Cobalamin is a water-soluble vitamin of the B group and an essential cofactor for nucleic acid synthesis and hematopoiesis. Because absorption of this vitamin is complex and requires intact pancreatic and ileal function, serum cobalamin concentration can be clinically used as a diagnostic and prognostic marker when assessing intestinal and pancreatic diseases in companion animals.1,2 Measurement of serum cobalamin concentration is generally considered a standard test in the diagnostic workup of cats and dogs with chronic gastrointestinal clinical signs.3
Traditionally, cobalamin has been considered a light-sensitive analyte.4–6 In the authors’ experience, serum samples submitted to diagnostic laboratories for cobalamin concentration measurement are frequently rejected when those samples have not been protected from light exposure. Even major commercial veterinary laboratories mandate that blood samples for cobalamin testing be protected from light after collection.a For this reason, requests for testing of stored samples are also often declined, which is inconvenient for patients, pet owners, and clinicians.
Information regarding the effects of sample storage conditions could help guide optimal preanalytical handling of samples submitted for cobalamin testing and be used in the development of recommendations on how to store specimens to ensure optimal results. However, to the authors’ knowledge, this information has not been reported in the veterinary literature. The purpose of the study reported here was to examine the effects of light exposure and storage temperature and duration on cobalamin concentration in serum samples from cats and dogs.
Materials and Methods
Animals
Cats and dogs of any age, breed, or sex evaluated at the Clinic for Small Animal Internal Medicine of the Vetsuisse Faculty, University of Zurich that had to be euthanized were considered eligible, irrespective of the reason for euthanasia. Animals were excluded if they had received cobalamin-containing products orally or parenterally within the past 6 months. The study protocol was approved by the cantonal animal experiments and ethics committee of the university (permission No. 211/2012). Verbal consent was obtained from all owners prior to blood sample collection.
Sample collection
Immediately before euthanasia, blood samples (7 to 10 mL) were collected from cats and dogs via cephalic or saphenous catheters that had been freshly placed for the purpose of euthanasia. Clotted blood samples were then centrifuged at 3,000 × g and separated into 11 serum aliquots of approximately 250 μL each. One of these aliquots was immediately transported in light-protected tubes to the laboratory in a routine manner to serve as a baseline serum sample. Five serum aliquots were stored in plastic tubes in a refrigerator at 6°C. This was done in a routine fashion, exactly the way leftover blood samples are stored at the authors’ institution in anticipation of possible additional test requests. The remaining 5 serum aliquots were kept on a shelf and exposed to daylight and room temperature (20°C).
After 24, 48, 72, 96, and 120 hours of storage, 1 serum aliquot from each of the 2 storage conditions was wrapped in aluminum foil and frozen at −20°C for a period not exceeding 31 days. All frozen serum samples were then transported together on dry ice to the laboratory for analysis. The delivery period did not exceed 90 minutes, and it was verified that all samples had remained frozen at the time of delivery.
Cobalamin assay
Only sets of aliquots with cobalamin concentrations > 150 ng/L (lower detection limit of the assay) in all 11 aliquots were considered for analysis. At the laboratory, all serum samples were assayed in the same run by means of an automated competitive binding chemiluminescence assay for cobalamin.b Intra- and interassay coefficients of variation for this assay were 2.1% and 3.4%, respectively. The reference range used for serum cobalamin in cats and dogs was 305 to 1,967 ng/L and 261 to 1,001 ng/L, respectively.1,7
Statistical analysis
The percentage decrease in serum cobalamin concentration over time (model 1) and the effect of the interaction of time between storage condition (model 2) were analyzed by generation of 2 linear mixed-effects models to account for the random effect of individual animals on cobalamin values.8,c The likelihood ratio was used to determine whether the fixed effect of storage condition on serum cobalamin concentration was significant (P < 0.05). The optimal model formulation was determined by inspection of the Akaike information criterion value.
Results
Animals
Serum samples were obtained from 10 cats and 9 dogs; however, the samples from 1 cat were excluded from analysis because all stored serum aliquots for that cat contained < 150 ng of cobalamin/L (baseline cobalamin value, 168 ng/L). Mean age of the remaining 9 cats (5 males and 4 females) was 10.5 years, and that of the 9 dogs (5 males and 4 females) was 9.5 years. Cats included 7 European Shorthairs, 1 European Longhair, and 1 Chartreux. Dogs included 3 mixed-breed dogs and 1 each of the following breeds: Schapendoes, Labrador Retriever, Newfoundland, Jack Russell Terrier, Shetland Sheepdog, and Miniature Schnauzer.
Serum cobalamin concentration
Median baseline serum cobalamin concentration was 424 ng/L (range, 178 to 1,880 ng/L). No difference in serum cobalamin concentrations was identified between cats and dogs (P = 0.45); therefore, data for both species were analyzed together. Data for individual animals were graphically displayed (Figure 1).
Cobalamin (Cbl) concentrations in serum samples from 9 individual cats (A through 1) and 9 individual dogs (J through R) before (0 hours) and after sample storage in the refrigerator (6°C; dark blue lines) or at room temperature (20°C) in daylight (light blue lines) for up to 120 hours. Note that the scale of the y-axes is not fixed but has been individually scaled to the represented animal.
