15 hete d8

Manufactured by Cayman Chemical

Sourced in United States

15-HETE-d8 is a stable isotope-labeled variant of 15-hydroxyeicosatetraenoic acid, a naturally occurring eicosanoid. It can be used as an internal standard or reference compound in analytical methods.

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15-HETE (16 citations) 15-hydroxyeicosatetraenoic acid (HETE)-d8 (2 citations) 15(S)-HETE-d8 (2 citations)

10 protocols using 15 hete d8

Evaluation of Cell Lines and Reagents

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A431 (Human epithelial carcinoma) and HEK293 (Human embryonic kidney)
cell lines were obtained from ATCC (Manassas, VA) and used at low passage within
four years of purchase. HUVECs (Human Umbilical Vein Endothelial Cell) were from
Lonza (Walkersville, MD). Recombinant c-Src active protein was from Millipore
(Billerica, MA). 15-HETE-d8, arachidonic acid-d8 and arachidonic acid (AA) were
from Cayman Chemical (Ann Arbor, MI). All other reagents were of analytical
grade and from SigmaAldrich (St. Louis, MO).

Dilly A.K., Tang K., Guo Y., Joshi S., Ekambaram P., Maddipati K.R., Cai Y., Tucker S.C, & Honn K.V. (2016). Convergence of Eicosanoid and Integrin Biology: Role of Src in 12-LOX activation. Experimental cell research, 351(1), 1-10.

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Quantification of Lipid Metabolites

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ARA-d8, 15-HETE-d8, LTB₄-d4 and PGE₂-d4 were obtained from Cayman Chemical (Ann Arbor, MI, USA). Highly purified EPA ethyl ester (EPA-E) (> 98%), DHA ethyl ester (DHA-E) (> 97%), and ethyl arachidonate (ARA-E) (> 99%) were obtained from Nippon Suisan Kaisha, Ltd. (Tokyo, Japan), Harima foods, Inc. (Osaka, Japan) and NuChek Prep, Inc. (Elysian, MN, USA), respectively. All solvents of LC/MS grade were obtained from Wako (Tokyo, Japan).

Naoe S., Tsugawa H., Takahashi M., Ikeda K, & Arita M. (2019). Characterization of Lipid Profiles after Dietary Intake of Polyunsaturated Fatty Acids Using Integrated Untargeted and Targeted Lipidomics. Metabolites, 9(10), 241.

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Comprehensive Eicosanoid Analysis Method

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A comprehensive analysis method for eicosanoids has been described previously (17 ). The method utilizes an LC-8060 device, consisting of a quantum triple-quadrupole mass spectrometer (Shimadzu, Kyoto, Japan) and a software method package for the simultaneous analysis of 196 products with 18 deuterium internal standards (supplemental Table S2): tetranor-PGEM-d6, 6-keto-PGF1α-d4, TXB2-d4, PGF2α-d4, PGE2-d4, PGD2-d4, PGA2-d4, LTB4-d4, 14,15-DiHET-d11, 1 5-HETE-d8, 12-HETE-d8, 5-HETE-d8, 11,12-EET-d11, LTC4-d5, LTD4-d5, PAF-d4, OEA-d4, and AA-d8 (Cayman Chemical, Ann Arbor, MI). The eicosanoids were identified by multiple reactions monitoring (MRM) and were analyzed using LabSolutions software (Shimadzu).

Morita Y., Kurano M., Sakai E., Sawabe M., Aoki J, & Yatomi Y. (2021). Simultaneous analyses of urinary eicosanoids and related mediators identified tetranor-prostaglandin E metabolite as a novel biomarker of diabetic nephropathy. Journal of Lipid Research, 62, 100120.

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Brain Lipid Extraction and Analysis

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Brain samples were homogenized with glass homogenizer on ice in such a way that the total volume of homogenate was 3 ml in cold MeOH. 5ul of internal standard mixture (5 ngs of LTB4-d4, 15-HETE-d8, PGD2-d4, EPA-d5 and 25 ng of AA-d8, all purchased from Cayman Chemical, Ann Arbor, MI), and sonicated in the water bath with ice for 30 minutes. Sample was stored at −80C overnight for the extraction. The following day, C18 column (BondElut 500 mg, Agilent Technologies) was equilibrated with 20ml cold MeOH followed by 20ml cold H₂O in a vacuum manifold for Solid Phase Extraction. The sample was diluted with cold pH 3.5 H₂O such that MeOH concentration is 10~15 % and pH of the sample became 3.5~4.0 before loading it to the column. The sample was loaded into the column with 1~2 drops per minute. The column was washed with H₂O and 2ml hexane, and lipid was eluted with 10ml methylformate. Sample was dried under N₂ flow, and re-suspended with 10 ul MeOH followed by 5 ul H₂O.

