Lipoprotein profiles in Miniature Schnauzer dogs with idiopathic hypertriglyceridemia and hypercortisolism

Abstract

Miniature Schnauzer dogs (MSs) are predisposed to both idiopathic hypertriglyceridemia (iHTG) and hypercortisolism (HCort). To our knowledge, the lipoprotein profiles of MSs with iHTG have not been compared to those with HCort. We analyzed cholesterol and triglyceride concentrations and lipoprotein fractions in 4 groups of MSs: normotriglyceridemia (NTG) without concurrent disease (Healthy-NTG), HCort and NTG (HCort-NTG), HCort and HTG (HCort-HTG), and iHTG. Lipoprotein fractions were assessed by lipoprotein electrophoresis and compared between groups. Fifty-one plasma samples were analyzed. Twenty-five dogs had NTG (16 Healthy-NTG, 9 HCort-NTG) and 26 dogs had HTG (7 iHTG, 19 HCort-HTG). Dogs with iHTG or HCort-HTG had significantly higher cholesterol concentrations than Healthy-NTG dogs. Dogs with HCort-HTG had higher cholesterol than HCort-NTG dogs. There was a significantly higher low-density lipoprotein (LDL) percentage in iHTG and HCort-HTG dogs than HCort-NTG dogs. HCort-HTG dogs also had lower high-density lipoproteins (HDL) than HCort-NTG dogs. It was not possible to readily distinguish MSs with iHTG from MSs with HCort-HTG or Healthy-NTG using lipoprotein electrophoresis fractions. The diagnosis of iHTG remains a diagnosis by exclusion.

Lipoproteins are carrier molecules in the blood that aid in the transport of various lipids.^14,35,53 The major classes are chylomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL).^14,44,53 These lipoprotein fractions have been rigorously studied in human medicine and help in predicting and investigating diseases in humans, especially coronary heart disease.^39,47 Lipoprotein profiling in dogs is not a routine clinical practice, although several studies have reported the profiles in both health and disease.^{1,9,18,23,29,31,33,36–38,46,51}

Fasting hypertriglyceridemia (HTG) in Miniature Schnauzer dogs (MSs) has a reported prevalence of 32.8%. ⁴⁸ MSs are thought to have a genetic alteration in lipid metabolism, predisposing them to fasting HTG, but the actual mechanism for the abnormality and the genetic basis have yet to be identified. ⁴⁶ A study utilizing both ultracentrifugation and electrophoresis to investigate the lipoprotein profiles of MSs with idiopathic HTG (iHTG) found that affected dogs had increased VLDL when compared to healthy dogs of other breeds, with or without an accompanying chylomicronemia. ⁴⁶ Another study used a novel lipoprotein density profiling method to compare lipoprotein profiles in MSs with iHTG to those in MSs and non-MS dogs with normotriglyceridemia (NTG). ⁵¹ The lipoprotein profile of MSs with iHTG was characterized by an increased proportion of triglyceride-rich lipoprotein (TRL), which includes both chylomicrons and VLDL, and a decreased proportion of LDL, compared to normotriglyceridemic MSs and dogs of other breeds with NTG. ⁵¹ Most major lipoprotein classes in MSs with NTG were similar to those of other dog breeds. However, most MSs with NTG had higher TRL concentrations and lower LDL fractions compared to other breeds. It was also found that MSs with NTG have lower cholesterol concentrations than dogs of other breeds. ⁴⁷ However the reported composition of lipoprotein classes was merely presumptive based on the density characteristics of the analyzed fractions.

HTG in dogs can also be secondary to other diseases such as diabetes mellitus, hypothyroidism, hypercortisolism (HCort), and pancreatitis. ⁵³ Hypothyroidism, pancreatitis, and diabetes mellitus were excluded by clinical evaluation and laboratory tests in previous lipoprotein profiling studies of iHTG.^48,51 HCort, however, has usually been excluded based on the absence of clinical signs with or without limited adrenal function testing. Studies evaluating the lipoprotein profiles in dogs with HCort have detected increased LDL,^1,3,17 increased VLDL,^3,17 and low HDL.^3,17 We searched databases, including PubMed and Google Scholar, using the following search terms: Miniature Schnauzers, lipoproteins, hypercortisolism, Cushing’s syndrome, hyperadrenocorticism and idiopathic hyperlipidaemia. Our search failed to reveal any papers comparing lipoprotein profiles of MSs with iHTG and HCort; thus, to our knowledge, the differences between lipoprotein patterns in MSs with iHTG and HCort have not been reported previously.

