Lipids have long been considered to be mere building blocks of cell membranes or energy-storage molecules. It is now evident, however, that these small, nonpolymeric molecules play several key roles in all areas of the biological system, acting as signaling molecules and regulatory messengers in endocrine actions, cell growth, morphogenesis and membrane trafficking.
Defects or imbalances in lipid levels or regulation can have a disastrous effect on many biological functions, culminating in disease development. Comprehensive analysis provides important insight into the functions of lipids in health and disease, and may lead to the discovery of potential molecular drug targets for cancer, diabetes, neurodegenerative disorders, inflammation, cardiovascular and other diseases.
It is difficult to analyze their composition because lipids vary substantially in structure and there are thousands of different molecular species. However, advances in mass spectrometry and bioinformatics provide improved analytical sensitivity and selectivity for investigating lipids having diverse chemical compositions, often in complex mixtures. Large-scale studies of lipid pathways and networks are being conducted to identify and quantify molecular lipid species of different structural and functional form, and to examine their interaction with other lipids, proteins and metabolites.
Although vast in number, lipids are generally the same from species to species. Thus, data obtained from animal studies can be a good predictor of potential success in later clinical settings.
The continual postsampling activity of enzymes (e.g., lipases and phosphatases) that cause substantial changes during sample preparation makes it difficult to accurately quantify lipid levels in tissue samples. When the sample quality deteriorates rapidly, vital information about the in vivo components is distorted or lost. This is problematic because reduced sample quality increases the risk of incorrectly interpreting data. The full potential of lipidomics can only be realized if the sample is stabilized directly after sampling.
The conventional method of snap freezing halts, and even reduces, enzymatic activity, but is not a permanent solution because thawing during sample handling leads to substantial postsampling effects.
Maintaining sample integrity
An additive-free technology that preserves sample quality throughout the entire workflow, the Stabilizor system (Denator AB, Gothenburg, Sweden) utilizes conductive heating to generate rapid, homogeneous and irreversible thermal denaturation of proteins. It permanently eliminates enzymatic activity and ex vivo lipolysis that causes variations during postsampling, such as rapid accumulation of free fatty acids. After heat stabilization of fresh or frozen tissue samples, lipids can be extracted and analyzed using traditional buffers and techniques.
Eliminating postsampling effects
Fatty acids are essential components of mammalian cells and participate in the development and maintenance of the nervous system. Free fatty acids (FFAs) are involved in pathological conditions of the nervous system, including neurodegenerative diseases, mental disorders and stroke. Lipid metabolism analysis helps to elucidate the mechanisms of these diseases and identify novel disease biomarkers.
Preanalytical sample handling is essential because some lipids are unstable and postmortem changes in the brain may cause postsampling effects (such as substantial release of FFAs), as demonstrated in a study from the University of Oxford (Oxford, U.K.) and Karolinska Institutet (Solna, Sweden). Intense phospholipase (PLA2) activity and other timedependent postsampling changes were detected in the lipid pool of nonstabilized tissue, with a significant increase after 2 minutes at room temperature (Figure 1).
Figure 1 – Effect of homogenization time on FFA levels at room temperature in a snap-frozen brain. To confirm the effects of ongoing phospholipase activity, a homogenate from frozen brain tissue was left at room temperature for 0–40 min before protein precipitation and analysis. All analyzed FFAs increased in concentration over time.
Figure 2 – Effect of brain and liver tissue heat stabilization on detected FFA concentrations.Due to continued lipase activity, postsampling effects induced a considerable release of FFAs from nonstabilized tissue (up to 3700%) in comparison to heat-stabilized tissue. The Stabilizor system was used to stabilize rat liver and brain samples, and PLA2 activity and ex vivo lipolysis were reduced (Figure 2). Heat stabilization enabled the researchers to obtain reproducible, consistent lipid profiles to demonstrate the effects of euthanasia on lipidomic studies.1
Preserving sphingolipids
Sphingolipids are key regulators in cell death and survival. Sphingolipid metabolites like ceramide (Cer) and sphingosine-1-phosphate (S1P) are important mediators in the signaling cascades of numerous biological functions such as apoptosis, proliferation, stress response, necrosis, inflammation, autophagy, senescence and differentiation. The ability to determine concentrations of sphingolipids and their metabolites that reflect the in vivo status as closely as possible is critical to understanding their role and is of particular interest in diabetes, cancer, microbial infections, cardiovascular disease, etc.
In a study conducted at Tohoku University (Sendai, Japan), the levels of sphingosine (Sph), S1P and Cer in mouse liver tissue were analyzed using mass spectrometry. The samples treated using snap freezing experienced substantial conversion from the phosphorylated Sph into both S1P and Cer over a time span of two hours. This happened as a result of the enzymatic activity of different lipases and phosphatases still present and active during sample handling. The heat-stabilized samples remained stable throughout.
As shown in Figure 3, heat stabilization prevented degradation of sphingolipids during sample preparation. This process will be useful for determining the localization of sphingolipids in tissue in an attempt to understand the involvement of sphingolipid mediators in human disease (Figure 4).2
Figure 3 – LC-MS determination of sphingolipid levels in mouse liver in room temperature. Heat-stabilized (HS, orange) samples were compared to snap-frozen samples (SF, gray) in a time study lasting up to 120 minutes. The results indicate a time-dependent shift in snap-frozen samples from phosphorylated sphingosine (S1P) into the nonphosphorylated and apoptosis-associated forms sphingosine (Sph) and ceramide (Cer).
Figure 4 – Applications for which heat stabilization can be used.Conclusion
Lipidomics will be very important in the development of the next-generation drug targets for many diseases. Heat stabilization provides improved sample quality and allows researchers to discover new, biologically relevant information.
References
- Jernerén, F.; Söderquist M. et al. Post-sampling effects of free fatty acids—effects of heat stabilization and methods of euthanasia. J. Pharmacol. Toxicol. Meth. 2015, 71, 13–20.
- Saigusa, D.; Okudaira, M., et al. Simultaneous quantification of sphingolipids in small quantities of liver by LC-MS/MS. Mass Spectrom. 2014, 3, S0046.
Marcus Söderquist, Ph.D., is senior scientist, Denator AB, Arvid Wallgrens Backe 20, 413 46 Gothenburg, Sweden; tel.: +46 (0) 31 41 28 41; e-mail: [email protected]; www.denator.com