Generation of Bioactive Oxylipins from Exogenously Added Arachidonic, Eicosapentaenoic and Docosahexaenoic Acid in Primary Human Brain Microvessel Endothelial Cells

Abstract

The human blood-brain barrier (BBB) is the restrictive barrier between the brain parenchyma and the circulating blood and is formed in part by microvessel endothelial cells. The brain contains significant amounts of arachidonic acid (ARA), and docosahexaenoic acid (DHA), which potentially give rise to the generation of bioactive oxylipins. Oxylipins are oxygenated fatty acid metabolites that are involved in an assortment of biological functions regulating neurological health and disease. Since it is not known which oxylipins are generated by human brain microvessel endothelial cells (HBMECs), they were incubated for up to 30 min in the absence or presence of 0.1-mM ARA, eicosapentaenoic acid (EPA) or DHA bound to albumin (1:1 molar ratio), and the oxylipins generated were examined using high performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). Of 135 oxylipins screened in the media, 63 were present at >0.1 ng/mL at baseline, and 95 were present after incubation with fatty acid. Oxylipins were rapidly generated and reached maximum levels by 2-5 min. While ARA, EPA and DHA each stimulated the production of oxylipins derived from these fatty acids themselves, ARA also stimulated the production of oxylipins from endogenous 18- and 20-carbon fatty acids, including α-linolenic acid. Oxylipins generated by the lipoxygenase pathway predominated both in resting and stimulated states. Oxylipins formed via the cytochrome P450 pathway were formed primarily from DHA and EPA, but not ARA. These data indicate that HBMECs are capable of generating a plethora of bioactive lipids that have the potential to modulate BBB endothelial cell function.

Comparative effects of high oleic acid vs high mixed saturated fatty acid obesogenic diets upon PUFA metabolism in mice

“…Hepatic and adipose oxylipin levels were determined using previously published methods [31]. Livers were homogenized in ice-cold Tyrode's salt solution (pH 7.6) in a 1:28 wt/volume ratio, after which Triton X-100 was added to achieve a final concentration of 0.01%.…”

“…Samples were loaded onto the columns, rinsed with 10% methanol, water (pH 3) and hexane before being eluted with methanol. Evaporated samples were then resuspended in solvent for analysis by HPLC-MS/MS (API 4000, AB Sciex, Canada) as described earlier [31], based on methods developed by Deems et al [32]. Detection and quantification limits were set at 3 and 5 levels above background, respectively [31,32].…”

“…Evaporated samples were then resuspended in solvent for analysis by HPLC-MS/MS (API 4000, AB Sciex, Canada) as described earlier [31], based on methods developed by Deems et al [32]. Detection and quantification limits were set at 3 and 5 levels above background, respectively [31,32]. Quantification of oxylipins was determined using a stable isotope dilution method and amounts expressed as nmol/g of tissue [33].…”

“…Prostaglandins, Leukotrienes and Essential Fatty Acids 119 (2017) [25][26][27][28][29][30][31][32][33][34][35][36][37] esterified LA by 30% (8.2 ± 1.1 µmol/g vs 11.7 ± 1.4 µmol/g for LF) and the content of ALA by 57% (0.22 ± 0.05 µmol/g vs 0.51 ± 0.05 µmol/g for LF) ( Table 4). The decrement in LA and ALA was nearly identical to the increase in OA (4.3 µmol/g) in this fraction in high OA-fed mice.…”

“…While PL-esterified levels of ARA were not changed by OA intake, ARA was elevated 64% (5.6 ± 0.8 µmol/g) by the high SFA diet vs the LF animals (3.4 ± 1.4 µmol/g). DHA content in the PL fraction was not altered between the diet [25][26][27][28][29][30][31][32][33][34][35][36][37] groups; however cardiac levels of DPAn-3 were lowered by intake of the high OA diet (38%) and elevated by the high SFA diet (44%). NL content of ALA and LA were also reduced by high OA intake 60% and 52%, respectively (Table 9).…”