New methodologies for studying lipid synthesis and turnover: Looking backwards to enable moving forwards

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.

Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry

“…The finding that palmitate N values in our C2C12 cells experiments ranged from 14–15 is similar to that previously reported in the literature in HepG2 and MCA cancer cells ( N = 16–18) [23]. Interestingly, a common feature and advantage of using 2 H 2 O to label endogenously-synthesized molecules, such as DNA, protein and lipids, lies in the amplification effect between precursor and product enrichments [16,17,24,30,31,35]. Specifically, the amount of 2 H enrichment (EM 1 ) in various molecules can exceed the 2 H 2 O level, thus enhancing analytical accuracy.…”

“…Following the administration of 2 H 2 O into the body water pool in vivo or into cell culture media in vitro, 2 H atoms become stably incorporated via enzyme catalyzed reactions into C-H bonds of nonessential amino acids, glycerol-3-phosphate, fatty acids, cholesterol, hexoses and pentoses (ribose and deoxyribose). Subsequent analysis of 2 H labelling of these constituents in macromolecules provides a highly sensitive measure of the rates of synthesis/turnover of lipids [15,16,17,18,19,20,21,22,23,24,25,26,27], proteins [15,28,29,30,31,32,33,34], DNA [15,35,36], RNA [15] and glucose/glycogen [25,26,37,38,39,40,41,42,43] and an objective assessment of fundamental cellular processes that include, but are not limited to, rates of cell and organelle proliferation, cell and tissue biomass turnover, de novo lipogenesis and gluconeogenesis. Such measurements have tremendous application to researchers in the field of cancer, immunology, cardiovascular disease, metabolism, diabetes, obesity, developmental biology, as well as the biotechnology and agricultural sectors.…”

“…Such measurements have tremendous application to researchers in the field of cancer, immunology, cardiovascular disease, metabolism, diabetes, obesity, developmental biology, as well as the biotechnology and agricultural sectors. In addition to measuring multiple metabolic processes simultaneously, 2 H 2 O labelling is advantageous because: (1) the even distribution of the 2 H 2 O in the total body/cellular pool makes it simple to determine the precursor:product labelling ratios that are required to calculate synthetic flux with confidence; (2) the 2 H 2 O tracer can be easily and safely administered for long periods of time in vivo and in vitro, permitting flux measurements in free living conditions or long-term culture experiments; and (3) the capacity to label for extended periods of time leads to efficient labelling of total cellular biomass, increasing the sensitivity of the analysis and reducing artefacts that are sometimes associated with incomplete quenching of cells/tissues during harvesting [3,24,32,35]. …”

Quantitation of Human Peptides and Proteins Via MS: Review of Analytically Validated Assays

“…Labeled water has the advantage that it can be administered orally and is incorporated into a wider range of molecules than just proteins, thus enabling kinetic measurements of metabolites such as lipids [85], however the variable incorporation rates of labeled water into different amino acids complicates the calculation of absolute turnover rates for most proteins. A summary of proteins, for which kinetics measurements (FSR and FCR) were performed are shown in Table 3.…”

Strategies for Extending Metabolomics Studies with Stable Isotope Labelling and Fluxomics

“…2 H 2 O can be administered to animals or humans in drinking water, or directly into the cell culture media in vitro , which results in the enrichment of the body/cellular water pool. A major advantage of using 2 H 2 O is that it can be used to probe multiple metabolic processes simultaneously and can be easily and safely administered for long periods of time in vivo and in vitro , permitting flux analysis in free living conditions or long term culture experiments [3,73,79,91]. However, it should be noted that high levels of 2 H 2 O enrichment can have toxic effects on cellular systems due to the slightly stronger bonds that deuterium forms with other atoms when compared to hydrogen, thus creating kinetic isotope effects on chemical reactions [92,93].…”