The effect of feeding on serum concentrations of cobalamin, folate, trypsin‐like immunoreactivity, and pancreatic lipase immunoreactivity in dogs with signs of chronic gastrointestinal disease

Abstract

Background

It is unknown if serum concentrations of cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin‐like immunoreactivity (cTLI) obtained postprandially are equivalent to measurements obtained after withholding food in dogs with suspected gastrointestinal disease.

Hypothesis/Objectives

Measurements of serum concentrations of cobalamin, folate, cPLI, and cTLI postprandially will be equivalent to measurements after 12 hours of withholding food in dogs with signs of chronic gastrointestinal disease. Changes observed will not alter clinical interpretation.

Animals

51 client‐owned dogs with signs of gastrointestinal disease.

Methods

Prospective single arm clinical trial. Serum concentrations of cobalamin, folate, cPLI and cTLI 2, 4, and 8 hours postprandially were compared by equivalence testing to values after withholding food for 12 hours (baseline).

Results

Mean serum cobalamin concentrations 2 hours (498.1 ± 213.1 ng/L; P = 0.024) and 4 hours (501.9 ± 207.4 ng/L; P = 0.008) postprandial were equivalent to baseline (517.3 ± 211.5 ng/L). Mean serum cTLI 2 hours (31.3 ± 14 μg/L; P < 0.001) and 4 hours (29.6 ± 13.1 μg/L; P = 0.027) postprandial were equivalent to baseline (31.1 ± 15 μg/L). Mean serum folate concentration 2 hours postprandial (15 ± 7.7 μg/L) was equivalent to baseline (13.7 ± 8.3 μg/L; P < 0.001). Equivalence could not be assessed for cPLI due to results below the lower limit of quantification. Feeding altered the clinical interpretation in 27% (cobalamin), 35% (folate), 20% (cTLI), and 12% (cPLI) of dogs.

Conclusions and Clinical Importance

The clinical interpretation for a substantial number of samples changed after feeding, therefore withholding food before sample collection is prudent.

Abbreviations

cPLI

canine pancreatic lipase immunoreactivity

cTLI

canine trypsin‐like immunoreactivity

EPI

exocrine pancreatic insufficiency

RER

resting energy requirement

RI

reference interval

SD

standard deviation

1. INTRODUCTION

Serum concentrations of cobalamin, folate, canine pancreatic lipase immunoreactivity (cPLI), and canine trypsin‐like immunoreactivity (cTLI) are commonly used as minimally invasive tools for diagnosing and monitoring small intestinal and exocrine pancreatic disease in dogs. ¹ , ² , ³ , ⁴ , ⁵ However, protocols for the measurement of these analytes recommend withholding food for 12 hours or overnight before sample collection. ⁶ , ⁷ , ⁸ Withholding food unnecessarily could be perceived as reducing dog welfare, logistic challenges for clinicians and clients, and in the acute hospital setting, delayed return to feeding. Importantly, these recommendations for withholding food were made based on assay methodology that is no longer typically used for measurement of these analytes in dog serum. Radioimmunoassay, in which lipemia can pose substantial assay interferences, was used previously for measurement of cTLI, necessitating a period of withholding food. Currently, measurement of serum cobalamin, folate, and cTLI is most commonly via chemiluminescence assays and cPLI via ELISA. Additionally, there are benefits of early return to enteral nutrition in dogs with primary enteropathies. ⁹

In humans, no clinically relevant differences in serum cobalamin concentrations were found among subjects fasted for various periods of time, from 1 to >16 hours. ⁶ In addition, serum folate concentrations do not increase significantly after a meal in humans. ⁷ In healthy dogs, withholding food has no clinically relevant effects on serum concentrations of cobalamin, folate, cTLI, or cPLI. ¹⁰ , ¹¹ , ¹² However, it is unknown if feeding affects serum concentrations of these analytes in dogs with signs of chronic gastrointestinal disease, which is the population to which these tests are applied.

