Identifying Static and Kinetic Lipid Phenotypes by High Resolution UPLC–MS: Unraveling Diet-Induced Changes in Lipid Homeostasis by Coupling Metabolomics and Fluxomics

Abstract

A novel method to differentiate diet-induced alterations in plasma lipid phenotypes "static (concentration of lipids) and kinetic (endogenous production, e.g., denovo lipogenesis)" was employed. C57Bl6 mice were randomized into 2 groups and fed either a high-carbohydrate, low-fat (HC) or a carbohydrate-free, high-fat diet (HF) diet for 13 days; D(2)O was administered via intraperitoneal injection and then adding D(2)O to the drinking water for 96 h. Principal component analysis (PCA) revealed differences in the plasma lipid content, for example, triglycerides (TG) 50:2, 50:3, and 52:2 were up-regulated in mice fed the HC diet, whereas TG 52:4, 52:1, 54:5, 54:3, 54:4, and 54:2 were higher in animals fed the HF diet. However, although the fractional contribution of synthesis was ~10-fold lower in HF vs HC fed mice, changes in TG concentration were not entirely mediated by altered de novo lipogenesis. In addition, the ability to couple isotope labeling measurements with PCA analyses revealed cases where there were no differences in the concentration of a compound but its source was substantially altered. In summary, this strategy determined (i) the presence/absence of differences in concentration and (ii) the contribution of different pathways and synthesis that could affect lipid biology in a mouse model respectively.

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

“…In contrast, 2 H is incorporated during amino acid metabolism, which could presumably be slower than the 18 O-labeling of amino acids. However, our data suggest that the 2 H-labeling of amino acids is rapid, and these observations are consistent with data that we recently obtained regarding the equilibration of ( 37,38 ). As both types of labeled water can be given to humans ( 10, 39 ), we suspect that it is possible to translate aspects of this work to clinical investigations.…”

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

“…Second, using the current methods, it is not possible to obtain clean signals for all lipid analytes. For example, although one can ascribe exact masses and retention times to virtually all of the analytes, the ability to reliably quantify the isotopic-labeling patterns in complex mixtures presents a diffi cult challenge ( 2,3 ).…”

“…Although the seminal studies that were compiled by Berman, Grundy, and Howard ( 1 ) have proved valuable, application of the previous analytical methods is less than ideal for high-throughput studies and/or for use in rodent models in which sample volumes may be limiting. Advances in mass spectrometry and stable isotope tracer methods now permit routine analyses of specifi c analytes ( 2,3 ). For example, whereas previous experiments studied the bulk pool of TG, it is now possible to examine individual species (2)(3)(4)(5)(6).…”

“…Advances in mass spectrometry and stable isotope tracer methods now permit routine analyses of specifi c analytes ( 2,3 ). For example, whereas previous experiments studied the bulk pool of TG, it is now possible to examine individual species (2)(3)(4)(5)(6).…”

Proteome Kinetics: Coupling the Administration of Stable Isotopes with Mass Spectrometry-Based Analyses

“…For example, the administration of a labeled precursor can present a challenge for in vivo studies. The administration of labeled water may be advantageous in these settings, the tracer can be given orally, it is relatively inexpensive and can be used to study multiple parameters simultaneously (this is especially important in studies of lipoprotein kinetics since questions regarding protein and lipid flux are often of equal importance) (Castro-Perez et al 2010;Castro-Perez et al 2011;Dufner and Previs 2003). In contrast, although we have demonstrated the ability to study protein synthesis in cell culture using labeled water (Dufner et al 2005), we believe that SILAC methods are generally superior for in vitro studies since it is trivial to completely substitute fully labeled amino acids for unlabeled amino acids in that setting.…”