parts of the gastrointestinal tract.[4-6] Consequently, gastrointestinal disease can lead to CBL deficiency. Subnormal serum cobalamin concentrations [CBL] most frequently have been reported in cats with suspected gastrointestinal disease, and clinical findings of weight loss, diarrhea, vomiting, anorexia, lethargy, or thickened intestines.[5, 7] Low [CBL] also has been linked to increased mean corpuscular volume (MCV) and decreased serum phosphorous concentration in some studies.[5, 7] The types of gastrointestinal disease associated with subnormal [CBL] include inflammatory bowel disease (IBD), intestinal lymphoma, cholangiohepatitis or cholangitis, pancreatitis and exocrine pancreatic insufficiency,[5, 7-10] reflecting the involvement of these organs in feline CBL homeostasis.
The reported prevalence of subnormal [CBL] in cats with gastrointestinal disease ranges from 0.1 to 78%.[5, 7-9, 11] This variation potentially is a consequence of the assay used, reference intervals, patient population, geographical location, or some combination of these factors.[5, 7-9, 11]
A potential solution to this problem is to determine the [CBL] that correlates with decreased activity of 2 CBL-dependent enzymes: methylmalonyl-CoA mutase and methionine synthase. In people, decreased activity of these enzymes causes increased serum and urine concentrations of methylmalonic acid [MMA] and homocysteine, respectively. Cats with undetectable or subnormal [CBL] have significantly increased [MMA], but not increased homocysteine concentrations.[5, 6, 13] Cats with increased [MMA] also had significantly increased serum concentrations of methionine and significantly decreased serum concentrations of cystathionine and cysteine. Furthermore, treating cats that had high [MMA] with parenteral CBL decreased the [MMA] and facilitated weight gain.
These findings suggest that measurement of [CBL] is a useful marker of gastrointestinal disease, and that treating cats diagnosed with CBL deficiency on the basis of an increased [MMA] confers a therapeutic benefit. Because MMA is not routinely measured in most clinical laboratories, it would be valuable to know the [CBL] at which CBL deficiency develops (ie, the point at which cats would benefit from supplementation). Initial studies conducted in cats with subnormal [CBL] (defined a priori as < 290 pg/mL) showed that a [CBL] ≤ 160 pg/L had a 74% sensitivity and 80% specificity for detecting [MMA] > 867 nmol/L. However, [MMA] has not been correlated with [CBL] > 290 pg/mL. Given that other laboratories have found substantially higher [CBL] in their healthy cat populations (290–900 pg/mL), it is possible that CBL deficiency, as defined by an increased [MMA], exists in cats with [CBL] > 290 pg/mL. Additionally, no data exist correlating gastrointestinal disease with CBL deficiency, or with the impact of CBL deficiency on clinicopathological variables.
Consequently, we sought to evaluate the ability of [CBL] to predict high [MMA], and the relationships of CBL and MMA to select clinical and clinicopathological variables.
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Our study provides novel information about CBL status in cats as determined by measurement of [CBL] and [MMA] in a relatively large cohort of cats, especially those with suspected or confirmed gastrointestinal disease. Our finding that [CBL] showed a significant but modest negative correlation with [MMA] supports the relationship previously observed between CBL and MMA in cats. However, our study calls into question the use of [CBL] as a sole indicator of CBL deficiency in sick cats, and highlights a group of cats with discordant results, in which [MMA] is high (supporting CBL deficiency), but [CBL] is above the lower limit of the reference interval (indicating adequate CBL status). Clear explanations for this discordance were not apparent, but could reflect changes in the availability and cellular uptake of CBL associated with discordance in people.[12, 16, 17] Additional studies are needed to study this relationship in more detail in cats. Given that treatment of CBL deficiency can provide measurable therapeutic benefits to cats with gastrointestinal disease, our study illustrates the complicated nature of assessing CBL status, where multiple methodologies and reference intervals exist, and do not necessarily agree. Clinicians should consider measuring MMA and CBL concurrently to better assess CBL status in cats, similar to recommendations in people.