There are numerous similarities between iHTG and HCort. Both cause HTG and occur frequently in MSs, both increase in prevalence with increasing age, and both have been associated with increased liver enzyme activity and proteinuria.^13,48,49 By performing a study with rigorous adrenal function testing, distinct lipoprotein fingerprints might be found for MSs with iHTG compared to MSs with HCort. We therefore investigated cholesterol and triglyceride concentrations and lipoprotein profiles in MSs with NTG and no concurrent disease, NTG and HCort, HTG and HCort, and iHTG.

Materials and methods

Animals and sample population

All samples were collected as part of a study evaluating HTG in Australian MSs with an approved Animal Ethics Permit from Murdoch University (R3174/19). MSs of all ages, sex, and neuter status were recruited across Australia. For this part of our study on lipoprotein profiling, only dogs >1-y-old were included. Exclusion criteria included: previously diagnosed HCort, diabetes mellitus, or hypothyroidism; treatment with drugs known to affect lipid metabolism (e.g., corticosteroids and phenobarbitone) within the last 2 mo; medical illness requiring veterinary attention in the last 4 wk; and current lactation or pregnancy. Dogs had general health assessed by an owner questionnaire and veterinary examination. The body condition score (BCS) was assessed as part of this examination using a 9-point scale according to descriptions and illustrations from the World Small Animal Veterinary Association (https://wsava.org/wp-content/uploads/2020/01/Body-Condition-Score-Dog.pdf). Fasting serum triglyceride and cholesterol concentrations were measured after a 15-h fast. Blood for triglyceride and cholesterol measurement was collected into a plain tube, centrifuged at 1,358g for 10 min at 4°C, then aliquoted and frozen at −80°C until processed. Fasting HTG was defined as fasting serum triglycerides above the laboratory RI (>1.80 mmol/L).

Dogs with fasting HTG underwent further investigati-ons including routine hematology and serum biochemistry (including 1,2-o-dilauryl-rac-glycero glutaric acid-(6′-meth-ylresorufin) ester [DGGR] lipase activity), and urinalysis. Triglyceride concentrations were also repeated in the routine biochemistry profile. Further investigations were performed ≤3 mo following the initial measurement of serum triglyceride concentrations, unless delayed because of the SARS-CoV-2 pandemic. Any dogs diagnosed with diabetes mellitus or hypothyroidism as a part of these follow-up investigations were excluded from the lipoprotein analysis. Thyroid function was assessed by measurement of total serum thyroxine (T4) concentration and, if decreased, canine thyroid-stimulating hormone (TSH) concentration was also measured.

To assess adrenal function, a standard low-dose dexamethasone suppression test (LDDST) was performed. If the results were negative for HCort, an adrenocorticotropic hormone (ACTH) stimulation test was performed within 2 mo of the LDDST. Owner’s consent was obtained for 30 MSs with NTG to undergo the same investigations as dogs with HTG. A subset of these dogs was included in our lipoprotein profile study. Based on the results of this testing, dogs were classified as Healthy-NTG (MSs with NTG and no concurrent disease affecting lipid metabolism), HCort-NTG (MSs with HCort and NTG), HCort-HTG (MS with HCort and HTG), or iHTG (MSs with idiopathic HTG). A total of 10 mL of blood from each dog was collected into EDTA for lipoprotein analysis. The sample was kept at 4°C before being centrifuged at 1,358 × g for 10 min at 4°C within 24 h. The plasma was aliquoted and stored at −80°C until analyzed.

