The overall goal of this procedure is to determine how either a xenobiotic treatment or a disease state alters cellular metabolism. This method has the potential to reveal how a disease state or an applied toxin affects cellular metabolism. And once this is known, our method has the ability to reveal whether some kind of intervention can correct the defect.
The main advantage of this technique is it doesn't rely on transformed cell lines, so it's more likely to provide answers that are directly relevant to human health. To begin this procedure prepare a 10 millimolar stock solution of rotenone in dimethyl sulfoxide. Perform a serial dilution of the 10 millimolar rotenone stock solution to obtain solutions with rotenone concentrations ranging from one nanomolar to 100 micromolar.
Next, add 50 microliters of each stock solution to five milliliters of a previously-prepared enriched Tyrode's buffer solution to prepare solutions with rotenone concentrations ranging from 10 picomolar to one micromolar. Add 50 microliters of DMSO to one of the buffer solution samples to serve as a vehicle control. To isolate platelets from whole blood, centrifuge whole blood samples at 175 x g for 15 minutes with no breaks.
When finished, transfer one milliliter of the upper platelet-rich plasma layer to a 1.5 milliliter microcentrifuge tube. When transferring the platelet-rich plasma, be careful not to disturb the buffy coat so the platelet pellet is not contaminated by lymphocytes. Centrifuge the platelet-rich plasma layer at 400 x g for five minutes.
Then, aspirate the supernatent. Using no fewer than three biological replicates for each condition, resuspend the platelet pellet in one milliliter of each previously-prepared rotenone solution. Following this, incubate the resuspended platelets in a water-jacketed carbon dioxide incubator set to 95%humidity and 5%carbon dioxide at 37 degrees Celsius for one hour.
At this point, pellet the platelets by centrifuging the samples at 3000 x g for three minutes. After aspirating the supernatent, add the appropriate stable isotope-labelled internal standard. Then, resuspend the platelets in 750 microliters of ice-cold 10%trichloroacetic acid.
Pulse sonicate each sample 30 times with 0.5 second pulses. Following centrifugation, affix C18 solid phase extraction columns to a vacuum manifold. Condition the columns with one milliliter of methanol.
Then, equilibrate the columns with one milliliter of double-distilled water. Run the sample-derived supernatent through the columns. When finished, wash the columns with one milliliter of water.
Next, load 10 milliliter glass centrifuge tubes into the vacuum manifold to collect the elution fractions. Elute the columns with one milliliter of 25 millimolar ammonium acetate and methanol. After drying the eluate under nitrogen gas, resuspend the dried residues in 50 microliters of 5%5-sulfosalicyclic acid and transfer the samples to HPLC files for analysis.
As previously reported in SH-SY5Y cells, changes in relative levels of aceyl CoA thioesters in response to rotenone are hypothesized to result from the inhibition of complex one. Specifically, there is a dose-dependent decrease in succinyl CoA with a simultaneous increase in beta-Hydroxybutyryl-CoA, while acetyl CoA levels were unchanged. The analysis of human platelets treated with rotenone reveals highly consistent results and provides a unique set of experimental data.
It is important to note that this observation underscores the mitochondrial dependence of the response to rotenone because platelets lack nuclei. Following this procedure, other methods like metabolic tracing can be performed in order to answer additional questions, such as:How does this treatment or disease state affect substrate utilization? If we identify changes in the levels of some metabolite, why is it changing?
Which steps in the pathway are affected, and in which way? After watching this video, you should have a good understanding of how to isolate platelets from whole blood, perform the controlled ex-vivo challenge and extract the aceyl CoA thioesters for LCMS anaylsis.
