The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species.
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.
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- "To circumvent the use of chronically catheterized animal models, Kurland and colleagues have used mini-osmotic pumps to deliver tracers whereas Bateman and colleagues have administered a single intraperitoneal bolus of labeled leucine to study protein synthesis  . While those approaches are somewhat easily implemented in rodent models, it is also possible to administer a more standard intravenous bolus which is suitable for studies in higher species as well  . "
ABSTRACT: Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.Biochimica et Biophysica Acta 05/2013; 1842(3). DOI:10.1016/j.bbadis.2013.05.019 · 4.66 Impact Factor
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ABSTRACT: We present a liquid chromatography/mass spectrometry (LC/MS) method for long-chain and very-long-chain fatty acid analysis and its application to (13)C-tracer studies of fatty acid metabolism. Fatty acids containing 14 to 36 carbon atoms are separated by C(8) reversed-phase chromatography using a water-methanol gradient with tributylamine as ion pairing agent, ionized by electrospray and analyzed by a stand-alone orbitrap mass spectrometer. The median limit of detection is 5 ng/mL with a linear dynamic range of 100-fold. Ratios of unlabeled to (13)C-labeled species are quantitated precisely and accurately (average relative standard deviation 3.2% and deviation from expectation 2.3%). In samples consisting of fatty acids saponified from cultured mammalian cells, 45 species are quantified, with average intraday relative standard deviations for independent biological replicates of 11%. The method enables quantitation of molecular ion peaks for all labeled forms of each fatty acid. Different degrees of (13)C-labeling from glucose and glutamine correspond to fatty acid uptake from media, de novo synthesis, and elongation. To exemplify the utility of the method, we examined isogenic cell lines with and without activated Ras oncogene expression. Ras increases the abundance and alters the labeling patterns of saturated and monounsaturated very-long-chain fatty acids, with the observed pattern consistent with Ras leading to enhanced activity of ELOVL4 or an enzyme with similar catalytic activity. This LC/MS method and associated isotope tracer techniques should be broadly applicable to investigating fatty acid metabolism.Analytical Chemistry 12/2011; 83(23):9114-22. DOI:10.1021/ac202220b · 5.83 Impact Factor
Article: Lipidomics[Show abstract] [Hide abstract]
ABSTRACT: Lipidomics characterizes the composition of intact lipid molecular species in biological systems and the field has been driven by some spectacular advances in mass spectrometry instrumentation and applications. This review will highlight these advances and outline their recent application to address clinical issues. This review first identifies recent advances in lipid detection and analysis by a variety of mass spectrometry techniques, then reviews specific application including stable isotope labelling of lipids, lipid mass spectrometry imaging, data analysis and bioinformatics, and finally presents examples of the application of lipidomics to selected disease states. Lipidomics so far has been principally concerned with identifying novel methodologies, but recent advances demonstrating applications in diabetes, neurodegenerative diseases, cystic fibrosis and other respiratory diseases clearly indicate the potential usefulness of lipidomics both to generate biomarkers of disease and to probe signalling and metabolic processes.03/2012; 15(2):127-33. DOI:10.1097/MCO.0b013e32834fb003