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In the brain, approximately 90% of oxylipins are esterified to lipids. However, the significance of this esterification process is not known. In the present study, we (1) validated an aminopropyl solid phase extraction (SPE) method for separating esterified lipids using 100 and 500 mg columns and (2) applied the method to quantify the distribution of esterified oxylipins within phospholipids (PL) and neutral lipids (NL) (i.e. triacylglycerol and cholesteryl ester) in rats subjected to head-focused microwave fixation (controls) or CO2 -induced hypercapnia/ischemia. We hypothesized that oxylipin esterification into these lipid pools will be altered following CO2 -induced hypercapnia/ischemia. Lipids were extracted from control (n = 8) and CO2 -asphyxiated (n = 8) rat brains and separated on aminopropyl cartridges to yield PL and NL. The separated lipid fractions were hydrolyzed, purified with hydrophobic-lipophilic-balanced SPE columns, and analyzed with ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. Method validation showed that the 500 mg (vs 100 mg) aminopropyl columns yielded acceptable separation and recovery of esterified fatty acid epoxides but not other oxylipins. Two epoxides of arachidonic acid (ARA) were significantly increased, and three epoxides of docosahexaenoic acid (DHA) were significantly decreased in brain NL of CO2 -asphyxiated rats compared to controls subjected to head-focused microwave fixation. PL-bound fatty acid epoxides were highly variable and did not differ significantly between the groups. This study demonstrates that hypercapnia/ischemia alters the concentration of ARA and DHA epoxides within NL, reflecting an active turnover process regulating brain fatty acid epoxide concentrations.

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

Linoleic acid (LA; 18:2 n-6), the most abundant polyunsaturated fatty acid in the US diet, is a precursor to oxidized metabolites that have unknown roles in the brain. Here, we show that oxidized LA-derived metabolites accumulate in several rat brain regions during CO2-induced ischemia and that LA-derived 13-hydroxyoctadecadienoic acid, but not LA, increase somatic paired-pulse facilitation in rat hippocampus by 80%, suggesting bioactivity. This study provides new evidence that LA participates in the response to ischemia-induced brain injury through oxidized metabolites that regulate neurotransmission. Targeting this pathway may be therapeutically relevant for ischemia-related conditions such as stroke.

Dried blood spots (DBS) by fingertip prick collection for fatty acid profiling are becoming increasingly popular due to ease of collection, minimal invasiveness and its amenability to high-throughput analyses. Herein, we assess a microwave-assisted direct transesterification method for the production of fatty acid methyl esters (FAME) from DBS. Technical replicates of human whole blood were collected and 25-μL aliquots were applied to chromatography strips prior to analysis by a standard 3-h transesterification method or microwave-assisted direct transesterification method under various power (variable vs constant), time (1-5 min) and reagent (1-10% H2SO4 in methanol) conditions. In addition, a standard method was compared to a 5-min, 30-W power microwave in 1% H2SO4 method for FAME yield from whole blood sphingomyelin, and sphingomyelin standards alone and spiked in whole blood. Microwave-assisted direct transesterification yielded no significant differences in both quantitative (nmol/100 µL) and qualitative (mol%) fatty acid assessments after as little as 1.5- and 1-min reaction times, respectively, using the variable power method and 5% H2SO4 in methanol. However, 30-W power for 5 min increased total FAME yield of the technical replicates by 14%. This increase appears largely due to higher sphingomyelin-derived FAME yield of up to 109 and 399% compared to the standard method when determined from whole blood or pure standards, respectively. In conclusion, microwave-assisted direct transesterification of DBS achieved in as little as 1-min, and 5-min reaction times increase total fatty acids primarily by significantly improving sphingomyelin-derived fatty acid yield.

