Four-dimensional trapped ion mobility spectrometry lipidomics for high throughput clinical profiling of human blood samples.

Lipidomics encompassing automated lipid extraction, a four-dimensional (4D) feature selection strategy for confident lipid annotation as well as reproducible and cross-validated quantification can expedite clinical profiling. Here, we determine 4D descriptors (mass to charge, retention time, collision cross section, and fragmentation spectra) of 200 lipid standards and 493 lipids from reference plasma via trapped ion mobility mass spectrometry to enable the implementation of stringent criteria for lipid annotation. We use 4D lipidomics to confidently annotate 370 lipids in reference plasma samples and 364 lipids in serum samples, and reproducibly quantify 359 lipids using level-3 internal standards. We show the utility of our 4D lipidomics workflow for high-throughput applications by reliable profiling of intra-individual lipidome phenotypes in plasma, serum, whole blood, venous and finger-prick dried blood spots.

Elevated n-3/n-6 PUFA ratio in early life diet reverses adverse intrauterine kidney programming in female rats.

Intrauterine growth restriction (IUGR) predisposes to chronic kidney disease via activation of proinflammatory pathways, and omega-3 PUFAs (n-3 PUFAs) have anti-inflammatory properties. In female rats, we investigated 1) how an elevated dietary n-3/n-6 PUFA ratio (1:1) during postnatal kidney development modifies kidney phospholipid (PL) and arachidonic acid (AA) metabolite content and 2) whether the diet counteracts adverse molecular protein signatures expected in IUGR kidneys. IUGR was induced by bilateral uterine vessel ligation or intrauterine stress through sham operation 3.5 days before term. Control (C) offspring were born after uncompromised pregnancy. On postnatal (P) days P2-P39, rats were fed control (n-3/n-6 PUFA ratio 1:20) or n-3 PUFA intervention diet (N3PUFA; ratio 1:1). Plasma parameters (P33), kidney cortex lipidomics and proteomics, as well as histology (P39) were studied. We found that the intervention diet tripled PL-DHA content (PC 40:6; P < 0.01) and lowered both PL-AA content (PC 38:4 and lyso-phosphatidylcholine 20:4; P < 0.05) and AA metabolites (HETEs, dihydroxyeicosatrienoic acids, and epoxyeicosatrienoic acids) to 25% in all offspring groups. After ligation, our network analysis of differentially expressed proteins identified an adverse molecular signature indicating inflammation and hypercoagulability. N3PUFA diet reversed 61 protein alterations (P < 0.05), thus mitigating adverse IUGR signatures. In conclusion, an elevated n-3/n-6 PUFA ratio in early diet strongly reduces proinflammatory PLs and mediators while increasing DHA-containing PLs regardless of prior intrauterine conditions. Counteracting a proinflammatory hypercoagulable protein signature in young adult IUGR individuals through early diet intervention may be a feasible strategy to prevent developmentally programmed kidney damage in later life.

Lipidomics and Transcriptomics in Neurological Diseases.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease. Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

Broad Lipidomic and Transcriptional Changes of Prophylactic PEA Administration in Adult Mice.

Beside diverse therapeutic properties of palmitoylethanolamide (PEA) including: neuroprotection, inflammation and pain alleviation, prophylactic effects have also been reported in animal models of infections, inflammation, and neurological diseases. The availability of PEA as (ultra)micronized nutraceutical formulations with reportedly no side effects, renders it accordingly an appealing candidate in human preventive care, such as in population at high risk of disease development or for healthy aging. PEA's mode of action is multi-facetted. Consensus exists that PEA's effects are primarily modulated by the peroxisome proliferator-activated receptor alpha (PPARα) and that PEA-activated PPARα has a pleiotropic effect on lipid metabolism, inflammation gene networks, and host defense mechanisms. Yet, an exhaustive view of how the prophylactic PEA administration changes the lipid signaling in brain and periphery, thereby eliciting a beneficial response to various negative stimuli remains still elusive. We therefore, undertook a broad lipidomic and transcriptomic study in brain and spleen of adult mice to unravel the positive molecular phenotype rendered by prophylactic PEA. We applied a tissue lipidomic and transcriptomic approach based on simultaneous extraction and subsequent targeted liquid chromatography-multiple reaction monitoring (LC-MRM) and mRNA analysis by qPCR, respectively. We targeted lipids of COX-, LOX- and CYP450 pathways, respectively, membrane phospholipids, lipid products of cPLA2, and free fatty acids, along with various genes involved in their biosynthesis and function. Additionally, plasma lipidomics was applied to reveal circulatory consequences and/or reflection of PEA's action. We found broad, distinct, and several previously unknown tissue transcriptional regulations of inflammatory pathways. In hippocampus also a PEA-induced transcriptional regulation of neuronal activity and excitability was evidenced. A massive downregulation of membrane lipid levels in the splenic tissue of the immune system with a consequent shift towards pro-resolving lipid environment was also detected. Plasma lipid pattern reflected to a large extent the hippocampal and splenic lipidome changes, highlighting the value of plasma lipidomics to monitor effects of nutraceutical PEA administration. Altogether, these findings contribute new insights into PEA's molecular mechanism and helps answering the questions, how PEA prepares the body for insults and what are the "good lipids" that underlie this action.

