F Synthetic StandardsFor each from the chlorinated lipid classes, RSK4 supplier steady isotope-labeled
F Synthetic StandardsFor every single on the chlorinated lipid classes, stable isotope-labeled internal standards are the best approach for quantitative evaluation. For TM-ClFALD evaluation, the internal standard employed is -ClFA evaluation, the internal 2-chloro-[d4-7,7,eight,8]-hexadecanal (2-Cl-[d4]HDA). For TMstandard utilized is 2-chloro-[d4-7,7,8,8]-hexadecanoic acid (2-Cl-[d4]HA). For 2-ClFOH analysis, the internal typical employed is 2-chloro-[d4-7,7,eight,8]-hexadecanol (2-Cl-[d4]HOH).Anal Biochem. Author manuscript; offered in PMC 2014 December 15.Wang et al.Page2-Cl-[d4]HDA has been previously synthesized [15] by the following measures: 1) synthesis of [7,7,8,8-d4]-hexadecanol from [7,7,eight,8-d4]-hexadecanoic acid (Healthcare Isotopes, Inc.) utilizing sodium bis(2-methoxyethoxy)aluminum hydride; 2) synthesis of [7,7,8,8-d4]-hexadecanal by partial oxidation at 70 utilizing oxalyl chloride-activated DMSO as catalyst (30); 3) synthesis of the dimethyl acetal of [7,7,eight,8-d4]-hexadecanal by acid methanolysis; four) synthesis in the dimethyl acetal of 2-Cl-[d4]HDA by acetal chlorination employing MnO2trimethylchlorosilane (31); and five) synthesis of 2-Cl-[d4]HDA by reflux in 1:1 trifluoroacetic acid/dichloromethane. The solution is purified by semi-preparative TLC and quantitated [15]. For the synthesis of 2-Cl-[d4]HA, [d4]-hexadecanoic acid ([d4]-16:0 FA) is subjected to TMchlorination with Cl2 making use of the Hell-Vollard-Zelinsky reaction and phosphorous as catalyst [21]. [d4]-16:0 FA is melted at 80 just before an equimolar quantity of phosphorous trichloride in dichloromethane is added for the reaction vial. Cl2 is then slowly bubbled into the reaction mixture for 1 h. The crude product is sequentially extracted and purified first by TLC then reversed phase HPLC as previously described [22]. For the synthesis of 2-Cl-[d4]HOH, 2-Cl-[d4]HDA is decreased with VitrideTM reagent (sodium bis(2-methoxyethoxy)aluminum hydride), and also the resultant alcohol is purified by TLC (petroleum ether/ethyl ether/acetic acid (70/30/1, v/v/v)) (Rf = 0.41).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptLipid extractionFigure three shows a flow chart for the extraction procedures employed for chlorinated lipids from either tissues, cells, cell culture media or plasma. For lipid extractions, derivatizations, and chromatography, HPLC grade solvents must be applied. For Bligh-Dyer extractions of cells or tissues the usage of chloroform and methanol bought from Fisher is recommended. Cellular or tissue lipids (e.g., from 1 106 neutrophils) are extracted by a modification from the approaches of Bligh and Dyer [13; 23] within the presence of 20 pmol every single of 2-Cl-[d4]HDA, 2-Cl-[d4]HA, and 2-Cl-[d4]HOH (e.g., in the case of 1 106 neutrophils extracted) that happen to be added as internal standards for TM-ClFALD, TM-ClFA, and TM-ClFOH quantitation, SGLT2 medchemexpress respectively. These cellular or tissue lipid extracts are then applied in subsequent analytical actions to quantify TM-ClFALD, cost-free TM-ClFA, and TM-ClFOH as described under. For cell culture media and plasma analysis of no cost TM-ClFA, a modified Dole extraction is routinely performed [11; 12]. Right after drying the organic phase extracts containing TM-ClFA, these samples are suspended in 150 TM… of methanol/water (85/15, v/v) containing 0.1 l formic acid and transferred to an autosampler vial with an insert, and these samples will subsequently be analyzed making use of LC-MS. Similarly Bligh-Dyer lipid extracts are suspended in 150 TM… of methanol/water (85/15, v/v) containing 0.1 f.