Assays

Hematology (XN-V hematology analyzers; Sysmex) was performed at 2 laboratories (Vetnostics, New South Wales; VetPath Laboratories, Western Australia). A manual differential count was included in each CBC. Serum biochemistry (including DGGR lipase) was performed at Vetnostics using Roche reagents on the Cobas 8000 (c502 and c702 models; Roche). Triglycerides and cholesterol were measured using enzymatic colorimetric assays on an automated spectrophotometer (Cobas INTEGRA 400 plus analyzer; Roche). Multistix 10 SG (Siemens) was used for urine dipstick analysis. Concurrent urine specific gravity measurement and sediment examination were performed in all dogs. Serum total T4 and TSH measurements were performed (Immulite 2000 immunoassay system; Siemens). Cortisol concentrations were measured (Advia Centaur XP immunoassay system; Siemens). The RIs for the adrenal function tests in healthy dogs were established by direct comparison with the radioimmunoassay (Coat-A-Count cortisol radioimmunoassay kit; Diagnostic Products) and RIs used by the University of Sydney (unpublished data); the upper RI for the ACTH stimulation test for this assay has also been assessed.^54,55 A diagnosis of hypothyroidism was made if there was a low total T4 concentration and concurrent increase in the TSH in the absence of HCort.

Lipoprotein electrophoresis

Lipoprotein electrophoresis was performed on buffered (pH 8.5) agarose gel on a semi-automated instrument (Hydrasys; Sebia), using a dedicated kit (Hydragel 15 lipoproteins; Sebia). Briefly, 10 μL of sample were loaded in each well of the applicator, then the applicator was placed into a wet chamber to allow samples to diffuse onto the applicator teeth for 5 min. After the agarose gel and the sample applicators were loaded into the instrument, the migration at 160 V for 25 min was started. When the migration was completed, the gel was dried at 60°C for 15 min, stained with Sudan black, washed with 45% ethanol, dried, and placed on the gel scanner. Gels were scanned at 570 nm and analyzed using the software Phoresis (Sebia), which calculated the area under the peaks corresponding to chylomicrons, VLDL, LDL, and HDL.

Statistical analysis

Statistical analyses were performed using SPSS (v.24; IBM), and p ≤ 0.05 was considered statistically significant. Data were tested for normality using the Shapiro–Wilk test. A Mann–Whitney U test was used for comparison of age, sex, and BCS. A Mann–Whitney U test was used for comparison of hematologic, biochemical, and urinalysis variables between groups. For dogs with a serum bilirubin <1 μmol/L, gamma-glutamyl transferase (GGT) <5 U/L, or >100 × 10⁶/L RBCs or WBCs in their urinary sediment, dogs were nominated as 1 μmol/L, 5 U/L, and 100 × 10⁶/L, respectively, for statistical analysis. Significant differences in lipid concentration between groups were assessed by a Kruskal–Wallis test followed by a Mann–Whitney U test.

Results

We included 51 MSs in our study. Samples were obtained from 16 Healthy-NTG, 9 HCort-NTG, 19 HCort-HTG, and 7 iHTG dogs. Median age was 8 y (range: 1–13 y). One dog was 10.5 mo when included in the study, but was >1-y-old when the sample for lipoprotein profiling was collected. Twenty-seven (53%) dogs were female (1 intact, 26 spayed), and 24 (47%) dogs were male (2 intact, 22 castrated; Suppl. Tables 1, 2). Median BCS was 5 (range: 3–7 on a 9-point scale). All dogs, except one from Queensland, resided in either Western Australia (n = 26) or New South Wales (n = 24). The only significant difference was a higher BCS in the iHTG group compared with the Healthy-NTG group (p = 0.008). The median time between measurement of fasting triglyceride concentrations and hematology, biochemistry, urinalysis, thyroid function, and LDDST was 91 d (range: 1–370 d). Median time between LDDST and ACTH stimulation test was 39 d (range: 1–138 d).

Monocytes were higher in HCort-HTG dogs compared to HCort-NTG dogs; however, absolute counts were within laboratory RIs (Suppl. Tables 2, 3). A platelet count was not available for one dog with iHTG and one with HCort-HTG because of platelet aggregation in the sample. Urine was unable to be collected in one dog from the Healthy-NTG group. Some dogs had serum glucose concentrations below the RI. None of these dogs had clinical signs of hypoglycemia, and this change was likely a storage artifact given that glucose was measured in serum. All dogs with fasting HTG at inclusion also had increased triglyceride concentrations. No dogs with NTG at inclusion had abnormal triglyceride concentrations on subsequent biochemistry analysis.