Belayev L., Hong S.H., Menghani H., Marcell S.J., Obenaus A., Freitas R.S., Khoutorova L., Balaszczuk V., Jun B., Oriá R.B, & Bazan N.G. (2018). Docosanoids Promote Neurogenesis and Angiogenesis, Blood-Brain Barrier Integrity, Penumbra Protection and Neurobehavioral Recovery after Experimental Ischemic Stroke. Molecular neurobiology, 55(8), 7090-7106.

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Resveratrol Modulates CYP1B1 Expression

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Resveratrol was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 15-HETE-D8 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Dulbecco’s Modified Eagle’s Medium/F-12 (DMEM/F-12) and DMEM were obtained from Gibco, Life Technologies (Grand Island, NY, USA). Fetal bovine serum was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). TRIzol reagent was purchased from Invitrogen Co. (Carlsbad, CA, USA). High-Capacity cDNA Reverse Transcription kit and SYBR® Green PCR Master Mix were purchased from Applied Biosystems (Foster City, CA, USA). Real-time PCR primers were prepared by Integrated DNA Technologies (Coralville, IA, USA). Immun-Blot® PVDF membrane was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Recombinant monoclonal anti-CYP1B1 antibody [ab185954] was purchased from abcam (Toronto, ON). Chemiluminescence Western blotting detection reagents (ECL) were obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA). All of the other chemicals used were obtained from Fisher Scientific Co. (Toronto, ON).

Shoieb S.M, & El-Kadi A.O. (2020). Resveratrol attenuates angiotensin II-induced cellular hypertrophy through the inhibition of CYP1B1 and the cardiotoxic mid-chain HETE metabolites. Molecular and Cellular Biochemistry, 471(1-2), 165-176.

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Quantification of Eicosanoid Metabolites

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Plasma and homogenized tissue (kidney and liver) samples were subjected to alkaline hydrolysis and solid-phase extraction was performed as described previously [41 (link)]. 10 ng of each 20-HETE-d6, 14,15-EET-d8, 14,15-DHET-d11, and 15-HETE-d8 (Cayman Chemicals, Ann Arbor, MI, USA) served as internal standards. Subsequent analysis of the endogenous eicosanoid profiles was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS; Lipidomix GmbH, Berlin, Germany) as established previously [42 (link)]. Results are given in ng metabolites per ml plasma or per g of organ wet weight.

Zhu Y., Blum M., Hoff U., Wesser T., Fechner M., Westphal C., Gürgen D., Catar R.A., Philippe A., Wu K., Bubalo G., Rothe M., Weldon S.M., Dragun D, & Schunck W.H. (2016). Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice. PLoS ONE, 11(1), e0145645.

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Comprehensive Lipid Mediator Profiling

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LC-MS/MS-based mediator lipidomics was performed as previously described (41 (link)). Lung tissues were homogenized in ice-cold methanol and kept in -20°C overnight. The methanolic extract was then diluted with water, acidified with HCl to a pH of 3.5, and applied to Sep-Pak C18 cartridges (Waters) for solid phase extraction. Deuterated internal standards (1 ng of leukotriene (LT) B₄-d4, LTD₄-d5, prostaglandin (PG) E₂-d4, and 15-HETE-d8 (Cayman Chemical, Ann Arbor, MI, USA) were added to the supernatants prior to extraction. For LC-MS/MS analysis, a triple quadrupole linear ion trap mass spectrometer (QTRAP 5500; AB Sciex, Foster City, CA) equipped with a 1.7-μm, 1.0 × 150 mm Acquity UPLC™ BEH C18 column (Waters Corp., Milford, MA) was used. MS/MS analyses were conducted in negative ion mode, and the eicosanoids and docosanoids were identified and quantified by multiple reaction monitoring. Calibration curves were obtained over a range of 1–1,000 pg. The LC retention times for each compound were determined using the corresponding synthetic standards. PD1 and PDX, stereoisomers, were not separable under this LC-MS/MS setting.

Miyata J., Yokokura Y., Moro K., Arai H., Fukunaga K, & Arita M. (2021). 12/15-Lipoxygenase Regulates IL-33-Induced Eosinophilic Airway Inflammation in Mice. Frontiers in Immunology, 12, 687192.

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Comprehensive Metabolite Profiling Protocol

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All unlabeled chemical standards (Supplementary Table S5) and stable deuterated isotope-labeled internal standards (IS), including PGE2 -d4, TXB2-d4, AA-d8, 15-HETE-d8, LTB4-d8, and Resolvin D1-d5, were purchased from Cayman Chemicals (Ann Arbor, MI, USA). LC-MS grade methanol (MeOH), acetonitrile (ACN), formic acid, and acetic acid were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Oasis HLB SPE cartridges (60 mg/3 mL) were from Waters Corporation (Milford, MA, USA). Centrifuge tube filters were from Corning Co. (Corning, NY, USA). Ultrapure water was obtained from a water purification system (ThermoFisher Scientific, NJ). All other chemical reagents from Sigma (St. Louis, MO, USA).

