Abstract
The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 μl of plasma.
Highlights
The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo
Oleic acid was used as the isotope rather than other possible forms so that the same tracer could be administered in experiments designed to investigate intestinal lipid assembly or hepatic synthesis of complex lipids from a nonesterified fatty acid (NEFA) precursor
The use of LC-MS to follow the disposition of stableisotopically labeled oleic acid in vivo provides all the tools required to interrogate biology and confidently assess pharmacological effects
Summary
The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The kinetics of synthesis and disposition can be ascertained by introducing a labeled substrate and measuring its incorporation into and decay from bulk lipid pools, which provides useful information on the flux of lipid through the relevant synthetic and metabolizing pathways. Such techniques have been used to study the kinetics of synthesis of VLDL triglycerides [1,2,3], cholesterol [4, 5], and phospholipids [6], as well as many other aspects of lipid disposition. When using a stable isotopically labeled tracer approach to evaluate the effects of drugs in vivo, it behooves the researcher to assess the labeling of the precursor pool (to evaluate comparability among subjects and between treated and control groups) in order to determine whether an observed effect can truly be attributed to action of the drug [9, 10]
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