Numerous statistical differences in sodium, potassium, chloride, anion gap, urea, aspartate transaminase activity, total protein, globulins, amylase, and creatine kinase were identified between groups (Suppl. Table 4). Most absolute values for these analytes were within RIs and, if abnormal, were clinically inconsequential (Suppl. Table 5). Lipase activities were higher in the iHTG group compared to Healthy-NTG (p = 0.033) and HCort-NTG (p = 0.031) dogs. Similarly, lipase activities were higher in HCort-HTG dogs than in Healthy-NTG (p = 0.007) and HCort-NTG (p = 0.012) dogs. Dogs with HCort-HTG had higher alkaline phosphatase (ALP) activities compared to dogs with Healthy-NTG (p = 0.029) and HCort-NTG (p = 0.028). Higher ALP activities were found in dogs with iHTG compared to dogs with HCort-NTG (p = 0.023) but not the Healthy-NTG group (p = 0.089). The only difference between dogs with iHTG and HCort-HTG was lower glucose concentrations in the iHTG group (p = 0.048).

Four dogs in the Healthy-NTG group had marginal increases in alanine transaminase (ALT) activity. One of these dogs had a concurrent mild increase in GGT activity. Three other dogs had a mild increase in GGT activity (n = 1) and marginal increases in ALP activities (n = 2). These dogs were reportedly healthy by the owners, and their lipoprotein profiles were not different from other dogs in this group.

Dogs with HCort-HTG had lower urine concentrations and higher urinary protein compared to Healthy-NTG dogs (p = 0.003 and p = 0.014, respectively) and HCort-NTG dogs (p = 0.002 and p = 0.012, respectively; Suppl. Tables 6, 7). Concurrent hypothyroidism was not identified in any of the included dogs. Three HCort-NTG dogs and 6 HCort-HTG dogs were diagnosed with HCort based on the results of the LDDST. HCort was diagnosed in the remaining 19 dogs based on an exaggerated response in the ACTH stimulation test (Suppl. Table 8).

Triglyceride and cholesterol concentrations, chylomicrons, VLDL, LDL, and HDL electrophoretic fractions displayed non-Gaussian distributions, and nonparametric tests were used to compare the groups. Cholesterol was significantly higher in dogs with iHTG (p = 0.022) and in dogs with HCort-HTG (p = 0.001) than in Healthy-NTG dogs. Dogs with HCort-HTG had significantly higher cholesterol (p = 0.037) than dogs with HCort-NTG. The LDL percentage was higher in dogs with iHTG (p = 0.01) and HCort-HTG (p = 0.009) than HCort-NTG. In addition, compared to HCort-NTG dogs, HCort-HTG dogs had lower HDL percentages (p = 0.03; Table 1, Suppl. Table 9). There was no significant difference in chylomicrons and VLDL percentages between the groups (Suppl. Table 10).

Table 1.

Descriptive data reporting the median, interquartile range, and minimum–maximum for each measured variable across different groups of Miniature Schnauzer dogs.

	Group
	Healthy-NTG			iHTG			HCort-NTG			HCort-HTG
	Median	IQR	Min.–max.	Median	IQR	Min.–max.	Median	IQR	Min.–max.	Median	IQR	Min.–max.
Cholesterol, mmol/L* † ‡ §	4.6	2.1	2.8–9.7	6.6	1.5	5.2–7.7	5.7	1.4	4.2–6.1	7.3	3.3	4.8–13.2
Triglycerides, mmol/L* † ‡ §	1.0	0.5	0.3–1.5	3.8	8.6	1.84–25.8	0.9	0.9	0.5–1.6	6.6	5.8	1.9–27.5
Chylo-EL, %	2.5	4.5	0.5–17.7	1.9	1.6	0.6–3.0	1.7	3.8	1.0–6.1	1.9	1.5	0.3–6.3
VLDL-EL, %	6.3	5.9	1.4–49.8	9.6	9.0	4.8–14.6	8.9	2.7	2.1–10.7	9.2	16.0	3.3–63.5
LDL-EL, %‡ §	8.6	5.9	1.1–19.2	9.1	16.3	6.6–31.3	5.3	4.3	2.3–12.4	9.7	8.1	2.9–26.7
HDL-EL, %§	78.4	13.0	27.3–95.3	75.1	21.7	58.5–86.5	82.7	8.0	77–91.8	75.2	18.9	29.5–89.3

