Understanding the kinetic relationship between adipose tissue lipolysis and hepatic VLDL-triglyceride production requires methods to precisely measure FFA flux and VLDL-triglyceride turnover. We have developed a technique for ex-vivo radiolabeling of VLDL-triglyceride. The procedure involves isolation of VLDL-lipoprotein particles by ultracentrifugation and incorporation of [3H]-triolein by sonication under sterile conditions. We have found such radiolabeled VLDL particles indistinguishable from native VLDL with regard to electrophoretic properties, cholesterol to triglyceride ratio and apo-B 100 concentration. Using size exclusion HPLC to separate individual lipoprotein fractions we have found that samples containing purified VLDL particles elute the same as to unprocessed VLDL particles in whole plasma. The labeling process can be conducted sterilely. This protocol investigates whether ex-vivo labeled VLDL-particles have kinetic properties similar to in-vivo produced VDL-triglycerides. These studies are completed. We demonstrated that it was possible to isolate, purify, and label human VLDL particles using both in vivo and ex vivo techniques. Because of difficulties with adequate control of diet during the volunteer's stay in the GCRC, two of the studies yielded less than ideal data. In general, however, we gained satisfactory results from both ex vivo and in vivo labeled VLDL. Additional studies have been planned to more carefully assess the metabolism of ex vivo labeled VLDL.
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