Novel lipoprotein density profiling in laminitic, obese, and healthy horses
Introduction
Lipoproteins are water-miscible macromolecules that provide transport for lipids in the blood. There are 5 classes of lipoproteins that are characterized by their hydrated density: very low–density lipoproteins (VLDL); intermediate-density lipoproteins; low-density lipoproteins (LDL); high-density lipoproteins (HDL); and chylomicrons [1]. In human medicine, altered proportions of lipoprotein classes are used to detect and classify diseases, such as insulin resistance, type 2 diabetes mellitus, and cardiovascular disease (CVD) [2], [3]. Differences in lipoprotein density distributions have also been described in obese dogs and dogs with metabolic diseases including hypertriacylglycerolemia [4]. Obesity is a growing concern in the equine population, with an estimated prevalence of 23%–31%. [5], [6] Importantly, obesity in horses has been associated with development of life-threatening conditions, including insulin dysregulation (ID) [7], [8], [9] and laminitis [10], [11], [12], [13]. In horses, the association between obesity and ID remains unclear as not all obese horses are ID and not all ID horses are obese [14]. A growing body of evidence supports that in both humans and horses, obesity is not simply the result of caloric intake relative to expenditure of energy but instead should be considered a clinical diagnosis that is mechanistically driven by a complex combination of poorly understood factors including impaired fat accumulation, altered insulin action, and immune response. Aside from diet and exercise, few targeted treatments are available to improve the body condition of overweight horses, emphasizing the critical need for early identification of at-risk individuals to implement preventative measures. However, in the absence of a complete understanding of the pathophysiology of obesity, early identification of horses at risk of obesity and development of effective therapeutics remain challenging.
The lipid and apolipoprotein compositions of various breeds of horses have been described confirming the existence of several subclasses of LDL and HDL [15], [16], [17], [18]. Disturbances in lipoprotein metabolism of critically ill and fasted ponies have also been described [19], [20]. The methodologies to study equine lipoproteins in the past have included electrophoresis, sequential density gradient centrifugation, and size-exclusion methods [7], [16], [17], [18], [19], [21], [22], [23]. The poor resolution of these methods results in limited detail related to the continuous density distribution of lipoproteins. Recently, a novel technique of continuous lipoprotein density profiling (CLPDP) to analytically assess an entire lipoprotein density distribution using advanced gradient-generating chemistry and imaging technology has been developed [24], [25]. This technique uses EDTA as a self-generating density gradient solution and ceramide as a fluorescent dye offering improved speed, resolution, and convenience. Given the use of these approaches for diagnosing metabolic diseases in people and other animals, an expedient, inexpensive, and robust method of lipoprotein profiling in horses could have utility in better characterizing horses with metabolic disease. Thus, the objectives of this study were to describe, characterize, and compare lipoprotein subfractions in healthy, laminitic, and obese horses using a novel method of density gradient ultracentrifugation.
Section snippets
Materials
Serum samples for the current project were obtained from a separate case-control project of laminitis [10]. For that study, veterinary members of the American Association of Equine Practitioners (AAEP) were asked to identify and collect data from 3 populations of horses: (1) a horse with acute laminitis not associated with grain overload, contralateral weight bearing, or a septic process within 4 wk of the onset of clinical signs; (2) a healthy horse residing at a different location than the
Continuous lipoprotein density profiling
Lipoprotein profiling was performed using CLPDP with modifications to a previously described method [29]. Briefly, 1,280 μL of 0.18 M bismuth sodium ethylenediaminetetraacetic acid was added to a 1.5-mL tube. The fluorescent probe 6-((N-(7-nitrobenz-2oxa-1, 3-diazol-4-yl)amino)hexanoyl) sphingosine was reconstituted with dimethyl sulfoxide (1 g/mL), and 10 μL of the 1 mg/mL solution was added to each tube to label each lipoprotein. Finally, 10 μL of serum was added, the sample was vortexed at
Study population
A total of 237 horses were included in the study, including LO (n = 66), LNO (n = 35), NLO (n = 41), and NLNO (n = 95). Signalment and physical characteristics of each group are reported in Table 1, Table 2. There were no significant differences in age (P = 0.2876) or sex (P = 0.7954). Groups differed significantly with respect to BCS (P < 0.0001) but did not differ by girth circumference (P = 0.1015), neck circumference (P = 0.1424), height (P = 0.1415) (Table 1), or whether they were engaged
Discussion
This study demonstrated a novel method of lipoprotein profiling in horses that might provide future utility in evaluating metabolic disease in horses. Lipoproteins comprise a heterogenous spectrum of particles that differ in size, density, and lipid and apolipoprotein composition. Evidence suggests that in humans, dyslipidemia may play a role in the pathogenesis of various conditions, including obesity and CVD [31], [32]. Well-established alterations associated with obesity include increased
Author Contributions
Michelle Coleman: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Visualization, Project administration, Rosemary Walzem: Conceptualization, Methodology, Investigation, Resources, Writing – review & editing, Supervision, Adam Kieffer: Methodology, Formal analysis, Investigation, Writing – review & editing, Visualization, Tomomi Minamoto: Conceptualization, Methodology, Formal analysis, Investigation, Writing – review & editing, Visualization, Jan
Acknowledgments
A special thanks to Jeannette Mawyer for her technical assistance and to the American Association of Equine Practitioners Foundation for providing serum samples.
This work was supported by the Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX and the American Association of Equine Practitioners Foundation, Lexington, KY.
There are no conflicts of interest for any of the authors.
The views and opinions expressed are those of the authors and do not necessarily
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Present Address: Army Medical Department Center & School, U.S. Military-Baylor University Graduate Program in Nutrition, JBSA-Fort Sam Houston, TX 78234, USA