Serum triglyceride and cholesterol concentrations and lipoprotein profiles in dogs with naturally occurring pancreatitis and healthy control dogs

Abstract Background Previous studies have reported an association between hyperlipidemia and pancreatitis in dogs, but details of this association remain poorly defined. Hypothesis/Objectives To compare serum triglyceride and cholesterol concentrations and lipoprotein profiles between dogs with naturally occurring pancreatitis and healthy dogs. Animals Seventeen dogs with a clinical diagnosis of pancreatitis (Group 1) and 53 healthy control dogs (Group 2). Methods Prospective case‐control study. Results In Group 1, 3/17 dogs (18%) had hypertriglyceridemia whereas in Group 2, 4/53 dogs (7.5%) had hypertriglyceridemia (odds ratio [OR], 2.63; 95% confidence interval [CI], 0.52‐13.14; P = .35). A significant difference was found in serum triglyceride concentrations between Group 1 (median, 67 mg/dL) and Group 2 (median, 54 mg/dL; P = .002). In Group 1, 4/17 dogs (24%) had hypercholesterolemia, whereas 1/53 (1.9%) dogs in Group 2 had hypercholesterolemia (OR, 16; 95% CI, 1.64‐155.5; P = .01). No significant difference was found in serum cholesterol concentrations between Group 1 (median, 209 mg/dL) and Group 2 (median, 227 mg/dL; P = .56). Lipoprotein profiles were significantly different between Group 1 and Group 2 dogs (Eigenvalues, 0.6719; R 2 = 1.0; P = .001). Conclusions and Clinical Importance Most dogs with pancreatitis (>70%) had serum triglyceride and cholesterol concentrations within reference intervals. In the small percentage of dogs that had hypertriglyceridemia, hypercholesterolemia, or both, increases were mild. Important differences were identified in lipoprotein profiles between dogs with pancreatitis and healthy control dogs. Dogs with pancreatitis had higher low‐density lipoprotein fractions and lower triglyceride‐rich lipoprotein and high‐density lipoprotein fractions than healthy dogs.


| INTRODUCTION
The nature of the association between hyperlipidemia and pancreatitis remains unclear in dogs, but it has been speculated to be bidirectional. 1,2 Hypertriglyceridemia, a form of hyperlipidemia, has been considered to be the cause of pancreatitis in some cases. This hypothesis is supported by the results of 2 clinical studies in dogs, 3,4 and is further supported by ex vivo studies 5 in dogs and clinical studies in humans. [6][7][8] On the other hand, hyperlipidemia also has been hypothesized to be the result of pancreatitis. 1,2 Although this association is widely believed to be true, scientific evidence supporting this hypothesis has not been documented in dogs with naturally occurring pancreatitis. Hypertriglyceridemia has been reported in dogs commonly with pancreatitis, but it remains unclear in these studies whether hypertriglyceridemia was the result of pancreatitis, a coexisting disease, a postprandial state, or a combination of these factors. [9][10][11][12][13] Furthermore, with the exception of the results of 1 study, 14 hypertriglyceridemia has not been reported to be a consequence of experimentally induced pancreatitis in dogs. 10,15,16 However, experimental models of pancreatitis do not always replicate the pathophysiologic mechanisms of spontaneous disease. Requirements for inclusion of dogs in Group 1 were: (1) a clinical diagnosis of pancreatitis; (2) absence of diseases that can cause secondary hyperlipidemia (eg, hypothyroidism, hyperadrenocorticism) 1,2 ; (3) not receiving or having received any medications known to affect lipid metabolism (eg, glucocorticosteroids) for at least 3 months before inclusion in the study 1,2 ; (4) being fed diets not labeled as "low-fat"; (5) withholding of food for at least 12 hours before blood collection; and (6)

| Group 2 (healthy control dogs)
Inclusion criteria for the control group were: (1) absence of any clinical signs at the time of blood collection and the preceding 2 months, (2) no major abnormalities on the serum biochemistry profile, (3) a serum Spec cPL concentration within the reference interval, (4) absence of diseases that may cause secondary hyperlipidemia (eg, diabetes mellitus, hypothyroidism, hyperadrenocorticism), (5) not receiving or having received any medications known to affect lipid metabolism (eg, glucocorticosteroids) for at least 3 months before inclusion in the study, and (6) being fed diets not labeled as "low-fat." 1 The healthy dogs belonged to students and staff of the College of Veterinary Medicine and Biomedical Sciences at Texas A&M University. Blood samples were collected from these dogs after food had been withheld for at least 12 hours. Blood samples were collected into glass tubes without additives, allowed to clot for 20 minutes, centrifuged, and serum aliquots were stored at −80 C until analysis. Owners were asked to complete a standardized questionnaire for each dog. Questions covered date of birth, sex and neuter status, body weight and BCS, current diet, current medications, and current and past health history of the dogs. Questionnaires from all dogs were reviewed to determine whether the dogs fit the inclusion criteria for the study.