The primary objective of the present study was to determine if serum concentrations of cobalamin, folate, cPLI, and cTLI obtained 2, 4, and 8 hours after feeding would be equivalent to values obtained after withholding food for 12 hours in dogs with chronic signs of gastrointestinal disease. A secondary objective was to determine if feeding changed the clinical interpretation of serum concentrations of cobalamin, folate, cPLI, or cTLI. We hypothesized that postprandial serum concentrations of cobalamin, folate, cPLI, and cTLI would be equivalent to values after withholding food in this population of dogs, and therefore the clinical interpretation of these analytes would remain the same before and after feeding.

2. MATERIALS AND METHODS

2.1. Study design

A prospective single arm clinical trial involving client‐owned dogs from the University of Wisconsin Veterinary Medical Center and local primary care practices in Wisconsin was carried out from June 2019 to October 2021.

2.2. Dogs

Dogs were eligible for enrollment if they had signs of chronic gastrointestinal disease, defined as a greater than 14‐day duration of owner‐reported vomiting, diarrhea, poor appetite, or unexplained weight loss. Enrollment criteria included adult, medium to large breed dogs (≥10 kg), consuming a nutritionist‐formulated dog food or commercially available dog food formulated to Association of American Feed Control Officials standards. Additionally, dogs had to be willing to eat in the clinic and be amenable to repeated venipuncture. Before enrollment, all dogs had a complete history and physical examination to assess for signs of anemia (pale mucous membranes, tachycardia, and tachypnea).

Exclusion criteria included dogs with moderate‐to‐severe anemia (hematocrit <30%), hypovolemia, coagulopathy, diseases not easily amenable to withholding food (ie, diabetes mellitus), or a history of supplementation with cobalamin or folate within the 30 days before sample collection. The study design was approved by the Institutional Animal Care and Use Committee at the University of Wisconsin School of Veterinary Medicine (Animal Use Protocol #V006113).

2.3. Sample collection and handling

Enrolled dogs were presented after food was withheld for 11 to 13 hours. After collection of 4 mL of whole blood (“baseline”), dogs were fed their normal diet or, if disinterested, a standardized prescription gastrointestinal diet (Purina Pro Plan Veterinary Diets EN Gastroenteric Canine Formula, Nestle Purina PetCare Co, St Louis Missouri, USA). Dogs needed to consume at least 50% of their calculated resting energy requirement (RER = body weight in kg^0.75 × 70 kcal) within the hospital to continue in the study. Blood samples (4 mL each) were then obtained from each dog at 2, 4, and 8 hours after feeding. Food was not permitted during this sampling period. However, free access to water was provided.

Blood samples were allowed to appropriately clot, then were centrifuged to collect serum, which was refrigerated within 1 hour of collection. Each sample was individually wrapped in aluminum foil to protect from light, and frozen at −80°C within 12 hours of collection as per previous work and the diagnostic laboratory recommendations. ¹³ , ¹⁴ All samples were shipped frozen and on ice via overnight courier to the Gastrointestinal Laboratory at Texas A&M University (TAMU), stored at −80°C, and analyzed as a single batch. Cobalamin, folate, cTLI, and cPLI have been demonstrated to be stable in serum for multiple years when frozen. ¹⁵ , ¹⁶ , ¹⁷

2.4. Laboratory tests

Cobalamin, folate, and cTLI assays were performed by commercially available automated chemiluminescence assays (Immulite 2000, Siemens Healthcare Diagnostics Inc, Deerfield, Illinois, USA). cPLI was measured by a commercial ELISA (Spec cPL, Idexx Laboratories, Westbrook, Maine, USA). ¹⁸ Clinical interpretations of values were defined as per the laboratory's recommendations (Table 1). Grossly lipemic samples were centrifuged before analysis.

TABLE 1.

Laboratory‐recommended interpretations of serum concentrations of cobalamin, folate, cPLI, and cTLI.