Citation: Journal of the American Veterinary Medical Association 252, 11; 10.2460/javma.252.11.1368
The statistical model with optimal fit to the study data included an adjustment for repeated measures by use of an animal-specific random slope. The effect of storage on serum cobalamin concentration was significant (P < 0.001; Figure 2). No significant change in concentrations from baseline over time was identified for refrigerated samples, whereas cobalamin concentrations in samples stored with daylight exposure at room temperature decreased significantly from baseline over time (0.14%/h; 95% confidence interval, 0.07% to 0.21%/h; Figure 3).
Mean cobalamin concentrations in serum samples from 9 cats and 9 dogs before (0 hours) and after sample storage in the refrigerator (6°C; black bars) or at room temperature (20°C) with daylight exposure (white bars) for up to 120 hours.
Citation: Journal of the American Veterinary Medical Association 252, 11; 10.2460/javma.252.11.1368
Mean percentage decrease from baseline of cobalamin concentrations in the serum samples represented in Figure 2 at various storage durations. See Figure 2 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 252, 11; 10.2460/javma.252.11.1368
Discussion
Findings of the present study indicated that the amount of cobalamin degradation over time was minimal (mean decrease, 0.14%/h) in daylight-exposed, room temperature-stored serum samples or nonsignificant in refrigerator-stored samples for up to 5 days. Similar results have been reported in the human medical literature, indicating no significant differences between serum cobalamin concentrations for light-protected and unprotected tubes after 7 days of storage.9 However, these findings for veterinary medicine, in which cobalamin is still largely considered as light sensitive by most laboratories, are new and provide clinically useful information. Indeed, this information is important given that serum cobalamin testing is performed more frequently now than a decade ago1,10–13 owing to the clinical relevance of cobalamin as a diagnostic and prognostic marker for gastrointestinal diseases in cats and dogs. In addition, continuous monitoring of serum cobalamin concentrations in cats and dogs during exogenous cobalamin administration is now routinely performed because it is generally accepted that providing such supplementation to cobalamin-deficient patients results in an improved quality of life and helps to improve the severity of clinical signs.13–15
We suggest that the results of the present study support the reasonableness of requesting serum cobalamin testing on previously collected canine and feline serum samples that may not have been light-protected or cooled before shipment. We would also suggest that cobalamin testing could indeed be considered for serum samples originally obtained up to 5 days previously for other diagnostic tests. In addition, stringent transport standards appear unnecessary to us should the cobalamin assay be performed within 5 days after sample collection. These interpretations would obviate the need for patients to return solely for a cobalamin testing appointment, making the process more convenient for owners and their pets.
In the study reported here, only 1 cat (third animal represented in Figure 1) had results that indicated storage conditions could have influenced the clinical decision regarding whether to provide cobalamin supplementation. The baseline cobalamin value (313 ng/L) for this cat was still within the reference range (305 to 1,967 ng/L), but all 10 values from subsequent assessment points for both storage conditions were < 305 ng/L, the lower reference limit. However, we believe this situation represented only a theoretical issue, given that it is unknown which cutoff value for serum cobalamin concentration reflects a true cobalamin deficiency that would benefit from cobalamin supplementation. Studies16–18 have shown that the ideal situation would be to also measure specific cellular cobalamin deficiency markers such as MMA in animals with low normal or subnormal cobalamin concentrations because such animals can still have unremarkable MMA values.
A serum cobalamin concentration at the lower end of the reference range does not necessarily correlate with low activity of the cobalamin-dependent enzyme methylmalonyl-CoA mutase. For instance, the cutoff value shown to provide the theoretical best combination of sensitivity and specificity for predicting cobalamin deficiency (defined as high serum MMA concentration) in cats is a serum cobalamin concentration of 160 ng/L (reference range, 290 to 1,500 ng/L).16 More recently, a serum cobalamin concentration < 209 ng/L (reference range, 290 to 1,500 ng/L) was shown to most accurately predict cobalamin deficiency (defined as high serum MMA concentration).17 Interestingly, in that study,17 high serum MMA concentrations were more common than low serum cobalamin concentrations in sick cats with gastrointestinal disease.17 Because the MMA assay is expensive and not routinely performed in most clinical laboratories, we currently recommend cobalamin supplementation for all cats and dogs with gastrointestinal disease and serum cobalamin concentrations near the lower reference limit.
The present study was not designed to evaluate the effects of various storage temperatures, particularly those of nonstandard storage temperatures, on cobalamin concentrations in serum samples from cats and dogs. In addition, no distinction could be made between the effects of light and those of storage temperature given the design of the study. Rather, our intention was to investigate the effects of real-life scenarios, and the main purpose was to determine whether it would be reasonable (and not ill-advised) to submit previously collected serum samples that had not been light-protected or cooled for serum cobalamin testing. Our findings indicated that cobalamin concentration could indeed be reasonably assessed in serum samples stored in a refrigerator for up to 5 days. Even without any protection of samples from light when stored at room temperature for 5 days, minimal cobalamin degradation occurred.
Acknowledgments
Presented in poster form at the 23th Congress of the European College of Veterinary Internal Medicine Companion Animals, Liverpool, England, September 2013.
ABBREVIATIONS
| MMA | Methylmalonic acid |
Footnotes
Idexx Diavet AG, Postfach 43, Bäch, Switzerland.
Vitamin B12, Immulite 2000, Siemens Healthcare Diagnostics Inc, Newark, Del.
R, version 3.3.2, The R Foundation for Statistical Computing, Vienna, Austria. Available at www.R-project.org. Accessed Nov 1, 2016.
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