Serial Measurement of Serum Pancreatic Lipase Immunoreactivity, Feline Trypsin-like Immunoreactivity, and Cobalamin Concentrations in Kittens

Simple Summary Serum concentrations of feline pancreatic lipase immunoreactivity (fPLI), feline trypsin-like immunoreactivity (fTLI), and cobalamin are commonly used for the diagnostic investigation of cats with gastrointestinal signs. No information on these parameters in healthy cats less than 1 year of age exists. We aimed to evaluate serum concentrations of fPLI, fTLI, and cobalamin in healthy cats at different time-points during their first 12 months of life. Fourteen healthy 2-month-old kittens were included. Blood was collected at 2, 3, 4, 6, and 12 months of age, and serum concentrations of fPLI, fTLI, and cobalamin were measured. Serum fPLI and fTLI concentrations did not show any statistically or clinically significant differences in young kittens. In contrast, serum cobalamin concentrations were commonly below the reference interval in kittens. Serum fPLI and fTLI concentrations are not practically affected by age in kittens as young as 2 months of age and could be used for the investigation of pancreatic diseases. Abstract Serum concentrations of feline pancreatic lipase immunoreactivity (fPLI), feline trypsin-like immunoreactivity (fTLI), and cobalamin are commonly used for the diagnostic investigation of cats with gastrointestinal signs. No information on these parameters in healthy cats less than 1 year of age exists. We aimed to evaluate serum concentrations of fPLI, fTLI, and cobalamin in healthy cats at different time-points during their first 12 months of life. Fourteen healthy 2-month-old kittens were included. Blood was collected at 2, 3, 4, 6, and 12 months of age, and serum concentrations of fPLI, fTLI, and cobalamin were measured. While there was a statistically significant difference in serum fPLI concentrations over time, there was no statistically significant difference between individual time-points. There was no significant difference in serum fTLI concentrations over time. Serum cobalamin concentrations were below the reference interval in 3/13 cats at 2 months of age and were significantly lower by 3 months, when 13/14 had hypocobalaminemia. By 12 months, serum cobalamin had significantly increased, yet 4/12 cats still had hypocobalaminemia. Serum fPLI and fTLI concentrations did not show any statistically or clinically significant differences in young kittens. In contrast, serum cobalamin concentrations were commonly below the reference interval in kittens. Serum fPLI and fTLI concentrations are not practically affected by age in kittens as young as 2 months of age and could be used for the investigation of pancreatic diseases.


Introduction
Serum concentrations of feline pancreatic lipase immunoreactivity (fPLI), feline trypsinlike immunoreactivity (fTLI), and cobalamin are commonly used for the diagnostic investigation of cats with gastrointestinal (GI) signs. Although GI and pancreatic diseases are (Purevax RCP and Purevax Rabies; Boehringer Ingelheim International GmbH) according to recent guidelines [20].

Sample Collection and Follow-Up Period
For kittens ≤ 3 months of age, food was withheld for 6 h, while for kittens > 3 months of age food was withheld for 12 h prior to blood collection. Upon enrollment into the study, a total of 3.5 mL of blood was collected from each kitten from the jugular vein, allowed to clot for 30 min, centrifuged at 3000× g for 10 min, and serum was collected, placed into Eppendorf tubes, and stored at −80 • C until analysis. In addition to the initial blood sample collected at approximately 2 months of age, additional samples were collected at 3, 4, 6, and 12 months of age and were handled in the same manner as for the initial sample. Samples were stored for no more than 12 months before all parameters were analyzed. The samples were sent by overnight courier, packed with icepacks, and were analyzed at the Gastrointestinal Laboratory at Texas A&M University. All serum samples were thawed at room temperature and serum concentrations of each parameter (i.e., fPLI, fTLI, cobalamin) were measured on the same day.
On each sampling day, a detailed clinical history was obtained, and cats underwent a thorough physical examination, like on enrollment.

Statistical Analysis
Data were tested for normality using the Kolmogorov-Smirnov test. Data were not normally distributed; therefore, overtime comparisons were performed with Friedman tests, while Dunn's post hoc tests were performed to determine which time-points were significantly different. Statistical significance was set at p < 0.05. All statistical analyses were performed using statistical software packages (SPSS 23 for Windows, IBM Corp., Armonk, NY, USA; and Prism 9, GraphPad Software Inc., San Diego, CA, USA).