Analyte	Value	Interpretation
Cobalamin	<250 ng/L	Low
	251‐400 ng/L	Gray zone
	>400 ng/L	Normal
Folate	<7.7 μg/L	Low
	7.7‐24.4 μg/L	Normal
	>24.4 μg/L	High
cPLI	0‐200 μg/L	Normal
	201‐399 μg/L	Gray zone
	>400 μg/L	High
cTLI	<2.5 μg/L	Low
	2.5‐5.7 μg/L	Gray zone
	5.7‐45 2 μg/L	Normal
	>45.2 μg/L	High

Abbreviations: cPLI, canine pancreatic lipase immunoreactivity; cTLI canine trypsin‐like immunoreactivity.

Analyte concentrations that were below or above the limits of quantitation were encoded as the next unit below or above that limit, respectively, as described previously. ¹¹

2.5. Statistical analysis

Sample size for this project was determined based on having 90% power in an equivalence test from paired differences with the primary outcome being the paired differences in serum cobalamin concentration at each postfeeding time point (2, 4, and 8 hours) compared with the baseline time point in a previous study. ¹¹ The margin of equivalence was set as ±50 ng/L rather than a percentage change to facilitate sample size estimation. This is narrower than the difference seen in Saver et al ¹¹ and was chosen because of the possibility that included dogs would likely have serum cobalamin concentrations close to the lower end of the reference interval and thus to be more conservative in possible conclusions. The previous data showed standard deviation (SD) estimates of paired differences near 70 ng/L, and for our study, we more conservatively assumed a larger SD of 90 ng/L. Lastly, because of evaluation of 3 different time points (2, 4, and 8 hours), the alpha level was split evenly (α = 0.017). Under these assumptions, the work was estimated to have 90% power in 3 equivalence tests with 50 enrolled dogs.

Two 1‐sided t tests (TOST), which are the preferred test for equivalence, were conducted on paired differences of each of the 4 serum outcomes (ie, cobalamin, folate, cPLI, and cTLI) at 2, 4, and 8 hours compared with 12 hours of fasting at baseline. Each TOST was Bonferroni adjusted for 3 tests within each outcome. The a priori set margins of equivalence for each variable were as follows: cobalamin ±50 ng/L, folate ±5 μg/L, PLI ±8 μg/L, and TLI ±5 μg/L. All analyses were conducted using R for statistical computing at a 5% significance level.

3. RESULTS

3.1. Animals

Fifty‐four dogs with clinical signs of gastrointestinal disease were enrolled based on the inclusion criteria. Three dogs did not complete the study protocol: 2 did not consume 50% of their RER and 1 had improper sample storage.

The mean (SD) age of the remaining 51 dogs was 5.8 (4.2) years (range, 0.8‐14 years), and the study group consisted of 19 (37.3%) spayed females, 25 (49.0%) castrated males, 5 (9.8%) intact males, and 2 (3.9%) intact females. The mean body weight was 26.8 (8.6) kg (range, 10.9‐55.3 kg). Mixed breed dogs were the most commonly included breed (n = 16 [31.4%]), and the remaining breeds are summarized in Table 2.

TABLE 2.

Number (n) of each breed enrolled and included for analysis.

Dogs (n)	Breed
16	Mixed breed
7	Golden retriever
3	English setter
3	Greyhound
2	German shorthaired pointer
3	Siberian husky
2	Rat terrier
1 each	Anatolian shepherd, Australian cattle dog, berger Picard, Bernese mountain dog, boxer, cocker spaniel, collie, Nova Scotia duck tolling retriever, German shepherd dog, giant schnauzer, Labrador retriever, Portuguese water dog, Rottweiler, St. Bernard, vizsla

3.2. Clinical signs

Clinical signs of chronic disease documented via the dogs' histories included diarrhea (n = 41 [80.4%]), weight loss (n = 12 [23.5%]), decreased appetite (n = 10 [19.6%]), and vomiting (n = 17 [33.3%]). Two owners also reported regurgitation in their dogs. The owner's description of diarrhea was consistent with small bowel diarrhea in 15 dogs, large bowel diarrhea in 6 dogs, and mixed bowel diarrhea in 20 dogs.

