The method aims to accurately quantify cellular bioactive lipid metabolites by the combination of stable isotope dilution, cnor phase, liquid chromatography, electron capture, atmospheric pressure, chemical ionization, and mass spectrometry. Begin by harvesting both media and cell samples and add a heavy isotope labeled internal standard mix. Extract the bioactive lipids with organic solvent, then derivate the bioactive lipids with an electron capture reagent proceed to precisely quantify the bioactive lipids by liquid chromatography.
Mass spectrometry through LCMS analysis of both cell lysate and media results precisely determine the concentration of targeted bioactive lipids and the levels of individual Anant ERs. The main advantage of this technique over existing methods like reverse phase liquid chromatography, mass spectrometry, and other LCMS methods, is that individual and anters are accurately quantified through the use of car normal phase chromatography and stabilized isotope dilution. Resulting data can provide valuable insight into the enzymatic pathways functioning in the cell.
For each 10 centimeter plate of a adherent, cells collect three milliliters of media in a 10 milliliter glass centrifuge tube. Next, wash the cells twice with PBS. Then add one milliliter of PBS, scrape the cells off the plate, and transfer the sample to a second to 10 milliliter glass centrifuge tube.
Prepare eight additional tubes with three milliliters of PBS for a calibration curve. Then add 10 microliters of the appropriate calibration standard to each tube. Align each test sample of the three milliliter collected cell culture media for the extraction of free bioactive lipids.
Add 10 microliters of internal standard mix to each calibration standard and sample equilibrate for 10 minutes at room temperature to process samples at five milliliters of dathyl ether and place on a shaker for 30 minutes. Centrifuge the samples and transfer the upper organic phase into 10 milliliter glass centrifuge tubes. Continue to dry the samples under nitrogen First, collect the harvested cell samples by centrifugation and resuspend each pallet in two milliliters of PBS by Vortex Reserve 25 microliters of each sample for cell lysate normalization.
Now equally divide each lysate into 10 milliliter glass centrifuge tubes. Add an additional two milliliters of PBS and 10 microliters of ISM to each sample in order to measure free cellular lipids and avoid alkaline degradation of lipids such as the prostaglandins, use one of each sample duplicates to perform dathyl ether extractions as shown earlier. To extract esterified lipids, add five milliliters of chloroform methanol mixture to each of the remaining samples.
Place on a shaker at low speed for 30 minutes, then centrifuge for 10 minutes. Now, collect the bottom organic layer and dry the samples under nitrogen to sify the esterified lipids. Add 0.5 milliliters of 0.4 normal potassium hydroxide in 80%methanol to each tube.
Incubate samples at 60 degrees celsius for one hour. Then add two milliliters of PBS and adjust the pH two six with concentrated hydrochloric acid. Finally, add 10 microliters of ISM to each sample and proceed to dathyl ether extractions to the dried down samples and calibration standards.
Add derivitization reagents in the specified order. Then vortex the samples and incubate at 60 degrees Celsius for one hour. Now, dry the samples under nitrogen prior to storage and analysis.
This method uses a combination of stable isotope dilution, chii, liquid chromatography, electron capture, atmospheric pressure, chemical ionization, and mass spectrometry analysis. Reconstitute the samples and calibration standards in 100 microliters of hexane ethanol, and transfer the entire samples to HPLC vials with insert place the samples in the chilled autos sampler. Create a sample list for analysis.
Next, set up the HPLC and MS methods using the parameters described in table one and table two in the accompanying written protocol for the normal phase separation. A chiral pack, a DH column to 30 degrees Celsius, inject sample and begin the run. Now set the MS in negative ion mode using the A PCI source.
Begin with a hexane blank to equilibrate the column. Next, run the calibration standard samples in order of increasing SM concentration. Continue to process test samples of culture media after the HBLC separation.
Add methanol post column to avoid deposition on the corona needle. Using data generated from the standards. Create a calibration curve plotting sample area on the Y axis, and standard concentration on the x axis derive the Y equals MX plus B equation.
Finally, determine the test sample concentrations of bioactive lipids for the cell.Lysates. Normalize these values by dividing the determined concentration of bioactive lipid by the number of cells per plate or the total protein normalized in previous steps. Data from multiple reaction monitoring indicate several linoleic and arachidonic acid.
Oxidized metabolites. 13 and nine, hold along with the corresponding 13 and nine Oxo ode are derived from linoleic acid. In addition, a arachidonic acid heat metabolites and their oxidized products are also present as expected.
There is a small shift in the retention time of the derated internal standard compared to the unlabeled standard. Importantly, this protocol also separates heat and anters and determines unique transitions for the stereo isomers that can result from enzymatic oxidation of a arachidonic acid or from reaction with reactive oxygen species. For example, this chromatogram shows the separation between the r and s stereo isomers of five.
Heat lipidomic analysis is a powerful technique for analyses of numerous lipid species. For example, this experiment evaluates and quantifies prostaglandins, isop senes, and leukotriene B four. In addition to the heats Oxo eats and omega hydroxylated metabolites, This method can be optimized for the extraction of bioactive lipids from other biological matrices such as plasma, urine, and tissue.
In some instances, bioactive lipids have been used as response biomarkers of oxidative stress.