Chylo-EL = chylomicron measured by electrophoresis; HCort-HTG = hypercortisolism with hypertriglyceridemia; HCort-NTG = hypercortisolism with normotriglyceridemia; HDL-EL = high-density lipoprotein when measured by electrophoresis; Healthy-NTG = normotriglyceridemia without concurrent disease affecting lipid metabolism; iHTG = idiopathic HTG; IQR = interquartile range; LDL-EL = low-density lipoprotein measured by electrophoresis; VLDL-EL = very-low-density lipoprotein measured by electrophoresis. Cholesterol concentrations RI: 3.4–10 mmol/L. Triglyceride concentrations RI: 0.2–1.8 mmol/L.

^* Statistical difference between Healthy-NTG and iHTG.

^† Statistical difference between Healthy-NTG and HCort-HTG.

^‡ Statistical difference between iHTG and HCort-NTG.

^§Statistical difference between HCort-NTG and HCort-HTG.

Discussion

We found that dogs with iHTG could not be distinguished readily from dogs with HCort using this method of lipoprotein profiling. Hypercholesterolemia is variably present in MSs with HTG. Although MSs in our study with HTG (iHTG and HCort-HTG) had significantly higher concentrations of cholesterol compared to the Healthy-NTG dogs, the absolute cholesterol concentrations were typically within laboratory RIs. This finding is similar to that reported previously. ⁴⁸

Studies have reported that dogs with iHTG have increased VLDL concentrations, with or without an accompanying chylomicronemia.^46,51 In some dogs with iHTG, a concurrent decrease in LDL concentrations has also been detected.^46,51 In contrast to these publications, our results did not detect significant differences in VLDL or chylomicron percentages between Healthy-NTG dogs and those with iHTG or HCort. The LDL percentages in iHTG dogs were higher than those dogs with HCort-NTG; however, no difference in LDL percentages was present when compared to Healthy-NTG and HCort-HTG dogs.

There are many possible explanations for the inability to demonstrate distinct lipoprotein fingerprints between dogs with iHTG and dogs with HCort. First, it could be that the dogs with HCort-HTG had concurrent iHTG. In humans, iHTG is a complex disease and both genetic and acquired risk factors often coexist in an individual patient. ²² Thus, it is theoretically possible that genetic defects in triglyceride production or clearance might not manifest as HTG in a healthy dog but when lipid metabolism is further disturbed by acquired risk factors, such as HCort and aging, HTG could result. Supporting this possibility is the fact that HCort was also diagnosed in patients with NTG. In addition, lipoprotein fractions differed between MSs with HCort-NTG and those with iHTG or HCort-HTG. If iHTG and HCort were concurrent, differentiating the 2 groups would likely be impossible with lipoprotein profiling and would require specific genetic assessment, currently unavailable.

Second, given that adrenal function testing has not been a prerequisite for the diagnosis of iHTG, it is possible that MSs reported previously as having iHTG could have had HCort. Hypercortisolism and iHTG share similar clinical and clinicopathologic abnormalities,^13,48,49 and in our study, the 2 disease entities could not be differentiated based on owner observations, clinical examination, or clinicopathologic data. False-positive adrenal function tests could confound analysis and are a possibility; however, specificities for the ACTH stimulation test and LDDST are very high in healthy dogs using comparative RIs.^11,12,27,43 The European Society of Veterinary Endo-crinology ALIVE Project “recognizes that demonstrable hypercortisolism is not always associated with or does not always lead to Cushing’s syndrome.” Therefore, lack of clinical signs of HCort is insufficient for exclusion of this disease. Increased VLDL concentrations have been reported for MSs with iHTG and dogs with HCort suggesting some similarity of lipoprotein abnormalities in these diseases.^3,17 Although we failed to detect any significant difference between VLDL percentages between groups, the median VLDL percentages were higher in iHTG and HCort groups compared to the Healthy-NTG group.