3.3. Feeding

The mean caloric intake (as the percentage of calculated RER) for the enrolled dogs was 62% (10; range, 50%‐90%). Twenty‐two (43.1%) dogs consumed the study diet alternative because of either lack of interest in or unavailability of their typical diet. Of the diets consumed by the dogs, the median grams of protein per 100 kcal was 6.69 g (range, 4.87‐8.38 g/100 kcal) and the median grams of fat per 100 kcal was 4.1 g (range, 1.7‐4.71 g/100 kcal). No dogs experienced vomiting, diarrhea, or regurgitation during the study period.

3.4. Analytes

Serum cobalamin, folate, and cTLI concentrations were normally distributed; however, cPLI was not normally distributed. All time points were available for all dogs except a single dog missing the 2‐hour postprandial sample because of tube breakage during transport.

3.4.1. Cobalamin

The mean serum cobalamin concentration after withholding food for 12 hours was 517 ng/L (212; reference interval [RI], 251‐908 ng/L; Table 3). Sufficient evidence for equivalence of mean serum cobalamin concentrations was present at 2 hours (P = .02) and 4 hours (P = .01), but not 8 hours postprandially (P = .36; Table 3) compared with the values after withholding food.

TABLE 3.

Mean (SD) serum concentrations of cobalamin, folate, and cTLI for 51 dogs with chronic gastrointestinal signs sampled after 12 hours of withholding food (time 0; baseline) versus at 2, 4, and 8 hours after feeding.

Variable	Time (h)	Mean (SD)	Difference (95% CI)	MoE	Equivalence P
Cobalamin (ng/L)	Baseline (0)	517.3 (211.5)	…	…	…
	Hour 2	498.1 (213.1)	−19.8 (−46.3 to 6.7)	50	.02 a
	Hour 4	501.9 (207.4)	−15.4 (−41.5 to 10.6)	50	.01 a
	Hour 8	482.6 (201.6)	−34.7 (−62.5 to −6.9)	50	.36
Folate (μg/L)	Baseline (0)	13.7 (8.3)	…	…	…
	Hour 2	15.0 (7.7)	1.2 (−0.5 to 3.0)	5	<.001 a
	Hour 4	17.3 (9.6)	3.6 (1.8 to 5.4)	5	.13
	Hour 8	16.4 (9.5)	2.7 (0.1 to 5.3)	5	.09
cTLI (μg/L)	Baseline (0)	31.1 (15.0)	…	…	…
	Hour 2	31.3 (14.0)	0.2 (−2.3 to 2.6)	5	<.001 a
	Hour 4	29.6 (13.1)	−1.5 (−4.6 to 1.7)	5	.03 a
	Hour 8	28.7 (13.2)	−2.3 (−5.6 to 0.9)	5	.12

Note: The variables were analyzed using a two 1‐sided t tests (TOST) procedure.

Abbreviations: CI, confidence interval; cTLI, canine trypsin‐like immunoreactivity; MoE, margin of equivalence; SD, standard deviation.

^a Sufficient evidence for equivalence between that measurement and baseline (0) for the selected analyte.

When compared with their serum cobalamin concentration after withholding food, interpretation was unchanged for 37/51 (73%) dogs (Figure 1). Of these dogs, 27 (73%) had serum cobalamin concentrations within the reference interval at all time points, whereas 6 dogs (22%) had a serum cobalamin concentrations within the gray zone (251‐400 mg/L; Table 1). Four dogs (11%) were persistently hypocobalaminemic across all time points. For 14/51 (27%) dogs, the clinical interpretation of serum cobalamin concentration was different at 1 or more postprandial time points when compared with the interpretation of serum cobalamin concentration after withholding food. In 12/14 (86%) of these dogs, the difference would have altered recommendations for cobalamin supplementation supplied by the TAMU Gastrointestinal Laboratory (Figure 2). For 8 of these dogs (67%), a normal cobalamin concentration after withholding food was followed by a single postprandial gray zone serum cobalamin concentration (n = 4), 2 postprandial gray zone serum cobalamin concentrations (n = 1), or all postprandial gray zone serum cobalamin concentrations (n = 3). For 4 of these dogs (33%), a gray zone cobalamin concentration after withholding food was followed by a single normal postprandial serum cobalamin concentration (n = 1), 2 normal postprandial serum cobalamin concentrations (n = 2), or all normal postprandial serum cobalamin concentrations (n = 1).

