Two lipid extraction protocols, including Bligh & Dyer method and a one-step methanol method, and two temperatures for sample transportation (room temperature or dry ice), were compared to in order to improve pre-analytical procedures of lipid analysis in reproductive cells. Lipid profiles obtained from room temperature or dry ice samples were similar, but they were affected by the extraction methods, when methanol and Bligh & Dyer were compared within RT or DI groups. Yet, it should be noted that the lipid profiles of BDP-RT samples were very comparable to BDP-DI, the classical or reference protocol requiring cold chain maintenance (Bligh & Dyer, 1959).
There is little information available regarding specific protocols that minimize analytical error and variability in samples to be evaluated for their lipid profiles, which is particularly important for minute and valuable samples. In the specific case of reproduction research, samples such as oocytes and embryos may often be from vulnerable populations or represent unique opportunities that would be difficult to replicate (e.g., in assisted reproductive technology procedures and clinical studies on reproductive medicine). Considering the importance of lipid research in reproduction, in the present study, we performed a simplified workflow for lipid analysis and investigated if the extraction method and/or the room temperature transportation could qualitatively impact the lipid recovery and profile, and ultimately the output data obtained from these types of samples.
In general, working with lipids demands careful handling and storage due to their high oxidation and hydrolysis rates [17]. In living organisms, oxidation is a normal process controlled by antioxidants; however, after death, this protective function decreases significantly and results in lipid degradation [18]. Taken into account that lipid oxidation proceeds very slowly at initial stages [19], and even though the time between lipid extraction and data acquisition was short in our study, considerably reducing the chances of oxidation, the samples were also kept in vacuum packaging as a strategy to avoid exposure to oxygen at room temperature.
It is known that the effectiveness of the lipid extraction protocol, to a large extent, depends on the chemical nature of the lipids and the type of associations in which they are found in the cell [20]. Hydrophobic lipids, for example, may be extracted with non-polar solvents such as ethyl ether or chloroform. Membrane associated lipids, on the other hand, require polar solvents such as ethanol or methanol to disrupt the hydrogen bond networks or electrostatic forces between lipids and proteins (Bou Khalil et al., 2010; Saini et al., 2021). Note that for the present study, after Bligh & Dyer extraction, we combined the aqueous and organic phases to recover most of the lipids in the sample.
For bovine oocytes, the choice of the sample processing protocol (one-step methanol vs. Bligh & Dyer) impacted the lipid profiles as indicated by PCA. Membrane lipids, including PC, PE and PI lipids, showed higher relative intensities when lipid extraction was done using only with pure methanol, even though the same was not observed for PG and PS lipids. In this way, one-step lipid extraction with methanol for analysis of membrane lipids in oocytes and embryos has been reported successfully and allowed accurate identification of lipid changes within individual lipid classes [22, 23]. Neutral lipids such as acyl-carnitines, cholesteryl esters and FFA were more efficiently extracted when a chloroform: methanol mixture was used. In a complex matrix such as the blood plasma, lipids of all classes can be recovered via chloroform/methanol extraction, but low recovery occurs for charged and non-polar lipids [24]. Another concern regarding lipid analysis is the fact that the samples often need to be stored or transported prior to analysis. Following a “gold standard” practice, lipids should be extracted immediately upon sampling and kept frozen at temperatures ranging from − 20 to -80°C until analysis [25, 26]. In the present study, we evaluated the effect of deviations from the gold standard storage/handling practice by transporting lipid extracts between Brazil and the USA at room temperature. It is important to mention that we did not monitor temperature variations during room temperature transportation, which can be considered as a limitation of this study, but also reinforces the robustness of the technical approach. Even if big temperature variations occurred, they did not have a pronounced impact the lipid profile, as evidenced by the unsupervised PCA analysis.
Although the overall the lipid profile was not impacted by the transportation at room temperature under vacuum, some significant changes were observed between DI and RT when considering the sum of the relative ion intensities for each lipid class. For PMP-RT, significant decreased abundance was observed for occurred for PE and TAGs and an increased abundance was observed for PC/SM compared to PMP-DI. For the BDP-RT protocol, higher relative abundance was observed for PI, PG, cholesteryl esters, and acyl-carnitines and lower abundance for FFA. We speculate that some of these differences are not necessarily related to the transportation at RT which may cause some lipid oxidation, but they might be related to the freeze-thaw cycles to which the samples were submitted before data acquisition. Unlike samples transported on dry ice that were only thawed prior to MS analysis, samples transported at room temperature were exposed to two freeze-thaw cycles. The first one occurred when the lipid extracts were removed from the − 80oC freezer to be shipped at room temperature. Then lipid extracts were stored at -80oC upon arrival. The second freeze-thaw cycle occurred when the lipid extracts were removed from the freezer for MS analysis. More studies are necessary to confirm this hypothesis.
The findings of this study suggest that the extraction method has more influence on the lipid profiling than the sample transportation at room temperature. In this way, considering that the Bligh & Dyer “gold standard” protocol requires maintenance of the cold chain of the samples, the room temperature transport allowed a more effective detection of PI, PG, cholesterol esters and acyl-carnitines, and other evaluated lipid classes, except FFA, in comparison with dry ice. Remarkably, TAG and FFA were the lipid classes most affect by sample transportation temperature, showing lower recovery in oocytes transported at room temperature after methanol or Bligh & Dyer extraction method, respectively.
If all samples in a given experiment are processed in the same way it is possible to obtain informative lipid profiles and consistent results in lipidomics of small-sized samples, hereby exemplified by the oocytes. Bligh & Dyer still seems to be more suitable for a comprehensive lipid profiling, since this approach allows the removal of the proteins and the detection of all lipid classes profiled in this study, including phosphatidylserines (PS), FFA and acyl-carnitines, which were not well detected otherwise. Although this modified pre-analytical approach based on the Bligh & Dyer method represents a viable option for lipid profiling, it will require further research and validation before it is proposed and widely used in different biological matrices. On the other hand, we acknowledge that for studies on specific subclasses (target lipid extraction), one-step extraction using pure methanol may represent a valid choice and a useful strategy for target lipid extraction tailored to specific biological matrices (e.g., oocytes, embryos, cell lines) and sampling (e.g., microextraction). Finally, the impact of transportation of lipid extracts under vacuum at room temperature was not observed by PCA analysis, meaning that lipid profiling can be performed after room temperature transportation without extensive consequences to the final results.