Abstract
Lipidomic profiling of plasma is an emerging field, given the importance of lipids in major cellular pathways, and is dependent on efficient lipid extraction protocols. Recent attention has turned to plasma lipidomics as a means to identify potential diagnostic and prognostic biomarkers related to dementia, neuropsychiatric health and disease. Although several solvent-based lipid extraction protocols have been developed and are currently in use, novel and more efficient methods could greatly simplify lipid analysis in plasma and warrant investigation. Human plasma from normolipidemic adult volunteers was collected to evaluate three different solvent extraction protocols, including the classical Folch method, the methanol/tert-butyl methyl ether (MTBE) (Matyash) method, and a recent single-phase methanol/1-butanol (Alshehry) method. Extracted lipids were analyzed using liquid chromatography mass spectrometry (LC-MS) in positive and negative ion mode. Overall, more than 500 different lipids were identified in positive and negative ion mode combined. Our data show that the single phase Alshehry method was as effective as the Folch and Matyash methods in extracting most lipid classes and was more effective in extraction of polar lipids. Normalized peak areas of the Alshehry method were highly and positively correlated with both the Folch and Matyash methods (r2 = 0.99 and 0.97, respectively). Within- and between- subject correlations were r = 0.99 and 0.96, respectively. Median intra-assay coefficient of variation (CV%) in positive mode was 14.1, 15.1, and 21.8 for the Alshehry, Folch and Matyash methods, respectively. Median Alshehry inter-assay CV (collected over 5 separate days) was 14.4%. In conclusion, the novel Alshehry method was at least as good as, if not better than the established biphasic extraction methods in detecting a wide range of lipid classes, using as little as 10 μL of plasma, and was highly reproducible, safer and more environmentally-friendly as it doesn't require chloroform.
Highlights
It is well-documented that lipids have multiple structural and functional roles, including signaling [1, 2], maintenance of membrane structure [3, 4], myelin sheath formation [5, 6], neurotransmission [7], and protein interactions in both plasma and organs including the brain [8, 9]
Peak areas of LPC, LPE, PI, PC and PG were higher in the Alshehry method than the Matyash method (p < 0.05, Tukey’s post-hoc test), while the peak area of PG and PI were greater in the Alshehry method relative to the Folch extraction
We confirm the findings of the originally published Alshehry single-phase protocol, and show that it is good as, if not better than the commonly used biphasic Matyash and Folch methods in the general extraction of plasma lipids, based on its ability to cover a broad range of lipid classes, including more polar lipids that may not be extracted readily using the Folch recipe
Summary
It is well-documented that lipids have multiple structural and functional roles, including signaling [1, 2], maintenance of membrane structure [3, 4], myelin sheath formation [5, 6], neurotransmission [7], and protein interactions in both plasma and organs including the brain [8, 9]. Recent advances in mass spectrometry have resulted in greater sensitivity, increased mass accuracy and faster scan speeds [15, 16] This has enabled greater sensitivity and better characterization of lipid changes in bodily fluids, cells, and tissue extracts, leading to renewed understanding of the role of different lipid classes in the pathobiology of diseases. Lipidomics has recently been applied to the study of dementia, where brain, CSF and plasma lipids have been identified as potential diagnostic and prognostic biomarkers for Alzheimer’s disease [17, 18], frontotemporal dementia [19], as well as other common neuropsychiatric disorders, such as schizophrenia and bipolar disorder [20, 21]
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