Expanding lipidomics coverage: effective ultra performance liquid chromatography-high resolution mass spectrometer methods for detection and quantitation of cardiolipin, phosphatidylglycerol, and lysyl-phosphatidylglycerol.

INTRODUCTION: Lipidomics can reveal global alterations in a broad class of molecules whose functions are innately linked to physiology. Monitoring changes in the phospholipid composition of biological membranes in response to stressors can aid the development of targeted therapies. However, exact quantitation of cardiolipins is not a straightforward task due to low ionization efficiencies and poor chromatographic separation of these compounds. OBJECTIVE: The aim of this study was to develop a quantitative method for the detection of cardiolipins and other phospholipids using both a targeted and untargeted analyses with a Q-Exactive. METHODS: HILIC chromatography and high-resolution mass spectrometry with parallel reaction monitoring was used to measure changes in lipid concentration. Internal standards and fragmentation techniques allowed for the reliable quantitation of lipid species including: lysyl-phosphatidylglycerol, phosphatidylglycerol, and cardiolipin. RESULTS: The untargeted analysis was capable to detecting 6 different phospholipid classes as well as free fatty acids. The targeted analysis quantified up to 23 cardiolipins, 10 phosphatidylglycerols and 10 lysyl-phosphatidylglycerols with detection limits as low as 50 nM. Biological validation with Enterococcus faecalis demonstrates sensitivity in monitoring the incorporation of exogenously supplied free fats into membrane phospholipids. When supplemented with oleic acid, the amount of free oleic acid in the membrane was 100 times greater and the concentration of polyunsaturated cardiolipin increased to over 3.5 µM compared to controls. CONCLUSIONS: This lipidomics method is capable of targeted quantitation for challenging biologically relevant cardiolipins as well as broad, untargeted lipid profiling.

Metabolomics Approach in the Study of the Well-Defined Polyherbal Preparation Zyflamend.

Zyflamend is a highly controlled blend of 10 herbal extracts that synergistically impact multiple cell signaling pathways with anticancer and anti-inflammatory properties. More recently, its effects were shown to also modify cellular energetics, for example, activation of fatty acid oxidation and inhibition of lipogenesis. However, its general metabolic effects in vivo have yet to be explored. The objective of this study was to characterize the tissue specific metabolomes in response to supplementation of Zyflamend in mice, with a comparison of equivalent metabolomics data generated in plasma from humans supplemented with Zyflamend. Because Zyflamend has been shown to activate AMPK, the "energy sensor" of the cell, in vitro, the effects of Zyflamend on adiposity were also tested in the murine model. C57BL/6 mice were fed diets that mimicked the macro- and micronutrient composition of the U.S. diet with and without Zyflamend supplementation at human equivalent doses. Untargeted metabolomics was performed in liver, skeletal muscle, adipose, and plasma from mice consuming Zyflamend and in plasma from humans supplemented with Zyflamend at an equivalent dose. Adiposity in mice was significantly reduced in the Zyflamend-treated animals (compared with controls) without affecting body weight or weight gain. Based on KEGG pathway enrichment, purine and pyrimidine metabolism (potential regulators of AMPK) were particularly responsive to Zyflamend across all tissues, but only in mice. Consistent with the metabolomics data, Zyflamend activated AMPK and inhibited acetyl CoA-carboxylase in adipose tissue, key regulators of lipogenesis. Zyflamend reduces adipose tissue in mice through a mechanism that likely involves the activation of AMPK.