Dear colleagues,
i am currently trying to oxidize Triacylglycerides (TG) in vitro by the fenton reaction to obtain standards that i want to use in later analysis of biological samples. I face the problem that TGs are quite hydrophobic so i need to use an organic solvent system. But i found that most organic solvents (MeOH, MeCN,CHCl3,...) have adverse effects on the kinetics of the fenton reaction. Do you have any experience with the oxidation of organic substrates in organic solvents by the fenton reaction? or can you help me with finding the right conditions/solvents for my reaction?
thanks in advance
regards Mike
i want to mimic lipid peroxidation that is induced by the hydroxyl radical (OH.) in vivo via the fenton reaction. This usually yields lipid hydroperoxides, hydroxides, epoxdies and a number of cyclic structures like furanes and prostanes. so it is pretty important to generate hydroxyl radicals in the first place
One option would be to use tert-butylhydroperoxide and UV light, but you are also going to get some additional photochemistry on the oxidation products. If you want to use Fenton, you can switch to fatty acid micelles, such as linoleic acid or some other PUFA in neutral to basic aqueous buffer. This also has the advantage that you don't need to do a hydrolysis later, just extract the fatty acids lowering the pH and using chloroform-methanol.
to form micelles was actually my initial plan. I wanted to synthesize micelles of phospholipids that contain TGs in their hydrophobic core and since i am studying lipid peroxidation in lipid droplets (micelles containing TGs) of adipocytes this would have been perfect. I just couldnt find a protocol on how to generate these specific micelles. Do you know how to do it or do you have any publications concerning their generation? the point of this experiment is to find oxidized TG species so the hydrolysis is unfortunately also not an option for me.
I can think of two options: the quick and dirty one, just make an emulsion with the triglycerides in water, add the oxidants and hope to find something. It has the problem that you don't know the size of the aggregates and the oxidants (especially *OH) will not penetrate, so you probably will not get much oxidation.
The other option would be to make those micelles you want, that you can find a protocol looking for synthetic lipoproteins, such as this one I just found that looks reasonable>
http://www2.lbl.gov/Science-Articles/Archive/sabl/2006/Oct/Nano-LDLs.pdf
without the peptides, of course.
Apparently you can generate micelles of a defined size, small size that is good for the oxidation. If the phospholipids bother you later, you can precipitate them with acetone, that should not precipitatte the TGs.
Great that is exactly what i was looking for. Thanks a lot. I will give you some feedback how it worked as soon as i've done it.
Best Regards Mike
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