FIGURE 1.

Modified error grid consensus illustrating comparison between baseline sample collected after withholding food for 12 hours (y‐axis) and 2‐, 4‐, and 8‐hour postfeeding samples (x‐axis) for cobalamin (row 1), folate (row 2), canine trypsin‐like immunoreactivity (cTLI; row 3), and canine pancreatic lipase immunoreactivity (cPLI; row 4). Values of cPLI below the limit of quantification of the assay (<30 μg/L) were recorded as 29 μg/L. Ranges of interpretation for each serum concentration are noted on the x‐ and y‐axes with capital letters and within the graph with dashed lines. Green open circles indicate no change in interpretation between baseline and the selected hours' serum concentration, blue triangles indicate a change that would not be considered clinically relevant, and orange closed circles indicate a change in clinical interpretation between the 2 measurements.

FIGURE 2.

Serum cobalamin concentrations in dogs (n = 14) where clinical interpretation of postprandial results differed from that when measured after withholding food for 12 hours. Each line represents an individual dog. The lighter gray region of the chart encompasses normal cobalamin concentrations, whereas the darker gray and white regions are gray zone and low cobalamin concentrations, respectively.

3.4.2. Folate

The mean fasted serum folate concentration was 13.7 μg/L (8.3), which fell within the reported reference interval (RI, 7.7‐24.4 μg/L). Sufficient evidence for equivalence of mean serum folate concentration was present at 2 hours (P < .001) but not at 4 hours (P = .13) or 8 hours (P = .09) postprandially compared with the values after withholding food (Table 3).

When compared with the serum folate concentration after withholding food, 33/51 (65%) dogs had an unchanged clinical interpretation of serum folate concentration at all time points postprandially (Figure 1). One of these dogs was persistently hypofolatemic (<7.7 μg/L) and 1 was persistently hyperfolatemic (>24.4 μg/L). In the remaining 18/51 (35%) dogs, clinical interpretation of serum folate concentrations at 1 or more postprandial time points differed from fasted values. For 9 of these dogs (50%), serum folate concentration varied between low (<7.7 μL/L) and normal, whereas for the other 9 dogs (50%), serum folate concentration varied between high (>24.4 μg/L) and normal (Figure 3).

FIGURE 3.

Serum folate concentrations in dogs (n = 9) where results varied between normal and low folate after feeding compared to measured after withholding food for 12 hours. Each line represents an individual dog. The lighter gray region of the chart encompasses normal folate concentrations, and the white region is low folate concentrations [Correction added after first online publication on 22 Apr 2024. Figure caption has been amended.].

3.4.3. Canine trypsin‐like immunoreactivity

The mean fasted serum cTLI concentration was 31.1 μg/L (15.0; RI, 5.7‐45.2 μg/L). Sufficient evidence for equivalence of mean serum cTLI concentration was present at 2 hours (P < .001) and 4 hours (P = .03), but not 8 hours (P = .12) postprandially compared with values after withholding food (Table 3).

For 41/51 (80%) dogs, clinical interpretation of cTLI serum concentration was unchanged postprandially compared with fasting values (Figure 1). Of these, 6/41 (15%) dogs had persistently high serum cTLI concentrations, and 1/41 (2%) dogs had persistently low (<1.00 μg/L) serum cTLI concentrations. For 10/51 (20%) dogs, clinical interpretation of serum cTLI concentrations differed postprandially compared with values after withholding food (Figure 4). All 10 dogs varied between normal and high serum cTLI concentrations.

FIGURE 4.

Serum canine trypsin‐like immunoreactivity (cTLI) concentrations in dogs (n = 10) where clinical interpretation of postprandial results differed from that when measured after withholding food for 12 hours. Each line represents an individual dog. The lighter gray region of the chart encompasses normal cTLI concentrations, whereas white regions are high or low cTLI concentrations.