Third, the failure to separate the populations could be a Type II error given the very small number of dogs identified with iHTG.

Fourth, some dogs had triglyceride concentrations only marginally above the RI at inclusion. Although the RI for triglyceride concentrations that we used is lower than that reported in other studies^48,51 or provided by other laboratories, our RI was validated in a healthy population of dogs. ⁵⁵ In addition, in our study, the subsequent biochemistry also included triglyceride measurement as part of the laboratory’s routine biochemistry profile. All dogs classified as NTG or HTG at inclusion remained classified within these groups when the triglycerides were repeated. Hence, the likelihood that the lower RI that we used led to the misclassification of dogs in the HTG groups is very unlikely. However, including dogs with only marginal increases in the fasting triglyceride concentrations may have affected our ability to separate differences in the lipoprotein profiles in dogs with HTG from healthy dogs. Dogs with mild HTG may not have as profound changes in the lipoprotein profiles.

Fifth, age is associated with increasing triglyceride concentrations, and older dogs of various breeds frequently have mild HTG.^{16,20,25,40,41} It is possible that dogs with mild HTG in our study may have increased triglyceride concentrations as a result of age rather than disease. However, to our knowledge, age-specific RIs are not available for triglyceride concentrations. Therefore, dogs in our study were assessed using the same RIs regardless of age.

Last, the failure to distinguish lipoprotein profiles between iHTG and HCort dogs could stem from methodology. A limitation of our study was the lack of a gold standard for the measurement of VLDL, LDL, and HDL concentrations in canine plasma. The methodologies used to study canine lipoproteins include electrophoresis, sequential density gradient centrifugation, and size exclusion methods.^{7,8,19,23,30,35,37,45,46} Sequential density gradient centrifugation is the gold standard in human medicine. In dogs, LDL and HDL span the density range of 26.05–28.15 mmol/L and 26.54–31.33 mmol/L, respectively, with a significant overlap precluding complete separation by ultracentrifugation. This overlap is the result of the presence of large, cholesterol-enriched HDL particles, called HDL₁, in the density range 26.54–28.49 mmol/L. Canine HDL contains a second, relatively smaller and denser subpopulation of particles in the density range 27.71–31.33 mmol/L, which although similar to human HDL₃, are usually referred to as HDL₂. ²³ HDL₂ are deficient in cholesterol and apolipoprotein E relative to HDL₁. Given the presence of HDL₁ in the LDL density range, ultracentrifugation is not the method of choice for dogs, and we selected lipoprotein electrophoresis. Although lipoprotein electrophoresis is only a semi-quantitative percentage evaluation for lipoprotein classes (i.e., it is not possible to properly quantify lipoproteins), electrophoretic methods separate canine lipoproteins efficiently and are generally accepted as accurate, even if differences between automated and manual electrophoresis have been demonstrated, particularly with their ability to identify canine VLDL. ²

In addition to the lack of gold standard methodology, there are no studies comparing the lipoprotein profiles of serum and plasma, to our knowledge.^{1,28,30,35,46} It is unlikely that plasma interferes with lipoprotein electrophoresis given that the main difference between plasma and serum is the presence of fibrinogen. Sudan black, the stain used for lipoprotein electrophoresis, is specific for lipids and does not stain fibrinogen; therefore, interference by fibrinogen is not expected. Furthermore, canine plasma lipoprotein fractions separated by lipoprotein electrophoresis have been shown to be accurate when compared to separation by ultracentrifugation. ²⁶

