Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics’ technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950–Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231–Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

Dietary mono-unsaturated fatty acids (MUFAs) are linked to human longevity and extend lifespan in several species1-12. But the mechanisms by which MUFAs promote longevity remain unclear. Here we show that an organelle hub involving lipid droplets and peroxisomes is critical for lifespan extension by MUFAs in C. elegans. MUFA accumulation increases lipid droplet number in fat storage tissues, and lipid droplet synthesis is necessary for MUFA-mediated longevity. Interestingly, the number of lipid droplets in young individuals can predict their remaining lifespan. MUFA accumulation also increases the number of peroxisomes, and peroxisome activity is required for MUFA-mediated longevity. By performing a targeted screen, we uncover a functional network between lipid droplets and peroxisomes in longevity. Interestingly, our screen also identifies ether lipids as critical components of the lipid droplet-peroxisome network. Using lipidomics, we find that the ratio of MUFAs to polyunsaturated fatty acids (PUFAs) in ether lipids is increased by MUFA accumulation. Ether lipids are involved in ferroptosis, a non-apoptotic form of cell death13-17, and MUFAs promote longevity in part via suppression of ferroptosis. Our results identify a mechanism of action for MUFAs to extend lifespan and uncover an organelle network involved in the homeostasis of MUFA-rich ether lipids. Our work also opens new avenues for lipid-based interventions to delay aging.

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Phosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

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The aging brain exhibits a decline in the regenerative populations of neural stem cells (NSCs), which may underlie age-associated defects in sensory and cognitive functions1-6. While mechanisms that restore old NSC function have started to be identified7-23, the role of lipids - especially complex lipids - in NSC aging remains largely unclear. Using lipidomic profiling by mass spectrometry, we identify age-related lipidomic signatures in young and old quiescent NSCs in vitro and in vivo. These analyses reveal drastic changes in several complex membrane lipid classes, including phospholipids and sphingolipids in old NSCs. Moreover, poly-unsaturated fatty acids (PUFAs) strikingly increase across complex lipid classes in quiescent NSCs during aging. Age-related changes in complex lipid levels and side chain composition are largely occurring in plasma membrane lipids, as revealed by lipidomic profiling of isolated plasma membrane vesicles. Experimentally, we find that aging is accompanied by modifications in plasma membrane biophysical properties, with a decrease in plasma membrane order in old quiescent NSCs in vitro and in vivo. To determine the functional role of plasma membrane lipids in aging NSCs, we performed genetic and supplementation studies. Knockout of Mboat2, which encodes a phospholipid acyltransferase, exacerbates age-related lipidomic changes in old quiescent NSCs and impedes their ability to activate. As Mboat2 expression declines with age, Mboat2 deficiency may drive NSC decline during aging. Interestingly, supplementation of plasma membrane lipids derived from young NSCs boosts the ability of old quiescent NSCs to activate. Our work could lead to lipid-based strategies for restoring the regenerative potential of NSCs in old individuals, which has important implications for countering brain decline during aging.

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