3.4.4. Canine pancreatic lipase immunoreactivity

Results for serum cPLI were <30 μg/L (below the limit of the working range of the assay) in 50% of the samples (n = 99); data were, therefore, converted to a 3‐category variable as per the laboratory's recommended interpretation (Table 1). Conclusions on equivalence could not be reached because of the large number of dogs with results <30 μg/L.

For 45/51 (88%) dogs, clinical interpretation of serum cPLI concentrations did not change at any time postprandially compared with values after withholding food (Figure 1). Of these, 4/45 (9%) dogs had persistently high serum cPLI concentrations, 40/45 (89%) dogs had persistently normal cPLI serum concentrations, and a single dog (2%) had persistently gray zone serum cPLI concentration (200 μg/L ≤ cPLI ≤ 400 μg/L). For the remaining 6/51 (12%) dogs, the clinical interpretations did vary from the value after withholding food at 1 or more postprandial time points (Figure 5). Four of these 6 dogs (67%) had serum cPLI values between the high and gray zone categories, whereas 2 dogs (33%) varied between normal and gray zone.

FIGURE 5.

Serum canine pancreatic lipase immunoreactivity (cPLI) concentrations in dogs (n = 6) where clinical interpretation of postprandial results differed from that when measured after withholding food for 12 hours. Each line represents an individual dog. The lighter gray region of the chart encompasses normal cPLI concentrations, whereas the darker gray and white regions are questionable or high cPLI concentrations, respectively.

4. DISCUSSION

Serum concentrations of cobalamin, folate, cPLI, and cTLI are important tools in the diagnosis of gastrointestinal and exocrine pancreatic disease in dogs, but the necessity of withholding food before their evaluation has not been assessed with the most utilized assay methodologies. Our results indicate that cobalamin and cTLI measurements 2 and 4 hours postprandially are statistically equivalent and usually clinically equivalent to a sample in which food was withheld for 12 hours. Additionally, only folate measurements 2 hours after feeding are statistically equivalent and usually clinically equivalent to a sample in which food was withheld for 12 hours. However, for many samples among all analytes, the overall clinical interpretation did differ between the sample drawn after withholding food and the sample collected at either 2, 4, or 8 hours after feeding.

In healthy dogs, there is a statistically significant but clinically unimportant change in serum cobalamin concentrations obtained 4 and 8 hours postprandially compared with samples collected after withholding food for 12 hours. ¹¹ This mirrored work in people demonstrating a small, clinically unimportant decrease in cobalamin with fasting in men. ⁶ In our study's cohort of dogs with signs of chronic gastrointestinal disease, whereas cobalamin concentration 2 and 4 hours postprandial was equivalent to values with food withheld for 12 hours, measurement 8 hours postprandially was not equivalent. Additionally, in a number of dogs (27%), the changes in postprandial serum cobalamin concentration would have equated to a difference in recommendations for supplementation from the reference lab, a potentially clinically important change not seen in healthy dogs.

Within both healthy dogs and the dogs within our study, mean postprandial serum cobalamin concentrations were lower than values after withholding food. This was unexpected given that in previous studies using oral ingestion of supraphysiologic doses of cobalamin, peak postprandial serum concentrations occurred at 4 hours in dogs and 1 to 7 hours in humans. ¹⁹ , ²⁰ However, dogs in the current study were only fed physiologic doses of cobalamin from a maintenance or gastrointestinal diet, which could contribute to differences in when peak concentrations would be observed.

Other potential explanations for the fluctuations in postprandial cobalamin concentrations observed may be a consequence of minor differences in enterohepatic recycling or biotransformation of cobalamin over time, assay variability, or altered nutrient absorption because of potential delayed gastric emptying. ²¹ , ²² , ²³ , ²⁴ , ²⁵ A previous study ²⁶ found that hospitalization of dogs could result in an up to a 3‐fold delay in gastric emptying, suspected to be because of stress. It is possible that the dogs in our study were stressed and as such had delayed gastric emptying. Appropriate cobalamin intake was controlled as each dog was fed their maintenance diet or a standardized diet that contained the recommended 35 μg of cobalamin/kg of dry matter. ²⁷ Finally, in humans, cobalamin and transcobalamin concentrations can vary by 10% over a 24‐hour period and were not associated with mealtimes, circadian rhythm, age, or meal composition. ²⁸ , ²⁹ , ³⁰ , ³¹