There are few studies investigating the lipoprotein profiles in dogs with HCort. When lipoprotein profiles assessed by chromatographic analysis were compared between healthy dogs, obese dogs, and those with HCort, dogs with HCort were found to have higher total concentrations of triglycerides, cholesterol, and VLDL and lower concentrations of HDL-cholesterol fractions. ¹⁸ Increases in VLDL fractions and concurrent increases in LDL percentages in HCort dogs have also been shown in other studies.^3,17,18 Our results revealed a higher LDL and lower HDL percentage in dogs with HCort-HTG compared to HCort-NTG. The reason why there was no significant difference observed in the VLDL and LDL percentages between the Healthy-NTG dogs and those with HCort remains unknown, although it may suggest insensitivities of this methodology. Another possible explanation for the lack of difference between the Healthy-NTG and HCort dogs could be false-positive adrenal function tests resulting in the misclassification of dogs as HCort. Specificity of the LDDST has been reported to be 67–73%^6,34,42; however, in the absence of nonadrenal disease, specificity increases to 93%, making this unlikely. ⁵⁶ The ACTH stimulation test has a reported specificity of >90%.^4,5,24 Most false-positive adrenal function test results are encountered with significant nonadrenal illness. Dogs included in our study were regarded as healthy by their owners and at veterinary examination, thus the possibility for false positives as a result of nonadrenal illness was low, rendering misclassification of dogs unlikely. The SARS-CoV-2 pandemic–related delay of subsequent investigations following the initial measurement of fasting triglyceride concentrations may have led to some dogs developing HCort after their initial sampling and misclassification in the HCort group or HCort-NTG group. However, only 1 dog in the HCort-HTG group and 4 dogs in the HCort-NTG group were potentially affected.

We found a significantly higher BCS in the iHTG group compared to the Healthy-NTG group. Although age between groups was not significant, the Healthy-NTG group did include younger animals, some of which were intentionally kept lean by the owners. In combination with the small numbers in the groups, this may have contributed to the statistical differences in BCS between the Healthy-NTG and iHTG groups. BCSs in all but 1 dog were determined by the same 2 authors (T. Bunn, G. Howard), thus minimizing variability of the scoring.

Limitations of our study include lack of gold standard methodology for lipoprotein analysis in dogs and the inherent issues with adrenal function testing as mentioned previously. In addition to these limitations, the effects of diet and age on our study variables were not assessed. The influence of diet on triglyceride concentrations, cholesterol, and lipoprotein profiles in MSs has been evaluated. ⁵² Low-fat diets significantly reduced triglyceride and cholesterol concentrations and altered lipoprotein density profiles, with a decrease in TRL and LDL in MSs with HTG. ⁵² Given that diet information was not collected for our study, it is possible that some dogs with iHTG that were being fed low-fat diets may not have been detected. However, triglyceride concentrations have been reported to be highly variable, even in dogs consuming the same diet.^50,52 It can be postulated that individual differences in lipid metabolism and disease are more likely to determine the triglyceride concentrations in dogs than diet alone.

A further limitation is that it was not possible to definitively exclude hypothyroidism in all dogs in our study; thus, concurrent hypothyroidism may have influenced lipoprotein profiles. Normal total T4 concentrations can occur in hypothyroid dogs, ¹⁵ and ~30% of dogs with hypothyroidism can have normal TSH concentrations. ³² Conversely, 40–60% of dogs with HCort are reported to have low total T4 and TSH concentrations, which may be increased or normal, ³¹ with free T4 concentrations also not a reliable measure in dogs with HCort. ¹⁰ Two dogs in our study, one in the Healthy-NTG group and one with HCort-HTG, had a low total T4 and a concurrent normal TSH. Neither dog displayed clinical signs consistent with hypothyroidism, and the lipoprotein profiles of these dogs did not differ from the other dogs in their respective groups. Whether the low T4 concentration in the dog with HCort-HTG was secondary to HCort or the result of hypothyroidism cannot be answered with certainty because the measurements were not repeated after treatment. ²¹ However, other than fasting HTG, this dog had no clinical or clinicopathological changes consistent with hypothyroidism, making hypothyroidism unlikely.

It was not possible to identify a specific lipoprotein pattern in dogs with iHTG or with HCort using this lipoprotein electrophoresis method. We could not establish the role of HCort in iHTG in MSs. Genetic analysis or lipoprotein profiling using a more sensitive method may be necessary to determine the relative roles of a primary lipid disorder and HCort in HTG of MSs. ⁵¹ Therefore, the diagnosis of iHTG remains a diagnosis by exclusion, requiring careful clinical assessment and extensive endocrine testing in addition to routine hematology and serum biochemistry.