This procedure enables the study of the intestinal digestion, absorption and lymphatic transport of dietary lipid in the conscious mouse. By infusing the lipid test meal intraduadenally at a constant rate, the lymphatic lipid output usually reaches a steady rate by the 3rd or 4th hour. Thus, lymphatic lipid output during the 5th or 6th hour represents the amount of lipid transported by the small intestine under steady-state conditions. At the end of the study, both luminal as well as mucosal radioactive lipids can be collected. Thus one can obtain a considerable amount of information regarding the digestion, uptake and mucosal esterification and the lymphatic transport of lipids.

Determinations of substrates in plasma/serum/lymph will be made using specific colorimetric assays. Reactions will be run in microtiter plates and analyzed on a plate reader. Using this method assays can be performed on small (5-25ul) samples of plasma or serum.

Lipoprotein fractions are isolated using columns arranged in tandem to achieve complete resolution of the major lipoprotein classes from plasma. The columns are equilibrated in 50 mM phosphate-buffered saline. Fractions (0.5 ml) are collected and the position of the major lipoprotein classes is determined by Cholesterol (C1083-C-FPLC) or Triglyceride (C1092-C-FPLC) assay on the column fractions using a microtiter plate enzyme-based assay.

The intestinal lymph duct and duodenum will be surgically cannulated. Chylomicrons are harvested and sized by negative staining. A small sample will be delipidated, and the apoprotein composition will be analyzed by polyacrylamide gel electrophoresis. Labeled CM is injected into mice and the plasma clearance rate is calculated as well as a determination of the uptake by the liver.

Determinations of free fatty acids in plasma/serum/lymph will be made using a specific colorimetric assay. Reactions will be run in microtiter plates and analyzed on a plate reader. Using this method assays can be performed on small (5-25ul) samples of plasma or serum.

Dietary fat containing 5% sucrose polybehenate, a non-absorbable food additive, is fed in a semi-synthetic diet containing absorbable fat to rodents. The animals eat the diet ad lib for 3 days. Two or more fecal pellets are collected on days 3 and 4. Fat absorption is calculated from the ratios of behenic acid to other fatty acids in the diet and in the feces as analyzed by gas chromatography of fatty acid methyl esters.

Determinations of phospholipids in plasma/serum/lymph will be made using a specific colorimetric assay. Reactions will be run in microtiter plates and analyzed on a plate reader. Using this method assays can be performed on small (5-25ul) samples of plasma or serum.

Total cholesterol will be measured using a colorimetric assay. Reactions will be run in microtiter plates and analyzed on a plate reader. Using this method assays can be performed on small (5-25ul) samples of plasma or serum.

Cholesterol is measured in FPLC fractions to determine the concentrations of VLDL-Cholesterol, LDL-Cholesterol, and HDL-Cholesterol. This service is completed in conjunction with C1054**.

Determinations of triglycerides in plasma/serum/lymph will be made using a specific colorimetric assay. Reactions will be run in microtiter plates and analyzed on a plate reader. Using this method assays can be performed on small (5-25ul) samples of plasma or serum.

Triglycerides are measured in lipid extracted via Folch extraction from tissue.  This service is completed in conjunction with C1104*.

Triglycerides are measured in FPLC fractions to determine the concentrations of VLDL-Triglycerides, LDL-Triglycerides, and HDL-Triglycerides. This service is completed in conjunction with C1054**.

Animals will be anesthetized and tissues harvested as requested. These tissue will be frozen in liquid nitrogen or fixed.

Lipids will be extracted from tissues using the Folch extraction method.

Samples are saponified and methylated for GC analysis. The extracted solution is injected into the GC and retention times are compared to known standards.