Lipid Analytics Core @ The Kohan Lab
Housed within the Division of Endocrinology and Metabolism, the Lipid Analytics Core is specially equipped for determining how dietary lipids are metabolized by the body. This core includes dedicated space within the 10th Floor Division of Endocrinology Research space for: Folch extraction, thin-layer chromatography (TLC), radio-tracer analyses, lipoprotein isolation, and a surgical suite dedicated to the intestinal lymph fistula mouse model (for analyzing intestinal lymph dynamics and dietary lipids absorption and metabolism). The Lipid Analytics Core complements the existing Glucose Metabolism Core in the Division (Directed by Dr. Michael Jurczak), which consists of Oroboros metabolic flux analyses, glucose clamp studies, and metabolic monitoring analyses.
In vivo Mesenteric Lymph Collection during Fat Feeding
Mesenteric lymph ducts collect chylomicrons after a fatty meal. Chylomicrons and associated intestinal antigens are pumped through the mesenteric lymph nodes to the thoracic vein prior to entering the systemic circulation. We use the conscious lymph fistual mouse model, first developed by Dr. Patrick Tso, to collect post-prandial mesenteric lymph during an acute or continuous small intestinal lipid infusion. This mouse model is a powerful tool for studies of the kinetics of dietary lipid absorption and metabolism.
Thin Layer Chromatography (TLC)
Quantifying triglyceride metabolism requires an analysis of the metabolic flux of fatty acids and triglycerides into and out of the cell. We use radio-labelled fatty acids and triglycerides to identify how lipids move through and are metabolized by enterocytes and immune cells. We first treat animals or isolated cells with physiological lipids (either micellar or lipoprotein) then perform a Folch extraction of isolated cells and standardize to total cell number. The amount of endocytosed 3H and 14C label is then determined by scintillation counting. We use Thin Layer Chromatography (TLC) of the extracted lipids to determine whether the dosed 3H and 14C cellular lipids are incorporated into stored lipids, cholesterol, the OXPHOS pathway, intracellular lipid droplets, or are excreted or used for beta-oxidation.
Isolation of Intestinal Lipoproteins
We routinely collect chylomicrons intestinal lymph and blood after a lipid infusion. Purified chylomicrons are isolated by ultracentrifugation, and the triglyceride, apolipoprotein, and 3H- and 14C- dose is determined by chemical assay and scintillation counting (respectively). We treat isolated immune cells and enterocytes with these physiological lipids to parse donor versus recipient metabolic effects of lipoproteins on key metabolic pathways.
