The metabolism of arachidonic acid (ARA) by cytochrome P450 /-1-hydroxylases network

The metabolism of arachidonic acid (ARA) by cytochrome P450 /-1-hydroxylases network marketing leads to the forming of 20-hydroxyeicosatetraenoic acid (20-HETE), which can be an important lipid-signaling molecule involved with regulation of vascular tone, angiogenesis, and inflammation. type 2 (MFE-2) gene Kaempferol in = H or COCH3. Components AND METHODS Components and chemical substances ARA was bought from Nu-Chek Prep (Elysian, MN). Solvents employed for display chromatography and HPLC had been bought from Fisher Scientific (Pittsburgh, PA). Criteria of 19-HETE, 20-HETE, 20-HETE-d6, and various other lipid mediators had been bought from Cayman Chemical substance (Ann Arbor, MI). CYP52M1 evaluation CYP52M1 (GenBank Accession “type”:”entrez-nucleotide”,”attrs”:”text”:”EU552419″,”term_id”:”189035856″,”term_text”:”EU552419″EU552419) was expressed in W(R) using pYeDP60 vector. Microsomes were isolated for the fatty acid hydroxylation assay by enzymatic digestion of the cell wall, followed by assisted hypotonic lysis and final CaCl2 precipitation as described (14). Hydroxylation assays were carried out by incubating 1 mg microsomal protein in 1 ml of 250 mM potassium phosphate buffer (pH 7.4) with 6.6 mM MgCl2, 6.6 mM glucose-6-phospate, 0.8 U glucose-6-phosphate dehydrogenase, 2.6 mM NADP+, and 0.2 mM fatty acid as substrate. The reaction was incubated at 30C under shaking at 120 rpm for 3 h and quenched by adding 0.5 ml 0.8% (v/v) H2SO4. The reaction mixture was extracted with ethyl acetate, and the resulting extract was analyzed by gradient HPLC (Varian Prostar) and evaporative light scattering detection (ELSD; Alltech) and LC-MS (Waters) as described (11, 15). Yeast fermentation ATCC 22214 (wild-type) and the MFE-2-knockout M30 strain (patented by Ecover Belgium NV) (15, 16) were cultured with a glucose-rich medium as described by Prabhune et al. (13) or Lang et al. (17). An overnight preculture (5 ml) Kaempferol was used to inoculate 200 ml medium. After 48 h incubation at 30C under shaking at 200 rpm, ARA dissolved in an equal volume of ethanol was added into the fermentation medium, and the incubation continued for another 5C12 days. At the end of the fermentation, sophorolipids were extracted twice with an equal volume of ethyl acetate. The extracts were combined and evaporated to dryness using a rotary evaporator, and the dried extract was washed with hexane to remove any residual fatty acids, such as added ARA. The yield of the sophorolipids was expressed as grams of sophorolipids per volume of the fermentation (g/l). HPLC and LC-MS analysis of the sophorolipids was performed as described above. Purification of 20-HETE The recovered sophorolipids were hydrolyzed in 1M aqueous HCl under reflux in a N2 atmosphere for 2 h to liberate the hydroxyl fatty acids. The reaction product was extracted with dichloromethane (CH2Cl2), and then the CH2Cl2 extract was dried over anhydrous MgSO4, loaded onto a silica gel flash column, and eluted with 4% methanol in CH2Cl2. The fractions were pooled based on TLC (silica gel, 4% methanol in CH2Cl2, stained with KMnO4) and 1H NMR analysis. After the silica gel chromatography, fractions made up of largely 19- and 20-HETE with minor impurities were obtained. The fractions made up of RDX 19- and 20-HETE were purified by HPLC using an Agilent 1100 HPLC system (Santa Clara, CA) equipped with a semipreparative silica gel HPLC column (Agilent Zorbax Sil, 250 9.4 mm, 5 m, Agilent catalog no. 880952-201), which was eluted with a mobile phase of 2-propanol/hexane/acetic acid (8:92:0.1, v/v/v), detected at 210 nm with flow rate of 2C3 ml/min. The collected HPLC eluents were evaporated to dryness using a rotary evaporator, redissolved in CH2Cl2, washed with aqueous solution of NaHCO3 to remove residual acetic acid, dried over anhydrous MgSO4, evaporated to dryness, and stored at ?80C under a N2 atmosphere. The structure of the purified 20-HETE was supported by Kaempferol NMR, as well as LC-MS/MS with coelution of standard 20-HETE and 20-HETE-d6. 1H NMR of 20-HETE (CDCl3, 400 MHz): 1.25C1.72 (m, 8H, H-3, 17, 18, and 19); Kaempferol 2.14 (m, 4H, H-4, and 16); 2.36 (tr, = 6.4 Hz, 2H, H-2); 2.82 (m, 6H, H-7, 10, and 13); 3.67 (tr, = 6.4 Hz, 2H, H-20); and 5.4 (m, 8H, H-5, 6, 8, 9, 11, 12, 14, and 15). The signal assignments were made by comparison with published NMR data (10). LC-MS/MS analysis The structure and purity of purified 20-HETE were further assessed by LC-MS/MS. The hydrolyzed sophorolipids or isolated 20-HETE was dissolved in methanol or acetonitrile to prepare a 1 mg/ml solution, then diluted 1,000C4,000 times for LC-MS/MS analysis. The solutions were injected Kaempferol into a LC-MS/MS system, including Agilent 1200SL (Santa Clara, CA) system coupled to AB Sciex 4000 QTrap system (Foster City, CA). The LC/MS/MS method was described earlier (18). The mass spectrometer was operated under unfavorable electrospray.