Cats
A total of 14 cats completed the 10-month study. Of these, 8 were males and 6 were females. All cats were neutered/castrated by the end of the study period and were tested negative for FeLV antigen, FIV antibodies, and canine parvovirus antigen.

Serum fPLI Concentrations
Serum was available for all time-points for measurement of fPLI concentrations for 12/14 cats, and only these cats were included in the over-time analysis. Two additional cats were missing one time-point each (at 12 months). Overall, there was a statistically significant Vet. Sci. 2022, 9, 469 4 of 11 difference for serum fPLI concentrations over time (p = 0.0168; Figure 1). However, post hoc testing revealed that there was no statistically significant difference between any of the individual time-points. Overall, 0% (0/14) of cats had increased serum fPLI concentrations at 2 months of age; 21.4% (3/14, including 1 cat that was excluded from the over-time analysis) at 3 months; 0% (0/14) at 4 months; 7.1% (1/14) at 6 months; and 0% (0/12) at 12 months of age. All the increased serum fPLI concentrations were in the equivocal reference range (i.e., between 3.6 and 5.3 µg/L). Finally, no cats had consecutively increased serum fPLI concentrations over time ( Figure 1).
females. All cats were neutered/castrated by the end of the study period and were tested negative for FeLV antigen, FIV antibodies, and canine parvovirus antigen.

Serum fPLI Concentrations
Serum was available for all time-points for measurement of fPLI concentrations for 12/14 cats, and only these cats were included in the over-time analysis. Two additional cats were missing one time-point each (at 12 months). Overall, there was a statistically significant difference for serum fPLI concentrations over time (p = 0.0168; Figure 1). However, post hoc testing revealed that there was no statistically significant difference between any of the individual time-points. Overall, 0% (0/14) of cats had increased serum fPLI concentrations at 2 months of age; 21.4% (3/14, including 1 cat that was excluded from the over-time analysis) at 3 months; 0% (0/14) at 4 months; 7.1% (1/14) at 6 months; and 0% (0/12) at 12 months of age. All the increased serum fPLI concentrations were in the equivocal reference range (i.e., between 3.6 and 5.3 µ g/L). Finally, no cats had consecutively increased serum fPLI concentrations over time ( Figure 1).

Serum fTLI Concentrations
Serum was available for all time-points for measurement of fTLI concentrations for 12/14 cats, and only these cats were included in the over-time analysis. One cat had two time-points missing (at 4 and 12 months) and another cat had one time-point missing (at 12 months). No significant differences of serum fTLI concentrations were found over time ( Figure 2). In addition, no cats had serum fTLI concentrations below the lower limit of the reference interval at any time-point. In 21.4% (3/14, including the 2 cats that were excluded from the over-time analysis) of cats, fTLI was above the upper limit of the reference interval at 2 months; in 14.3% (2/14, including 1 cat that was excluded from the over-time analysis) at 3 months of age; while in 7.7% (1/13) and 7.1% (1/14) at 4 and 6 months of age, respectively. One cat had an increased fTLI concentration at both 2 and 3 months of age (this cat was not included in the over-time analysis) and one cat from 2 to 6 months of age ( Figure 2). reference interval at any time-point. In 21.4% (3/14, including the 2 cats that were excluded from the over-time analysis) of cats, fTLI was above the upper limit of the reference interval at 2 months; in 14.3% (2/14, including 1 cat that was excluded from the over-time analysis) at 3 months of age; while in 7.7% (1/13) and 7.1% (1/14) at 4 and 6 months of age, respectively. One cat had an increased fTLI concentration at both 2 and 3 months of age (this cat was not included in the over-time analysis) and one cat from 2 to 6 months of age ( Figure 2).

Serum Cobalamin Concentrations
Serum was available for all time-points for measurement of cobalamin concentrations for 11/14 cats, and only these cats were included in the over-time analysis. One cat had two time-points missing (at 4 and 12 months) and 2 cats had one time-point missing each (at 2 and at 12 months, respectively). Serum cobalamin concentrations significantly decreased (p = 0.005) from 2 (median, 494 ng/L; range, <150-1704 ng/L) to 3 months of age (median, 178 ng/L; range, <150-611 ng/L), and this was followed by a significant increase (p < 0.001) by 12 months of age (median, 404 ng/L; range, 169-938 ng/L, Figure 3). Overall, 30.8% (4/13, including the three cats that were excluded from the over-time analysis) of cats had serum cobalamin concentrations below the lower limit of the reference interval at 2 months of age; 92.9% (13/14, including the 3 cats that were excluded from the over-time analysis) at 3 months of age; and by the age of 12 months, 33.3% (4/12, including one cat that was excluded from the over-time analysis) of cats still had cobalamin concentrations below the lower limit of the reference interval (Figure 3). 30.8% (4/13, including the three cats that were excluded from the over-time analysis) of cats had serum cobalamin concentrations below the lower limit of the reference interval at 2 months of age; 92.9% (13/14, including the 3 cats that were excluded from the overtime analysis) at 3 months of age; and by the age of 12 months, 33.3% (4/12, including one cat that was excluded from the over-time analysis) of cats still had cobalamin concentrations below the lower limit of the reference interval ( Figure 3).

