I'm looking to titrate total brain lipid extracts with hydrogen peroxide. Then under fluid AFM introduce amyloid-beta to determine changes in aggregation profiles with respect to the extent of oxidation. I would like to determine the extent of oxidation at least prior to peptide introduction and post 48hrs but have been unable to find a sufficient method that would allow me to do so. FTIR and MS seem to be the most viable but have their limitations, not to mention TBLE's content is 56% unknown. If I could find a method that would show even a change from 10% lipid oxidation to 12% lipid oxidation this I feel would be suitable.
UV/VIs at 234 nm
http://www.avantilipids.com/index.php?option=com_content&view=article&id=1694&Itemid=413
The 56% number is not totally unknown. They are known to be non-phospholipids:
∼20% cholesterol, ∼10% sphingomyelin as well as ∼20% ganglioside and less than 10% ceramide
from http://www.sciencedirect.com/science/article/pii/S0928098708001759
Numbers for individual batches may vary
I'm not sure a change from 10% lipid oxidation to 12% lipid oxidation will make a statistically significant difference considering the other variables
Yeah UV vis at 234nm isn't going to work very well. With TBLEs content being what it is I will have no idea about what is actually being oxidized considering 234nm is monitoring the conjugated dienes. I think after much reading that MS or IMS is gonna be my best choice. By settings up a profile for the unoxidezed TBLE and observing shifts in spectra accordingly with oxidation. It won't be exact either but would provide a result I would feel much more confident about
You may want to look at a series of papers commissioned by NIEHS to assess the best markers of oxidative stress. They did four double blinded analysis of peripheral stress, central stress and inflammation. All suggest that the non enzymatic oxidation of arachidonic acid into F2tIsoprostanes was the most senstive and reproducible.
http://www.niehs.nih.gov/research/resources/databases/bosstudy/
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