The synthesis rates of n‐3 and n‐6 polyunsaturated fatty acids (PUFAs) in rodents and humans are not agreed upon and depend on substrate availability independently of the capacity for synthesis. Therefore, we aimed to assess the activities of the enzymes for n‐3 and n‐6 PUFA synthesis pathways in liver, brain, testicle, kidney, heart, and lung, in relation to their protein concentration levels. Eight‐week‐old Balb/c mice (n = 8) were fed a standard chow diet (6.2% fat, 18.6% protein, and 44.2% carbohydrates) until 14 weeks of age, anesthetized with isoflurane and tissue samples were collected (previously perfused) and stored at −80°C. The protein concentration of the enzymes (Δ‐6D, Δ‐5D, Elovl2, and Elovl5) were assessed by ELISA kits; their activities were assayed using specific PUFA precursors and measuring the respective PUFA products as fatty acid methyl esters by gas chromatographic analysis. The liver had the highest capacity for PUFA biosynthesis, with limited activity in the brain, testicles, and kidney, while we failed to detect activity in the heart and lung. The protein concentration and activity of the enzymes were significantly correlated. Furthermore, Δ‐6D, Δ‐5D, and Elovl2 have a higher affinity for n‐3 PUFA precursors compared to n‐6 PUFA. The capacity for PUFA synthesis in mice mainly resides in the liver, with enzymes having preference for n‐3 PUFAs. [ABSTRACT FROM AUTHOR]

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is emerging as an important target in the brain for the treatment or prevention of cognitive disorders. The identification of high-affinity ligands for brain PPARα may reveal the mechanisms underlying the synaptic effects of this receptor and facilitate drug development. Here, using an affinity purification–untargeted mass spectrometry (AP-UMS) approach, we identified an endogenous, selective PPARα ligand, 7(S)-hydroxy-docosahexaenoic acid [7(S)-HDHA]. Results from mass spectrometric detection of 7(S)-HDHA in mouse and rat brain tissues, time-resolved FRET analyses, and thermal shift assays collectively revealed that 7(S)-HDHA potently activated PPARα with an affinity greater than that of other ligands identified to date. We also found that 7(S)-HDHA activation of PPARα in cultured mouse cortical neurons stimulated neuronal growth and arborization, as well as the expression of genes associated with synaptic plasticity. The findings suggest that this DHA derivative supports and enhances neuronal synaptic capacity in the brain. Omega-3s for an active brain: Activation of the nuclear receptor PPARα in mice is neuroprotective and boosts memory. Liu et al. found an endogenous ligand for PPARα in the brain that may promote these effects. Proteomic analysis of rodent brain lysates and biophysical assays revealed that 7(S)-HDHA, a hydroxylated derivative of the omega-3 fatty acid DHA, bound to PPARα with high affinity. Treating primary cortical neurons with 7(S)-HDHA promoted dendritic outgrowth and arborization, as well as the expression of PPARα target genes that are associated with synaptic plasticity. Supplementing the diet of young male rats with DHA increased the amount of 7(S)-HDHA in the cortex. The findings further support the notion that omega-3 fatty acids may support synaptic function in the brain and show a potential mechanism underlying such benefits from dietary DHA. [ABSTRACT FROM AUTHOR]

Previously, results from studies investigating if brain palmitic acid (16:0; PAM) was maintained by either dietary uptake or de novo lipogenesis (DNL) varied. Here, we utilize naturally occurring carbon isotope ratios (13C/12C; δ13C) to uncover the origin of brain PAM. Additionally, we explored brain and liver fatty acid concentration, brain metabolomics, and behavior. BALB/c dams were equilibrated onto either a low PAM diet (LP; <2%) or high PAM diet (HP; >95%) prior to producing one generation of offspring. Offspring stayed on the respective diet of the dam until 15‐weeks of age, at which time the Open Field test was conducted, prior to euthanasia and tissue lipid extraction. Although liver PAM was lower in mice fed the LP diet, as well as female mice, brain PAM was not affected by diet or sex. Across mice of either sex on both diets, brain 13C‐PAM revealed compared to dietary uptake, DNL from dietary sugars contributed 68.8%–79.5% and 46.6%–58.0% to the total brain PAM pool by both peripheral and local brain DNL, and local brain DNL alone, respectively. DNL was augmented in mice fed the LP diet, and the ability to up‐regulate DNL in the liver or the brain depended on sex. Anxiety‐like behaviors were decreased in mice fed the LP diet and were correlated with markers of LP diet consumption including increased liver 13C‐PAM, warranting further investigation. Altogether, our results indicate that DNL from dietary sugars is a compensatory mechanism to maintain brain PAM in response to the LP diet. [ABSTRACT FROM AUTHOR]