| Ethics approval
The study protocol was reviewed and approved by the Clinical Research Review Committee at Texas A&M University (TAMU-CRRC# 2008-37).
The owners of each healthy dog enrolled in the study signed an informed owner consent form. For dogs with pancreatitis, only residual serum was used and therefore informed consent was not obtained.

| Lipoprotein profile analysis
Lipoprotein profiling was carried out using a bismuth sodium ethylenediaminetetraacetic acid (NaBiEDTA) density gradient ultracentrifugation method as previously described. 19 This method has been T A B L E 1 Characteristics of dogs with pancreatitis and healthy control dogs included into the study validated in dogs 20 and used to evaluate lipoprotein profiles in healthy dogs 21 and in dogs with idiopathic hyperlipidemia 21 and exocrine pancreatic insufficiency. 20 This method also has been used to evaluate lipoprotein profiles in cats with hepatic lipidosis. 22 The sodium salt of BiEDTA has been described as a novel solute forming a self-generating density gradient during ultracentrifugation of serum samples for the separation of lipoproteins. 23 The methodology used in our study previously was shown to identify 11 distinct lipoprotein fractions in dogs based solely on density characteristics. 20  and Group 2 (median, 227 mg/dL; range, 97-338 mg/dL; P = .56; Figure 2).  Table 2). Figure 4A shows the lipoprotein density profile of a representative dog with pancreatitis. Figure 4B shows the lipoprotein density profile of a representative healthy dog. These results suggest that the main differences between these 2 representative lipoprotein profiles involved the LDL fractions, whereas the differences in TRL and HDL fractions were less prominent.

| DISCUSSION
The purpose of our study was to investigate serum lipid and lipoprotein abnormalities associated with naturally occurring pancreatitis in dogs.
Mild increases in serum triglyceride or cholesterol concentrations or both were seen in a relatively small proportion of dogs with pancreati-  Our findings are in agreement with those of studies of experimentally induced pancreatitis in dogs. 10,15,16 Most of those studies have concluded that hypertriglyceridemia is not a consequence of experimentally induced pancreatitis in dogs. 10,15,16 In 1 study, statistically significant increases in serum triglyceride concentrations were noted after induction of pancreatitis, but serum triglyceride concentrations remained within the reference interval throughout the study period. 16 Similar findings were reported for hypercholesterolemia in those studies. 10,15,16 Although major changes in serum triglyceride and cholesterol concentrations were not found in dogs with pancreatitis in our study,  [25][26][27][28][29] These changes in lipids and lipoproteins that occur during inflammation are part of the innate immune response and are believed to benefit the host through various mechanisms such as redistribution of nutrients to immune cells and binding of endotoxins and other biological substances. 29,31 Similar changes were observed in our study with respect to HDL and triglycerides. Inflammation in humans appears to induce a shift in LDL profiles leading to the appearance of small dense LDL molecules that correspond to fractions LDL 4 and LDL 5 , a finding that was observed also in the dogs of Group 1 in our study. [25][26][27][28][29] It seems likely that, as in humans, changes in serum lipid concentrations and lipoprotein profiles in dogs with pancreatitis might be the result of a general inflammatory response rather than a pancreatitis-specific change.
Our study had some limitations. The major limitation is related to the small number of dogs with pancreatitis included in our study. This was partly a consequence of the strict inclusion criteria of our study that required exclusion of dogs that had evidence of concurrent diseases or conditions that can affect lipid metabolism. Previous studies have shown that pancreatitis and endocrine diseases that affect lipid metabolism commonly coexist. [11][12][13]32 In accordance with those studies, approximately 40% of the dogs initially enrolled in our study eventually were excluded because of the presence of concurrent disease that could have affected lipid metabolism. Another limitation was that the diagnosis of pancreatitis ideally should have been based on a combination of diagnostic tests in addition to clinical presentation and Spec cPL concentration. However, serum Spec cPL concentration has been shown to be very specific for pancreatitis. [33][34][35] Although confirmation of the presence of pancreatitis usually requires histopathology, it is not commonly used in a clinical setting.
Finally, the severity of pancreatitis in the dogs included in our study was not evaluated. It is likely that different degrees of severity corresponding to different degrees of inflammation might lead to differences in the degree of hyperlipidemia, although this has not been proven.
In conclusion, the majority of dogs with naturally occurring pancrea-