Washed RBCs need to be stored for at least 2 months. Washing solutions differ. Is the addition of BHT critical at this stage or if liquid nitrogen processing is used, can it be omitted? Then, would the addition of BHT still be required during the solvent extraction stage?
No experience with liquid nitrigen. I usually add BHT during storage and then again in the solvent but all in reduced amounts as it will interfere, under my chromatographic conditions, with the separation of C14:0.
Steck, TL and Kant, JA (1974). Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods in Enzymology 31 (Part A): 172-180.
Burton, GW, Ingold, KU, and Thompson, KE (1981). An improved procedure for the isolation of ghosts membranes from human red blood cells. Lipids 16: 946.
For storage for 2 months, you don't require BHT if storing at -80C or colder (LN2), but it doesn't hurt and can prep and run samples with it present - just identify the BHT peaks (and break down products peaks - if using an agressive transesterification protocol) on chromatogram. Reed et al. J. Lab. & Clin Med 1960 is often used, but others use Rose and Oaklander J Lipid Res 1965. As far as prepping the cells, people do analyze them as collected (plasma contaminanety has minor effect on final compositiion), others do a saline wash, while others prepare ghost membranes.
Is there some reason why the cell have to be stored? Perhaps processing fresh and storing the FAME would be better. If you are only interested in total FAME and not FAME within lipid classes a direct methylation of red cells could work. We use Rise et al 2005. A trick is that when adding the methanolic HCl, the cells tend to clump, and it is best to add reagent while vortexing.
Risé, P., Salvetti, F., Galli, C. Application of a direct transmethylation method to the analysis of fatty acid profile in circulating and cultured cells. Analytical Biochemistry (2005) 346:182–184.
Well the objective is to do population profiling for many parameters and it makes sense to hold samples until lab time is available. We are aiming for total FAME and worried about the quality of the chromatogram.
We have analysed red blood cells using the one-step extraction and methylation procedure of Lepage and Roy following washing with saline.
Clayton, E. H., Gulliver, C. E., Piltz, J. W., Taylor, R. D., Blake, R. J. and Meyer, R. J. (2012). Improved extraction of saturated fatty acids but not omega-3 fatty acids from sheep red blood cells using a one-step extraction procedure. Lipids 47 (7): 719-727.
Hi,
In FAME studies, adding BHT antioxydant is commonly recommended during storage and/or extraction in organic solvent to prevent lipid degradation. I'm used to working with BHT at the rate of 0,1% (w/v) in solvent, and to following Lepage & Roy (1984) methodology for direct esterification (Edward's comment).
As mentioned by Susana and Ken, BHT peak(s) have to be previously identified on chromatograms in order to discriminate it from "true" FA peaks.
Hope this helps.
It is interesting the Lepage&Roy and the recent Clayton et al direct methylation yield more FA, particulaly saturates then Folch extraction followed by methylation. The direct methylation of Rise et al yields comparable amounts of FA versus Folch extraction followed by methylation. Perhaps keys may be that the Folch extraction may be less effcient at extrating saturate rich sphingomyelin, and the slightly harsher methylation conditions of Lepage&Roy are boosing the saturates (i.e. from sphingomyelin which is difficult to methylate).
The analysis is very tricky. Oxidation is common and changes the FA profile.
working with BHT at the rate of 0,1% (w/v) in solvent, and to following Lepage & Roy (1984) methodology for direct esterification (see above). Then one has derivatives of BHT and cholesterol (among others). Depending on the column and method used, they are likely to elute superimposed with other FAME.