Discussion
Measurement of noninvasive markers of GI disease in cats with GI signs is routine in clinical practice. However, the concentrations of markers of GI and pancreatic disease have not been previously evaluated in clinically healthy cats less than 1 year of age. Thus, the aim of our study was to investigate the concentrations of three serum markers commonly used in the diagnostic investigation of GI diseases, namely serum fPLI, fTLI, and cobalamin concentrations, in young healthy cats. Overall, serum fPLI and fTLI concentrations were usually within the established reference intervals and were not affected by the age between 2 and 12 months. In contrast, serum cobalamin concentrations were significantly affected by age and different proportions of cats were hypocobalaminemic at different ages.
Serum fPLI concentrations were not associated with any clinically meaningful changes during the first year of life in our cats. In a small number of cats, serum fPLI concentrations were slightly above the upper limit of the reference interval at 3 and 6 months of age. However, the abnormally high serum fPLI concentrations in all these cats ranged between 3.6-5.3 µg/dL, concentrations that fall below the cutoff for a diagnosis of pancreatitis [21]. Serum fTLI concentrations did not change significantly during the first year of life in cats, and no cats had decreased serum fTLI concentrations, which are of interest for the diagnosis of EPI. A small percentage of cats had abnormally high serum fTLI concentrations, mainly at 2 and 3 months of age. From a total of 7 (of 67 measurements) increased serum fTLI concentrations, only one was associated with an abnormally high serum fPLI concentration (3 months of age, serum fPLI, 5 µg/L and fTLI, 144.3 µg/L). This cat was clinically healthy during physical examination, with no concerns reported by the owner, and no abnormalities of serum fPLI and fTLI concentrations were noted thereafter.
The activities and/or concentrations of pancreatic lipase and trypsin have not been thoroughly investigated in humans during early life, and different assays are used for evaluating these parameters [25]. The exocrine pancreas in humans has been described to mature slowly following birth, which is associated with low activities of lipase and trypsin in the pancreatic juice as well as with reduced response to secretagogues in 1-monthold infants [26]. In one study, the activities of lipase and trypsin measured in duodenal aspirates increased significantly in children from 1.7 to 8 years of age, suggesting an ongoing pancreatic secretory maturation process [27]. In our study, fPLI and fTLI concentrations did not significantly change with age, and the cats with concentrations higher than the upper limit of the reference interval did not have clinical signs of pancreatitis. Therefore, these values may represent the individualized maturation process of the secretory exocrine pancreas. Alternatively, since serum PLI concentrations show important intraindividual variability in dogs, this may also be the case in cats and may have been a contributing factor for the increased concentrations noticed in our study [28].
Serum cobalamin concentrations were subnormal in a considerable percentage of cats at all time points (ranging between 30% and 93%). In addition, serum cobalamin concentrations significantly decreased between 2 and 3 months of age, and this was followed by a gradual and significant increase by the age of 1 year. In the only study published so far as a full paper where the relationship between age and serum cobalamin concentrations was investigated, only adult (between 3 and 9 years of age) healthy colony cats were included [29]. In that study, an inverse relationship between serum cobalamin and age was identified, with increasing age being significantly associated with decreasing serum cobalamin concentrations.
In humans, serum cobalamin concentrations undergo significant changes during development. During early life, serum cobalamin decreases in humans, with the lowest concentrations observed from 6 weeks to 6 months of age, and this is followed by a gradual increase with a peak between 3 and 7 years of age, while a reduction is observed thereafter, which reaches a plateau towards adulthood [30,31]. Several studies have been published in humans suggesting the necessity to establish different reference intervals for serum cobalamin concentrations in infants and children, instead of the ones used in adults [30,[32][33][34][35], because low serum cobalamin concentrations (based on adult reference intervals) can be a common and normal finding in breast-fed infants [33,36]. Similarly, based on the present study, the use of the cobalamin reference interval of adult cats may not be appropriate in kittens and young cats. This should be taken into consideration when deciding whether hypocobalaminemia in young cats reflects a clinically relevant deficiency that requires cobalamin supplementation or whether it is a physiological process.
