Visualization by mass spectrometry of 2‐dimensional changes in rat brain lipids, includingN‐acylphosphatidylethanolamines, during neonatal brain ischemia

Abstract

Spatial synthesis of N-acyl-phosphatidylethanolamines (NAPEs) and N-acylethanolamines (NAEs) during ischemia-reperfusion in neonatal rats has been investigated and compared to the spatial degradation of other phospholipids. Ischemia was induced in anesthetized Wistar P7 rat pups by left middle cerebral artery electrocoagulation combined with a transient and concomitant occlusion of both common carotid arteries. Pups were sacrificed after 24 and 48 h. Sham-treated animals were sacrificed after 48 h. The frozen brains were sliced and subjected to desorption electrospray ionization imaging mass spectrometry. There was a remarkable increase in the levels of many species of NAPEs in the whole injured area at both time points, and a clear but minor increase in selected NAEs. In the ischemic area, the sodium adducts of phosphatidylcholine and of lyso-phosphatidylcholine accumulated and the potassium adduct of phosphatidylcholine disappeared, indicating breakdown of the Na(+)/K(+) pump. Free fatty acids, e.g., arachidonic and docosahexaenoic acids, tended to be more abundant in the periphery than in the center of the ischemic area and showed different spatial distribution. NAPEs are synthesized in the whole ischemic area where the cells seem to be dead and other phospholipids are degraded. Free fatty acids can be found in the periphery of the ischemic area.

Quantitative analysis of N‐acylphosphatidylethanolamine molecular species in rat brain using solid‐phase extraction combined with reversed‐phase chromatography and tandem mass spectrometry

“…A total lipid extract from 100 mg brain, which is an extremely lipid‐rich tissue, would have to be diluted in a volume of 10 mL or more for routine lipidomic analysis. While other analytical methods applied to NAPE skip extensive sample preparation methods 6, 7, 30, 32, 34, the presented sample cleanup by SPE eliminates many highly abundant lipid classes such as triacylglycerols that consequently enables reconstitution of the NAPE‐containing fraction in a much smaller volume (200 μL). Preliminary testing during method development showed no analyte losses caused by SPE sample preparation.…”

“…1A), similarly to N ‐acylphosphatidylserine 42. While other approaches to NAPE determination rely on either full scan, SIM, or a single SRM transition per analyte 6, 7, 25, 26, 27, 28, 29, 30, 31, our method uses these two N ‐acyl specific transitions for each analyte, leading to a more selective identification and minimizing the risk of false positive results, as shown in Supporting Information Fig. S1.…”

“…NAPEs not only serve as precursors to NAEs, but also have biological functions of their own. Increasing NAPE levels together with their product NAEs after neuronal damage 5, 6, 7 are indicative of their involvement in a neuroprotective mechanism 8, 9. Postprandial increase of NAPE levels and inhibition of food intake by their administration, may suggest a potential anorectic role 10, 11, although this is subject of controversy and has not yet been clearly shown 1.…”

“…However, this approach groups all species with the same N ‐acyl chain together without the possibility for individual detection of intact NAPE molecular species. Full scan methods 6, 7, 25 or single ion monitoring methods 26 can only deliver elemental compositions, with no identification of substructures such as the N ‐acyl chain. Selected reaction monitoring (SRM) in negative ion mode, with the carboxylate anion 27, 28 or neutral loss 29, 30 of the sn ‐2 fatty acyl chain as the product ion, is only able to identify the sn ‐2 fatty acyl, and further targeted tandem MS experiments are necessary to determine the N ‐acyl chain 31.…”

Mass spectrometry imaging of biomarker lipids for phagocytosis and signalling during focal cerebral ischaemia

“…MSI has already illustrated that during the initial ischaemic phase lysophosphatidylcholine (LysoPC) will accumulate in the ischaemic area11121314, as well as a cessation of Na + /K + -ATPase activity during cell death can be visualized as a decrease in the abundance of the potassium adduct of intracellular phosphatidylcholine (e.g. PC(34:1)) and an increase in the abundance of the sodium adduct of the same PC species1314.…”