Chhonker Y.S., Kanvinde S., Ahmad R., Singh A.B., Oupický D, & Murry D.J. (2021). Simultaneous Quantitation of Lipid Biomarkers for Inflammatory Bowel Disease Using LC–MS/MS. Metabolites, 11(2), 106.

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Quantitative Lipid Analysis by LC-MS/MS

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HPLC-grade methanol, acetonitrile, isopropanol and formic acid used for sample purification and LC-MS/MS analysis were JT Baker-brand (ThermoFisher Scientific, Waltham, MA). Authentic lipid standards of 6-keto-PGF_1α, PGE₂, PGD₂, PGF_2α, PGJ₂, 15-hydroxyeicosanoic acid (15-HETE), 2-arachidonoylglycerol (2-AG), anandamide (AEA), arachidonic acid (AA), oloeylglycerol (OG), and oleoylethanolamide (OEA) were purchased from Cayman Chemicals (Ann Arbor, MI). 16:0 lysophosphatidylcholine (LPC) and 17:0 LPC were purchased from Avanti Polar Lipids (Alabster, AL). The following deuterated internal standards were purchased from Cayman: 6-keto-PGF_1α-d4, PGF_2α-d4, PGE₂-d4, PGD₂-d4, PGJ₂-d4, 15-HETE-d8, 2-AG-d5, AEA-d4, AA-d8, and OEA-d4. OG-d5 was purchased from US Biological (Salem, MA).
All LC-MS/MS analysis was performed on a Shimadzu Nexera system in-line with a SCIEX 6500 QTrap; except the LPC analysis, which was performed on a Shimadzu LC system in-line with a SCIEX 3200 QTrap mass spectrometer. The 6500 QTrap was equipped with a TurboV Ionspray source and operated in positive and negative ion modes. The 3200 QTrap was equipped with an ionspray source and operated in positive ion mode. For both LC-MS systems, SCIEX Analyst software (ver 1.6.2) was used to control the instruments and acquire and process the data.

Morrison V.E., Houpert M.G., Trapani J.B., Brockman A.A., Kingsley P.J., Katdare K.A., Layden H.M., Nguena-Jones G., Trevisan A.J., Maguire-Zeiss K.A., Marnett L.J., Bix G.J., Ihrie R.A, & Carter B.D. (2023). Jedi-1/MEGF12-mediated phagocytosis controls the pro-neurogenic properties of microglia in the ventricular-subventricular zone. bioRxiv.

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Oxylipidomic Measurements Utilization

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Chemicals used for this study were of following origin: Arachidonic acid (AA) and standards of 15-HETE, 12-HETE, 5-HETE, 12-HEPE, 15-HEPE, 14-HDHA, 17-HDHA, 13-HODE, 13-HOTrE) from Cayman Chem.; sodium borohydride from Life Technologies, Inc. (Eggenstein, Germany); restriction enzymes from ThermoFisher (Schwerte, Germany); isopropyl-β-thiogalactopyranoside (IPTG) from Carl Roth GmbH (Karlsruhe, Germany); E. coli (strain Rosetta2 DE3 pLysS) from Novagen (Merck-Millipore, Darmstadt, Germany). Oligonucleotide synthesis was carried out at BioTez Berlin Buch GmbH (Berlin, Germany). Nucleic acid sequencing was performed at Eurofins MWG Operon (Ebersberg, Germany). HPLC grade solvents and water were purchased from Fisher Scientific (New Hampshire, USA). The origin of other chemicals employed in this study is specified in the description of the methods. The following chemicals were used for oxylipidomic measurements: Deuterated standards (LTB4-d4, 20-HETE-d6, 15-HETE-d8, 13-HODE-d4, 14,15-DHET-d11, 9,10-DiHOME-d4, 12,13-EpOME-d4, 8,9-EET-d11, PGE2-d4; 10 ng/ml each) from Cayman Chem. (Ann Arbor, USA); acetonitrile, solvents from Merck (Darmstadt, Germany) and Fisher Scientific (Schwerte, Germany).

Reisch F., Heydeck D., Schäfer M., Rothe M., Yang J., Stehling S., Püschel G.P, & Kuhn H. (2023). Knock-in mice expressing a humanized arachidonic acid 15-lipoxygenase (Alox15) carry a partly dysfunctional erythropoietic system. Cellular & molecular biology letters, 28(1).

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