The current TAMU Gastrointestinal Laboratory recommendations for cobalamin supplementation do not necessitate the need to make exact distinctions between hypocobalaminemia and low normal “gray zone” serum cobalamin concentrations. However, in the 14/51 of dogs that had a serum cobalamin status change at a postprandial time point compared with fasted values, 12/14 changed in terms of clinical interpretation (ie, necessity of cobalamin supplementation). Therefore, although most dogs in the current study do not appear to have large fluctuations in serum cobalamin after feeding, and statistical equivalence was established between samples after food was withheld, 2 h postprandial and 4 h postprandial samples, there was a population of dogs for which feeding did alter the clinical interpretation of cobalamin status.

In the present study, serum folate concentrations 2 hours postprandially, but not at 4 and 8 hours postprandially, were equivalent to baseline values. As with cobalamin, the variability noted at 4 and 8 hours could result from differences in gastric emptying and folate biotransformation over time. In rats, plasma concentrations of an active folic acid metabolite, 5‐methyltetrahydrofolic acid, were 2‐fold higher at baseline than at the fed state. This suggests a circadian rhythm of folate metabolism that might be further influenced by intestinal metabolism and microflora. ³²

In the dogs in our study, although there was no statistical equivalence to baseline samples at 4 and 8 hours postprandially, clinical interpretation would have changed in several dogs. A change from normofolatemia to hyperfolatemia might have little effect on treatment recommendations for a given dog. Alternatively, a change between normofolatemia and hypofolatemia in 9/51 dogs might have impacted folate supplementation recommendations and future diagnostic decisions such as choosing colonoscopy, esophagogastroduodenoscopy, or both. Despite this finding, the postprandial changes in serum folate, when present, were unlikely to substantially alter clinical recommendations for the majority of dogs.

In the current study, serum cTLI concentrations were equivalent to baseline concentrations 2 and 4 hours postprandially, but not at 8 hours postprandially. A previous study evaluating postprandial cTLI in healthy dogs receiving diets with variable fat content noted that serum cTLI increased, up to 20% from baseline, in dogs after feeding. ¹² This finding might have contributed to the recommendation to withhold food for 12 hours when evaluating cTLI. ¹¹ However, in our study, the mean cTLI concentration after withholding food was higher than mean postprandial measurements. TLI has been found to be affected by lipemia but not by hemolysis or hyperbilirubinemia. There were no dogs in the current study in which a postprandial lipemia was noted. ³³ , ³⁴

Only 1 dog in the present study was diagnosed with exocrine pancreatic insufficiency (EPI; cTLI <1 at all 4 time points) and was included in the 28% of dogs with hypocobalaminemia. Because of the low sample size, we are unable to comment on the necessity of withholding food for the measurement of cTLI in dogs with suspected EPI and further evaluation in this subset of dogs is warranted. ⁴ , ³⁵ , ³⁶

Although 10/51 dogs had a change in interpretation of the serum cTLI concentration between the postprandial samples and the sample after withholding food, none were below the lower limit of the reference interval. The half‐life of trypsinogen in canine serum is very short (14 minutes), likely accounting for the cTLI changes in dogs for which clinical interpretation postprandially would have varied (Figure 4). ³⁷ Additionally, given the relative lack of specificity for cTLI for the diagnosis of pancreatitis compared with cPLI, it is unlikely that this would change clinical treatment of those dogs. Thus, in terms of clinical interpretation, withholding food appears unnecessary for measurement of cTLI in dogs where EPI is not highly suspected.

