Abstract
Typical lipidomics methods incorporate a liquid–liquid extraction with LC–MS quantitation; however, the classic sample extraction methods are not high-throughput and do not perform well at extracting the full range of lipids especially, the relatively polar species (e.g., acyl-carnitines and glycosphingolipids). In this manuscript, we present a novel sample extraction protocol, which produces a single phase supernatant suitable for high-throughput applications that offers greater performance in extracting lipids across the full spectrum of species. We applied this lipidomics pipeline to a ruminant fat dose–response study to initially compare and validate the different extraction protocols but also to investigate complex lipid biomarkers of ruminant fat intake (adjoining onto simple odd chain fatty acid correlations). We have found 100 lipids species with a strong correlation with ruminant fat intake. This novel sample extraction along with the LC–MS pipeline have shown to be sensitive, robust and hugely informative (>450 lipids species semi-quantified): with a sample preparation throughput of over 100 tissue samples per day and an estimated ~1000 biological fluid samples per day. Thus, this work facilitating both the epidemiological involvement of ruminant fat, research into odd chain lipids and also streamlining the field of lipidomics (both by sample preparation methods and data presentation).
Highlights
Lipids are generally understood as a class of molecules that have a high solubility in organic solvents and typically contain or originate from fatty acids
This lipidomics method was tested, validated and applied in a rat model investigating ruminant fat biomarkers via a beef tallow dose response dietary investigation. This lipidomics liquid chromatography with mass spectrometry detection (LC–MS) method incorporating both of the described sample preparation protocols: protein precipitation and Folch liquid–liquid, were utilised for the quantitation of lipids in liver samples from Sprague–Dawley rats who received one of four experimental diets overfed at 17% above matched growth
A rise in liver ceramides is typically associated with aggravated non-alcoholic fatty liver disease (NAFLD) and insulin resistance [23], this in conjunction with a decrease in cardiolipins may suggest that the changes in the experimental diets here are detrimental for these pathologies
Summary
Lipids are generally understood as a class of molecules that have a high solubility in organic solvents and typically contain or originate from fatty acids. Lipids may be commonly derived, research has shown that there is a huge variety both structurally and functionally Lipids are emerging as biomarkers of dietary/nutritional intakes [7] as well as indicators of pathophysiological status [8,9,10,11]. As a reaction of lipid-pathophysiological involvements, the field of lipidomics has emerged as a discipline that examines and quantifies a large proportion of the lipids present in a given sample set. Lipidomics requires an effective isolation protocol that comprehensively extracts lipids from the sample as well as an analytical method that allows their identification and quantitation. The typical analyte isolation protocols (with/without minor adaptations) that are often used in the literature include
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