Chapter One

Ying-Yong Zhao1,2,∗, Xian-long Cheng4 and Rui-Chao Lin3     1Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, The College of Life Sciences, Northwest University, Shaanxi, China     2Division of Nephrology and Hypertension, School of Medicine, University of California, Irvine, CA, USA     3School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China     4National Institutes for Food and Drug Control, State Food and Drug Administration, Beijing, China
∗ Corresponding author: E-mail: zyy@nwu.edu.cn or zhaoyybr@163.com 

Lipids are the fundamental components of biological membranes as well as the metabolites of organisms. Lipids play diverse and important roles in biologicals. The lipid imbalance is closely associated with numerous human lifestyle-related diseases, such as atherosclerosis, obesity, diabetes, and Alzheimer’s disease. Lipidomics or lipid profiling is a system-based study of all lipids aiming at comprehensive analysis of lipids in the biological system. Lipidomics has been accepted as a lipid-related research tool in lipid biochemistry, clinical biomarker discovery, disease diagnosis, and in understanding disease pathology. Lipidomics will not only provide insights into the specific functions of lipid species in health and disease, but will also identify potential biomarkers for establishing preventive or therapeutic programs for human diseases. This review presents an overview of lipidomics followed by in-depth discussion of its application to the study of human diseases, including extraction methods of lipids, analytical technologies, data analysis, and clinical research in cancer, neuropsychiatric disease, cardiovascular disease, kidney disease, and respiratory disease. We describe the current status of the identification of metabolic biomarkers in different diseases. We also discuss the lipidomics for the future perspectives and their potential problems. The application of lipidomics in clinical studies may provide new insights into lipid profiling and pathophysiological mechanisms.

With the development of “omics,” lipidomics, a branch of metabolomics, was first put forward by Han and Gross (Han and Gross, 2003). Lipidomics has been defined as “the full characterization of lipid molecular species and of their biological roles with respect to expression of proteins involved in lipid metabolism and function, including gene regulation” (Spener et al., 2003). Based on research purposes, lipidomics can be divided into three analytical objectives (Navas-Iglesias et al., 2009): focused lipidomics (lipid profiling), targeted lipidomics (targeted lipid analysis), and untargeted lipidomics (global lipid profiling). The aim of focused lipidomics is applied to analyze a specific group of lipid metabolites, a certain class or pathway using tandem mass spectrometry (MS/MS). Product-ion scanning, precursor-ion scanning, and neutral-loss scanning are used to identify lipid molecules focusing on limited categories. The targeted lipidomics approach, aiming at determining a few important lipids, can be carried out by using multiple reaction monitoring or selected reaction monitoring, as the fragmentation patterns of analysis lipids are known. The untargeted lipidomics approach focuses on analyzing a very wide range of lipids in biological samples. In other words, lipidomics seeks to identify and quantify lipids within a biological system; it is also concerned with elucidation of individual molecular species in lipid metabolism and the normal function or dysfunction of the biological system. Lipidomics has emerged as a crucial component in the broader push to arrive at an integrated picture of the role of genes, proteins, and metabolites that fully describes cellular function. The emergence of lipidomics and its rapid increase in systems biology has been summarized in several reviews (Brown and Murphy, 2009; Henriksene et al., 2014; van Meer, 2005; Wenk, 2005). From the literature, first, new functional interactions of illumination have uncovered the specific nature of lipid–protein and lipid–lipid interactions in biochemical systems. Thus, lipids could no longer be regarded as simple lipid–protein and lipid–lipid interactions; rather, they have appeared as important participators with unusual biophysical properties and biochemical roles. Second, the development of new analytical tools is providing the ability to track changes in individual lipids at very low levels and to take quantitative snapshots of important portions of the lipidome. This review will cover (1) the biological function and extraction methods of lipids; (2) separation techniques for lipid classes and species; (3) methods of lipid detection and data analysis; and (4) some practical uses of lipidomics in clinical chemistry.

Lipids, the fundamental components of biological membranes, are structurally and functionally a diverse class of metabolites. Lipids play diverse and important roles in biological system including composing membrane bilayer, storing energy, producing signal transduction, providing functional implementations of membrane proteins and their interactions, etc (Subramaniam et al., 2011). The main difference between lipids and carbohydrates, proteins, and nucleic acids is their solubility in organic solvents. Historically, lipids are defined either by these physical properties, specifically solubility in non polar solvents, or by the presence of long hydrocarbon chains; however, not all lipids satisfy both definitions. Recently, investigators have attempted to refine this definition. A new nomenclature system has been proposed for lipids based on lipid biosynthesis, namely “hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion-based condensations of thioesters and/or by carbocation-based condensation of isoprene units.” Lipids were classified into eight major categories: fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides (Figure 1.1) (Fahy et al., 2005). Even within this more specific definition, lipids include a broad range of molecular structures. Unlike other biomolecules, complex lipids such as sphingolipids and glycerophospholipids include a wide range of building blocks that can give rise to a bewildering array of combinations. Permutations that may arise only from common eukaryotic lipid motifs give rise to more than 180,000 theoretical phospholipid structures that could be present in a given cell or tissue extract (Yetukuri et al., 2008). However, this number does not include complexity that may arise from consideration of isomeric lipids that differ only in double-bond position, backbone substitution, or stereochemistry (Mitchell et al., 2009). The need to develop analytical tools that can readily tackle such a diverse range of molecular structures is a key reason that lipidomics has lagged behind genomics, proteomics, and metabolomics.

Because lipids embedded in complex biological matrixes, but not appeared in their free form, an extraction procedure is indispensable for further analysis. The general procedures are lipids separation from the matrix; removal of any nonlipid components, such as saccharides, proteins or other small molecules, and fractionation and isolation of lipids from the extract (Ekroos, 2012).

The obtained components from lipid extraction depend on the extraction method especially the used solvent. Nonpolar solvents such as petroleum ether, hexane, or supercritical CO2 can be used for simple neutral lipid extraction such as acylglycerols and esters of fatty acids. More polar lipids such as phospholipids, glycolipids, lipoproteins, and free fatty acids need more polar solvents including CH3CN, CH3OH, and C2H5OH. Different extraction methods have been used in lipidomics and the choice of the technique must depend on the analytical matrix. Typically, liquid–liquid extraction and solid-phase extraction are mainly two extraction methods for lipid extraction in lipidomics. Generally, a phase separation is produced between immiscible solvents with the lipids partitioning into the hydrophobic phase. Various single solvents or mixed solvents have been suggested as extracted solvents. The CHCl3 and CH3OH was often used as mixed solvent in a two-step extraction, which was developed by Folch and coworkers in 1957 (Shahidi and Wanasundara, 2002) and this method used CHCl3:CH3OH (2:1) and large volumes of saline aqueous solution for washing out the nonlipid compounds (Folch et al., 1951). Binary solvent was the...