Within the present study, conclusions on postprandial equivalence of serum cPLI concentration could not be reached because of the high number of dogs with results <30 μg/L. However, most dogs had postprandial serum cPLI concentrations that did not differ from concentrations after withholding food. Previous studies found no statistically significant differences in serum cPLI concentrations in healthy dogs after feeding when compared with concentrations after withholding food. ¹⁰ , ¹¹ Although there was variability in serum cPLI concentration between measurements within the same dog, no general pattern could be observed. ¹⁰ An individual's dietary fat percentage is not anticipated to influence serum cPLI concentration. ¹² However, the individual variability could be explained by inherent circadian oscillation in analytes, as previously noted for both serum lipase and serum alpha‐amylase activity. ³⁸

Clinically, the interpretation for most dogs' serum cPLI concentration did not vary between postprandial samples and samples collected after withholding food. Many dogs for which clinical interpretation did change (Figure 5) had decreases over time, which is most likely attributable to the relatively short 2‐hour half‐life of cPLI. ⁵ This is important because it means dogs with suspected pancreatitis should have consistently increased serum cPLI based upon ongoing pancreatic damage, although 1 recent study calls that dogma into question. ³⁹ Only 2/51 dogs had increases from a normal serum cPLI concentration to gray zone values between food withheld and postprandial samples, changes that could have altered the clinical diagnoses. However, interpretation of serum cPLI concentration and a diagnosis of pancreatitis should always be made considering clinical signs. ⁴⁰ Additionally, although our study aimed to evaluate the equivalence of the assay over time in a population of dogs, rather than a repeated‐measures test for individual dogs, intraindividual variation for cPLI is known to be high. ⁴¹ A small proportion of dogs here (4/51) had persistently increased serum cPLI concentrations; thus, further investigation into the impact of feeding on serum cPLI concentration is warranted.

One limitation of our study was the requirement for a dog to consume 50% of their RER before enrollment. This likely resulted in selection for dogs having milder disease processes. In addition, a definitive diagnosis was not required for enrollment, preventing the ability to make correlations between findings and specific etiologies. However, the inclusion criteria were designed to mimic the clinical selection of dogs in which measurement of serum concentrations of cobalamin, folate, cTLI, and cPLI would be obtained. Unfortunately, we were unable to assess equivalence of cPLI because of the large quantity of dogs with cPLI results of <30 μg/L. Further evaluation utilizing dogs diagnosed with active pancreatitis would be indicated to explore the necessity of withholding food for the measurement of this analyte. This is expected to be challenging as a common clinical presenting complaint in dogs with acute pancreatitis is anorexia. ⁴² The size of dogs included was limited because of the total blood volume required; thus, if breed or size differences exist, they could have been missed. Finally, we tested equivalence against a fixed value for each analyte, rather than a percentage of variance from baseline. This may have influenced our findings; however, in an effort to minimize type I error, we chose even smaller fixed margins of equivalence than used in previous studies based on our collective clinical experience and judgment. ¹²

In conclusion, our study demonstrated that 2‐hour postprandial measurement of serum cobalamin, folate, and cTLI concentrations was equivalent to fasted values in dogs with signs of chronic gastrointestinal disease. In addition, both cobalamin and cTLI were equivalent at 4 hours postprandially to fasted values. None of the analytes had statistical equivalence at 8 hours postprandially. However, for a substantial number of dogs, clinical interpretation of serum cobalamin, folate, or both changed after feeding, suggesting that withholding food before collection of blood samples would be prudent.

CONFLICT OF INTEREST DECLARATION

Two authors of this study (Joao Pedro Cavasin and Jörg M. Steiner) are employed by or affiliated with the Gastrointestinal Laboratory at Texas A&M University, which provides analyses of the analytes in this study on a fee‐for‐service basis.

OFF‐LABEL ANTIMICROBIAL DECLARATION

Authors declare no off‐label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

Approved by the University of Wisconsin‐Madison, Animal Use Protocol #V006113.

HUMAN ETHICS APPROVAL DECLARATION

Authors declare human ethics approval was not needed for this study.

Melco A, Pritchard JC, Hetzel SJ, Saver A, Cavasin JP, Steiner JM. The effect of feeding on serum concentrations of cobalamin, folate, trypsin‐like immunoreactivity, and pancreatic lipase immunoreactivity in dogs with signs of chronic gastrointestinal disease. J Vet Intern Med. 2024;38(3):1465‐1474. doi: 10.1111/jvim.17064