The mechanisms of cobalamin absorption, transport, and metabolism have not been elucidated in cats during early life. In humans, cobalamin is stored in the liver of the fetus during pregnancy, and subsequently, hepatic cobalamin stores play a major role in determining serum cobalamin concentrations during the first months of newborn life [36,37]. Following birth, cobalamin is supplemented through the milk along with cobalamin binders, especially haptocorrin or R-protein, that prevent cobalamin uptake by gastrointestinal bacteria [38].
During infancy in humans, the IF is either not produced in sufficient quantities by the stomach or it is not functional until a certain age [39][40][41]. In addition, IF receptors in the ileum are not adequately expressed due to the immaturity of the GI tract [42]. Finally, gastrin and pepsin, which decrease the gastric pH allowing the release of cobalamin from Vet. Sci. 2022, 9, 469 8 of 11 ingested proteins, are also not produced in sufficient amounts [42]. Therefore, cobalamin transport in infants is reported to be mediated by haptocorrin, and the absorption of cobalamin is suspected to take place by alternative receptors until the IF and IF-receptors are adequately developed [42]. In cats, IF is almost exclusively produced by the ductal cells of the exocrine pancreas [15] and it is unknown when the pancreatic IF production and ileal IF-receptor expression start playing major roles for cobalamin transport and absorption following birth. All cats remained clinically healthy over the 10-month followup period in our study, and no concerns were reported by their owners. Therefore, the reduction in cobalamin between 2 and 3 months of age as well as the high prevalence of hypocobalaminemia until the age of 6 months may reflect an age-related process driven by the immaturity of the GI tract and/or reduced pancreatic IF secretion, and the transition from breastfeeding/milk-replacement formula to a solid diet.
Serum cobalamin concentrations are also known to be affected by the intestinal microbiota [43]. The intestinal microbiota and metabolome have recently been shown to change significantly from 2 to 12 months of age [44,45]. The immaturity of the intestinal microbiome in young kittens until they reach an adult-like state might be an additional contributing factor that could be related to the changes in serum cobalamin concentrations during the first year of life in cats.
Markers of cellular deficiency of cobalamin, such as serum methylmalonic acid (MMA) concentrations, have been previously investigated in cats [46][47][48]. Measurements of serum MMA concentrations could have been useful in our study, although they have not been evaluated in young kittens and it is unknown if they represent an accurate marker of cellular cobalamin deficiency. However, MMA is measured by gas chromatography-mass spectrometry (GC-MS) and requires a large volume of serum that was not available in our study.
The main source of cobalamin in cats is considered to be the diet [29]. All cats in our study were fed the same diet, and therefore, differences in dietary cobalamin can be excluded as a possible cause of the differences in cobalamin concentrations among cats. In addition, the fact that serum cobalamin concentrations fluctuated over time without any diet changes suggests that dietary factors are unlikely to be responsible for these changes.
Food was withheld for 6 h in kittens < 3 months of age and for 12 h for kittens > 12 months of age. This was in order to prevent problems such as hypoglycemia, caused by prolonged fasting in kittens of a very young age. However, this is not expected to have influenced our results, because in a recent study it was shown that feeding did not affect serum fPLI and cobalamin concentrations [49]. Although feeding can affect serum fTLI concentrations, no differences were found in our study for fTLI over time.
Our study had some limitations. The number of cats that completed the study was relatively small. This was a prospective study with a relatively long follow-up period, and it was challenging to engage owners to regularly come back for blood collection. Even with these small numbers, our results were rather consistent in each group, and therefore, they likely reflect the general population.

Conclusions
In conclusion, our results indicate that serum fPLI and fTLI concentrations are not significantly affected by age in young kittens, between the ages of 2 and 12 months, and can be used for the investigation of pancreatic diseases in these animals. In contrast, serum cobalamin concentrations are commonly below the established reference interval in kittens. Whether this represents a physiological or a pathological condition is not clear, and further studies are needed to investigate this finding. In any case, an age-specific reference interval for serum cobalamin concentration should be established for kittens. However, given that cobalamin supplementation is not associated with any known side effects, overdiagnosis of hypocobalaminemia in kittens would not be expected to be detrimental.