A Robust Protocol for Extracting Aqueous Metabolites of High Lipid Sera

Abstract

Background: With the increasing focus of metabolomic methods on obesityrelated diseases, it is important to consider how sample handling may need to be adapted for the high compositions of lipids that can occur in such subjects. Introduction: High-lipid (cloudy, milky appearances; a.k.a. lipemic) biofluids are common in very high BMI subjects. Organic extractions of biofluids are useful for removing protein backgrounds, inactivating capsid viruses, and yielding relatively stable samples with excellent spectroscopic characteristics. This work considered how acetonitrile extractions, which are widely used, perform on lipemic sera. Results: In this technical note, we report the observation and remediation of a liquid-liquid phase separation in acetonitrile extractions of many lipemic sera. This unexpected behavior can be challenging to identify, especially if working with small volumes. The liquid-liquid separation shows a high miscibility of proteins in both liquid phases that impairs NMR data quality. We also report a simple temperature-based adaption of the acetonitrile extraction procedure that consistently results in a single aqueous phase and eliminates unwanted constituents. Conclusion: A robust approach to achieving reproducible, high quality samples of aqueous metabolites from lipemic sera from very high BMI subjects should be of utility in expanding metabolomics applications to lipemic biofluids.

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“…Although acetonitrile and water are miscible in all proportions, an interesting result connected to this work was the discovery of a liquid-liquid phase separation during the extraction of the majority of the samples [24], where high lipids likely constitute a third component and cause the observed phase separation. To reproducibly and reliably obtain aqueous metabolites, a slightly modified extraction protocol was implemented for the sera used in this study that includes a brief room-temperature step to break the liquid-liquid phase separation; this simple modification preserves the removal of proteins and may be of interest in metabolomics of obesity-related diseases [24]. [25]; a demographic statement is included in Methods.…”

“…Samples were always on ice or refrigerated during handling, with one exception noted below. Each sample was re-vortexed for 30-45 s at 4 • C to ensure proper mixing and disruption of debris prior to centrifuging for 10 minutes at 4 • C and 10,600 rcf on a benchtop refrigerated centrifuge, after which it was often found that the supernatant consisted of two liquid phases, an intriguing behavior since these solvents are miscible in all proportions [24]. We have reported and resolved the phase separation separately [24].…”

“…Each sample was re-vortexed for 30-45 s at 4 • C to ensure proper mixing and disruption of debris prior to centrifuging for 10 minutes at 4 • C and 10,600 rcf on a benchtop refrigerated centrifuge, after which it was often found that the supernatant consisted of two liquid phases, an intriguing behavior since these solvents are miscible in all proportions [24]. We have reported and resolved the phase separation separately [24]. Briefly, the two-phase region is attributed to a third component (lipids), and this region can be avoided by working briefly at room temperature.…”