We found that a [CBL] of < 189 pg/mL could reliably identify cats with increased [MMA], with no false positive diagnoses below this [CBL], and that an [MMA] ≥ 1,343 nmol/L was a better indicator of CBL deficiency in the study population than the historical [MMA] of > 867 nmol/L, because it provided fewer false negative results without increasing the rate of false positive results. Using ROC analysis, we found that a [CBL] of 209 pg/mL most accurately discriminated cats with high and low [MMA], when measured on the Immulite 1000 system. However, we recognize that ROC values typically are optimized on the basis of combined sensitivity and specificity, although exceptions exist in specific circumstances. Because CBL therapy is considered to have minimal risk, it could be argued that increasing the threshold for [CBL] above 290 pg/mL could provide clinical benefit to a greater number of cats. Nevertheless, when we examined the predictive values of various [CBL] at various disease prevalences with a [MMA] < 1,343 nmol/L indicating normal CBL status, we found that increasing the [CBL] cutoff to 900 pg/mL lowered the positive predictive value considerably, without impacting the negative predictive value (Table 2). The specific threshold values (189, 209, and 1,343 nmol/L) we identified in this study are based on the results obtained from our sample population. Whether decreasing the precision of our estimates, by rounding the threshold values to the nearest multiple of ten or hundred would produce similar outcomes remains to be determined.
Therefore, on the basis of our results, we formulated a strategy for evaluating cats with suspected CBL deficiency (Fig 3). In this strategy, cats with [CBL] ≤ 209 pg/mL would receive parenteral CBL supplementation and subsequent monitoring of response. Cats with [CBL] > 209 pg/mL would need to have [MMA] measured. Those with [MMA] < 1,343 nmol/L would be monitored, with periodic assessment of [CBL], whereas those with [MMA] ≥ 1,343 nmol/L would receive parenteral CBL supplementation and monitoring of clinical response and [MMA].
Figure 3. Treatment decision strategy based on serum cobalamin [CBL] and serum methylmalonic acid [MMA] concentrations.
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Similar to previous studies, we observed a high prevalence of increased [MMA] or low [CBL] in cats presenting with signs of gastrointestinal disease (eg, vomiting, diarrhea, weight loss, anorexia, icterus, palpably thickened intestines). Of the cats in which a diagnosis was ultimately made, those with alimentary lymphoma had lower [CBL] than cats with IBD or other diseases. This observation agrees with previous studies of alimentary lymphoma in cats, in which investigators also observed an association between CBL status and clinical outcomes. However, whether CBL supplementation of cats with alimentary lymphoma would alter outcome in these patients remains unknown.
Of particular interest were cats with discordant [MMA] and [CBL] results, particularly those cats with high [MMA] and normal [CBL]. Of the 23 cats identified with discordant results, clinical records were available for 13 cats. In the 10 cats without clinical records, we cannot speculate about the potential explanations for the discordance. In the 13 cats with medical records, possible explanations for such discordance included hepatobiliary disease (8 cats), diabetes mellitus (1 cat), and renal disease (2 cats), which all have been identified as factors in human patients.[12, 16, 17] Two discordant cats had a history of hyperthyroidism, which recently has been associated with low [CBL] in cats; both of these cats had been treated before measuring [CBL] and [MMA], 1 had iatrogenic hypothyroidism after radioiodine treatment and 1 was euthyroid on methimazole. Because subnormal [CBL] persists in people after treatment of hyperthyroidism, the relationship of hyperthyroidism to CBL status is unclear. The presence of concurrent diseases in many cats with discordant results made it difficult to determine the mechanisms underlying discordance (eg, concurrent intestinal and hepatobiliary disease, concurrent chronic kidney disease, iatrogenic hypothyroidism). The inability of [CBL] to reliably predict increased [MMA] in sick cats with [CBL] > 209 pg/mL coupled with the limited availability of MMA assays, suggest that routine administration of parenteral CBL to all cats considered at risk for CBL deficiency is the optimal solution. However, given that in humans, mechanisms associated with discordant [MMA] are attributable to alterations in CBL distribution and decreased cellular CBL uptake, rather than malabsorption, parenteral administration might fail to overcome this problem.[12, 16, 17] Monitoring [MMA] and clinical response after CBL supplementation of cats with discordant results might help resolve this dilemma.
