The method presented here was easy to perform and proved to be a quick and accurate screening method for lipoprotein analysis in dogs. Important differences in lipoprotein profiles between different groups of dogs were detected with this method. An important and novel finding of the present study is that Miniature Schnauzers with serum TAG and cholesterol concentrations within the reference interval have significantly different lipoprotein profiles than those of dogs of various other breeds. In addition, the present study confirmed and expanded the findings of previous studies reporting that specific lipoprotein classes are associated with hypertriacylglyceridemia in Miniature Schnauzers.
To our knowledge, this is the first study showing that Miniature Schnauzers with normal serum TAG and cholesterol concentrations differ significantly in certain lipoprotein fractions (R1 and R5 possibly corresponding to TRL and LDL4) from dogs of various other breeds. A previous study  identified a small number of healthy Miniature Schnauzers (4 out of 11 studied) that differed in some lipoprotein fractions (LDL and VLDL) compared with dogs of other breeds. However, in that particular study, Miniature Schnauzers were classified as non-lipemic based on the gross appearance of the plasma rather than a measured TAG concentration. Indeed, all 4 of the non-lipemic Miniature Schnauzers classified as “different” had mild increases in plasma TAG concentrations, and therefore, mild lipid metabolism alterations were present.
The R1 fraction (likely TRLs) was significantly higher in Miniature Schnauzers of group 2A than in dogs of other breeds, despite the fact that there was no significant difference in serum TAG concentrations between the two groups and serum TAG and cholesterol concentrations were within the reference interval. Interestingly, serum cholesterol concentrations were found to be significantly higher in dogs of other breeds compared to Miniature Schnauzers. This difference might be related to the fact that the R5 fraction was significantly higher in dogs of other breeds compared to Miniature Schnauzers. However, similarly to serum TAG concentrations, serum cholesterol concentrations were all within the reference interval. The density interval of R5 was d = 1.039-1.050 g/mL, a density region that can include LDL4 but also large buoyant HDL . The marked differences in lipoprotein profiles between Miniature Schnauzers and dogs of other breeds despite the normal serum TAG and cholesterol concentrations clearly suggest that serum TAG and cholesterol concentrations are insensitive markers for detecting differences in lipoprotein metabolism in dogs. These differences in lipoprotein profiles could be identified in the vast majority of dogs, as about 90% of them could be classified to the correct group based on their lipoprotein profile alone.
The reason for the differences in lipoprotein profiles between Miniature Schnauzers and dogs of other breeds with normal serum TAG and cholesterol concentrations is unknown. The clinical importance of such finding is also unknown. One plausible scenario for such difference is that some Miniature Schnauzers might have an early disorder in lipoprotein metabolism that has not yet affected the overall serum TAG and cholesterol concentrations, but these concentrations might be affected in the future. This hypothesis is supported by the findings of a previous study that suggested that hyperlipidemia is an age-related condition in Miniature Schnauzers . However, this does not seem a likely explanation in this study because all Miniature Schnauzers with serum TAG concentrations within the reference interval enrolled into this study were above 7 years of age (median age: 9.3 years). Therefore, these dogs were already of a rather advanced age and unlikely to develop hypertriacylglyceridemia later in life . Another possibility is that the majority of Miniature Schnauzers differ in their basic lipoprotein metabolism from dogs of other breeds but only a portion of these dogs have severe enough lipid metabolism disorders leading to hyperlipidemia. Also, differences in diet composition or may have accounted, at least in part, for the differences in lipoprotein profiles. Differences in the sexual status of the dogs between groups may also have played a role although this has not been demonstrated in previous studies. Clearly, further studies are needed to determine the reason for this finding and its clinical significance. Moreover, the ability to rapidly screen the entire density profile of serum lipoproteins has identified specific density regions amenable to further compositional studies that may identify the mechanism for the distinctive lipoprotein metabolism that occurs in Miniature Schnauzers.
Lipoprotein profiles of Miniature Schnauzers with hypertriacylglyceridemia were in agreement with the findings of previous studies . It is interesting to note that the main difference between hyperlipidemic and normolipidemic Miniature Schnauzers was a significant increase of the R1 (TRL) fraction in hyperlipidemic Miniature Schnauzers, and there was a strong correlation between TRLs and serum TAG concentrations. Similarly to previous studies, there was no difference in the HDL fractions . Interestingly, the present study showed that a specific fraction (R3 likely corresponding to the density of LDL2) was significantly decreased in Miniature Schnauzers with hyperlipidemia.
Another interesting observation is that the CLPDP of Miniature Schnauzers with hyperlipidemia, although different in their lipoprotein profiles from Miniature Schnauzers without hyperlipidemia, was rather diverse and there were some distinct differences among dogs in the same group. These differences were not always related to the different degrees of hyperlipidemia in these dogs. For example, many hyperlipidemic Miniature Schnauzers had 2 distinct peaks in their TRL fractions (Figure 7), while others only had one (Figure 6). These 2 peaks likely represent chylomicrons and VLDLs, which have slightly different densities. This is in agreement with findings of an older study , in which it was shown that some hyperlipidemic Miniature Schnauzers had increases in VLDLs only, while others had increases in both VLDLs and chylomicrons. It is not known why some of the hyperlipidemic Miniature Schnauzers have only one TRL fraction affected while others have 2. In addition, as shown in previous studies [10, 21], a fraction if these dogs had increases in serum cholesterol concentrations. Thus, it is obvious that hyperlipidemia in Miniature Schnauzers is not a phenotypically uniform disease. This might be the result of the effect of environmental factors or maybe due to genetic heterogeneity.
Miniature Schnauzers of either group were significantly older than the dogs of other breeds used in this study. This was the result of specific selection criteria. Hyperlipidemia in Miniature Schnauzers is known to be an age-related condition and, therefore, dogs in group 2 had to be of older age in order to have developed their phenotype with a high degree of probability if in fact they were to develop hypertriacylglyceridemia. For example, in one study, only 16% of Miniature Schnauzers between 1 and 3 years of age were hypertriacylcerolemic, while 78% of Miniature Schnauzers >9 years of age were hypertriacylglycerolemic. No association between age and serum TAG or cholesterol concentrations have been reported or suspected in dogs of breeds other than Miniature Schnauzers.
It needs to be pointed out that the present method as described in the present study does not allow detailed characterization of the components of each lipoprotein fraction. As mentioned above, the lipoprotein fractions were identified solely based on density characteristics and not on their functional properties or composition, which are to a large degree unknown in dogs. Therefore, all assignments within the lipoprotein fingerprint to traditional functional classes, e.g. LDL or HDL, were strictly considered nominal. Further studies are warranted to investigate the exact content and functional properties of all canine lipoprotein fractions.