Application of Lipid Class Ratios for Sample Stability Monitoring—Evaluation of Murine Tissue Homogenates and SDS as a Stabilizer

Abstract

Lipids are a ubiquitous class of structurally complex molecules involved in various biological processes. In the fast-growing field of lipidomics, preanalytical issues are frequently neglected. Here, we investigated the stability of lipid profiles of murine liver, brain, lung, heart, and spleen homogenates by quantitative flow injection analysis using tandem mass spectrometry and high-resolution mass spectrometry. Storage of tissue homogenates at room temperature showed substantial alterations of the lipid profiles reflecting lipolytic action. Therefore, ratios of ceramide to sphingomyelin, lysophosphatidylethanolamine to phosphatidylethanolamine, lysophosphatidylcholine to phosphatidylcholine, and diglyceride to triglyceride were applied to monitor sample stability and the effect of sodium dodecyl sulfate (SDS) as a potential stabilizing agent. The addition of SDS led to a concentration-dependent stabilization of lipid profiles in liver, brain, and heart homogenates, while in lung and spleen homogenates, in particular, the lysophosphatidylethanolamine to phosphatidylethanolamine ratio increased upon addition of SDS. In conclusion, we demonstrated that lipid class ratios reflecting lipolytic activity could be applied to evaluate both the stability of samples and the influence of stabilizers.

Accurate Lipid Quantification of Tissue Homogenates Requires Suitable Sample Concentration, Solvent Composition, and Homogenization Procedure—A Case Study in Murine Liver

“…Given these points, we apply bead-based homogenization to generate fluidic liver homogenates. Our results demonstrate that lipid recovery and preanalytical stability [21] is sufficient in H 2 O/MeOH = 1/1 supplemented with 1% SDS at a concentration of 0.05 mg wet weight/µL. However, we would like to emphasize that these conditions should not simply be transferred to other tissues without proper evaluation of preanalytics.…”

“…However, the addition of organic solvents could be advantageous for quenching of enzymatic activity and subsequent inhibition of lipid degradation [9]. In an accompanying study, the ratios of lipid classes, such as Cer/SM or LPE/PE, reflecting lipolytic activities, were used to investigate sample stability in various murine tissues [21]. Substantial lipolysis was observed in liver homogenates homogenized in H 2 O/MeOH = 1/1.…”

“…However, we would like to emphasize that these conditions should not simply be transferred to other tissues without proper evaluation of preanalytics. For example, SDS addition led to LPE generation in murine lung and spleen samples [21]. Respective evaluations should include both recovery and stability of lipids, which could be provided also by other stabilization methods, such as heat inactivation [23].…”

Evaluation of Lipid Extraction Protocols for Untargeted Analysis of Mouse Tissue Lipidome

“…Liver tissue, in particular, can present some challenges due to its high lipid content and the potential for lipid degradation during the extraction process. To address some of these challenges and improve the reproducibility of lipid extractions, researchers have explored modifications to the CHCl 3 /MeOH/H 2 O lipid extraction methods, such as adding antioxidants or surfactants to prevent lipid oxidation during the extraction process [ 47 , 48 ]. The same principle can be applied to the small intestine tissue, which also could have some limitations during lipid extraction because it is mainly a muscular tissue, and the ability of the extraction system to allow a uniform and superior dispersion of the tissue during extraction would affect the reproducibility of the analytical method.…”

Recommendations for good practice in MS-based lipidomics

“…Inappropriate preanalytic conditions may change the concentration dramatically like for lysolipids, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), which are generated instantly after drawing blood samples ( 15 ) and therefore require special precautions to preserve in vivo concentrations ( 18 ). Lipolytic activity ( 19 ), which may continue even after the addition of organic solvents like alcohol and could be monitored by lipid class ratios reflecting degradation ( 20 ), is highly undesirable in lipidomics. Generally, the analysis of potential degradation products like oxidized lipids, lysophospholipids, or phosphatidic acid needs a particular precaution to ensure that they do not just arise artificially from sample processing.…”