Antiepileptogenic Effect of Subchronic Palmitoylethanolamide Treatment in a Mouse Model of Acute Epilepsy.

Research on the antiepileptic effects of (endo-)cannabinoids has remarkably progressed in the years following the discovery of fundamental role of the endocannabinoid (eCB) system in controlling neural excitability. Moreover, an increasing number of well-documented cases of epilepsy patients exhibiting multi-drug resistance report beneficial effects of cannabis use. Pre-clinical and clinical research has increasingly focused on the antiepileptic effectiveness of exogenous administration of cannabinoids and/or pharmacologically induced increase of eCBs such as anandamide (also known as arachidonoylethanolamide [AEA]). Concomitant research has uncovered the contribution of neuroinflammatory processes and peripheral immunity to the onset and progression of epilepsy. Accordingly, modulation of inflammatory pathways such as cyclooxygenase-2 (COX-2) was pursued as alternative therapeutic strategy for epilepsy. Palmitoylethanolamide (PEA) is an endogenous fatty acid amide related to the centrally and peripherally present eCB AEA, and is a naturally occurring nutrient that has long been recognized for its analgesic and anti-inflammatory properties. Neuroprotective and anti-hyperalgesic properties of PEA were evidenced in neurodegenerative diseases, and antiepileptic effects in pentylenetetrazol (PTZ), maximal electroshock (MES) and amygdaloid kindling models of epileptic seizures. Moreover, numerous clinical trials in chronic pain revealed that PEA treatment is devoid of addiction potential, dose limiting side effects and psychoactive effects, rendering PEA an appealing candidate as antiepileptic compound or adjuvant. In the present study, we aimed at assessing antiepileptic properties of PEA in a mouse model of acute epileptic seizures induced by systemic administration of kainic acid (KA). KA-induced epilepsy in rodents is assumed to resemble to different extents human temporal lobe epilepsy (TLE) depending on the route of KA administration; intracerebral (i.c.) injection was recently shown to most closely mimic human TLE, while systemic KA administration causes more widespread pathological damage, both in brain and periphery. To explore the potential of PEA to exert therapeutic effects both in brain and periphery, acute and subchronic administration of PEA by intraperitoneal (i.p.) injection was assessed on mice with systemically administered KA. Specifically, we investigated: (i) neuroprotective and anticonvulsant properties of acute and subchronic PEA treatment in KA-induced seizure models, and (ii) temporal dynamics of eCB and eicosanoid (eiC) levels in hippocampus and plasma over 180 min post seizure induction in PEA-treated and non-treated KA-injected mice vs. vehicle injected mice. Finally, we compared the systemic PEA treatment with, and in combination with, pharmacological blockade of fatty acid amide hydrolase (FAAH) in brain and periphery, in terms of anticonvulsant properties and modulation of eCBs and eiCs. Here, we demonstrate that subchronic administration of PEA significantly alleviates seizure intensity, promotes neuroprotection and induces modulation of the plasma and hippocampal eCB and eiC levels in systemic KA-injected mice.

Simultaneous lipidomic and transcriptomic profiling in mouse brain punches of acute epileptic seizure model compared to controls.

In this study, we report the development of a dual extraction protocol for RNA and lipids, including phospholipids, endocannabinoids, and arachidonic acid, at high spatial resolution, e.g., brain punches obtained from whole frozen brains corresponding to four brain subregions: dorsal hippocampus, ventral hippocampus, basolateral amygdala, and hypothalamus. This extraction method combined with LC/multiple reaction monitoring for lipid quantifi-cation and quantitative PCR for RNA investigation allows lipidomic and transcriptomic profiling from submilligram amounts of tissue, thus benefiting the time and animal costs for analysis and the data reliability due to prevention of biological variability between animal batches and/or tissue heterogeneity, as compared with profiling in distinct animal batches. Moreover, the method allows a higher extraction efficiency and integrity preservation for RNA, while allowing concurrently quantitative analysis of low and high abundant lipids. The method was applied for brain punches obtained 1 h after kainic acid-induced epileptic seizures in mice (n = 10) compared with controls (n = 10), and enabled the provision of valuable new insights into the subregional lipid and RNA changes with epilepsy, highlighting its potential as a new viable tool in quantitative neurobiology.