We found significant correlations of [CBL] with MCV and [MMA] with HCT that parallel findings in people in whom macrocytosis correlates with [CBL] and is considered the forerunner of the anemia of clinical CBL deficiency.
In contrast to a previous study, we found no relationship of [MMA] or [CBL] with serum phosphorus concentration. However, in that study, most cats had both low folate concentrations and low [CBL], whereas in our study only 5 cats met these criteria. Of these 5 cats, only 1 cat had serum phosphorus concentration measured and it was within reference interval for our laboratory. Additionally, in contrast to a previous study, we also found no relationship of cysteine or methionine with [CBL]. The reason for these differences in the 2 studies remains unknown.
In the first study to examine CBL deficiency in cats, which used an RIA that correlated with the bioassay, 49/80 (61%) of cats had a [CBL] below the lower limit of the reference interval (< 900 pg/mL). Conversely, in a recent British study, using an automated chemiluminescence assay, only 11/39 cats (28.2%) had [CBL] below the lower limit of the reference interval (< 290 pg/mL). Applying the 290 pg/mL cutoff to the original study yielded a prevalence of 27% (21/78), suggesting that differences in reference intervals among laboratories impact the identification of cats with subnormal [CBL] and CBL deficiency, similar to observations in human patients.[12, 21] In our study, we used the Immulite 1000 system to measure [CBL] because the RIA methodology used for previous studies is no longer available. Our clinical laboratory validated the assay by a direct comparison with the RIA and found that the Immulite 1000 system showed high correlation with RIA. This allowed us a direct comparison of data from several previous studies that also used the Immulite 1000 system to examine [CBL] in cats. The initial report of subnormal [CBL] in 80 cats, when measured by the RIA, reported a prevalence of subnormal [CBL] of 60% with the 900 pg/mL cutoff and 27% with the 290 pg/mL cut-off. This concurs with our findings in this study using the Immulite 1000 assay that yielded prevalences of 57% and 29% using the same cutoff values, respectively, and a prevalence of 28.2% in 39 British cats using the 290 pg/mL cutoff and Immulite 1000 assay. These observations infer that differences in reference intervals, rather than assay performance, underlie the variability in prevalence of CBL deficiency reported in cats with gastrointestinal disease.[5, 7, 9] In 50 healthy cats in Australia [CBL] determined with the Immulite 1000 system assay ranged from 345 to 3,668 pg/mL with median values of 1,609 pg/mL in cats aged 0–5 years and 1,200 pg/mL in cats older than 5 years (P = .39). Notably, 75% of cats had [CBL] > 1,000 pg/mL. These ranges are substantially higher than those reported by Ruaux et al using the Immulite 1000 assay in 24 control cats that ranged from 600 to 1100 pg/mL. It is not clear why there is such a difference in the reference ranges among different studies that employ the same CBL analysis method. It could reflect geographic variability or factors such as diet, presence of subclinical disease, and potentially the age of reference population. Similar issues of variable reference intervals have been the focus of much discussion in humans, where the reference intervals can dramatically affect perceived prevalence rates of CBL deficiency.[12, 21]
Our study has several limitations inherent with observational studies. Not all patients had complete clinical and biochemical data available; however, we evaluated a relatively large cohort of cats for each statistical association. We cannot discount the possibility of unidentified CBL supplementation before sampling in the samples sent in by first opinion clinicians. However, the populations were similar with regard to [CBL] and [MMA]. In some cats, no definitive diagnosis (as defined by histopathology) was obtained; however, these cats were presumptively diagnosed and treated based on the clinician's best evidence and judgment. Cats without a diagnosis were coded “open.” We did not evaluate therapeutic interventions.
We elected to use [MMA] as the reference standard to define CBL status in our cats, based on human medical and veterinary historical precedents. Our data suggest that [MMA] might not entirely accurately reflect CBL status in all cats (similar to observations in humans). However, this does not invalidate the use of [MMA] as the reference standard, until a better indicator of CBL status emerges.