When plasma proteins, including the apolipoproteins, must be preserved from denaturation during the extraction of lipids a specific solvent system must be used (Cham BE et al., J Lipid Res 1976, 17, 176).
The most common procedure used for delipidation of plasma, protein solutions or cell culture medium involves the extraction of all kinds of lipids with a mixture of butanol and di-isopropyl ether. The proteins remain in solution in the aqueous phase, while the organic phase contains the dissolved lipids.
Procedure : One volume of serum or plasma containing 0.1 mg/ml of ethylenediamine tetraacetate (EDTA) is added to 2 volumes of a mixture of butanol/di-isopropyl ether (40/60, v/v). The vials are tightly closed and fastened on a mechanical rotator providing end-over-end rotation at about 30 rpm for 0.5 h.
After extraction, the mixture is centrifuged at low speed (2000 rpm) for 2 min to separate the aqueous and organic phase. The aqueous phase containing the delipidated proteins is removed by careful suction with needle and pump or syringe.
Traces of butanol remaining in the aqueous solution may be removed if necessary by washing that phase with 2 volumes of di-isopropyl ether. Residual solvent may be removed by an extraction with a water pump aspirator at 37°C for some minutes.
When proteins from plant oilseeds are studied by electrophoresis, lipids must be removed to prevent important interferences and thus to obtain good resolution. It was demonstrated that, in the presence of chloroform methanol, lipid contaminants can be thoroughly removed by the combination of two precipitation steps (10% TCA/acetone and acetone) and aqueous TCA wash steps (Wang W et al., Anal Biochem 2004, 329, 139).
A reliable extraction procedure was described by Dole (J Clin Invest 1956, 37, 350) for the extraction of free fatty acids and non polar acylglycerols.
To 1 ml plasma (or aqueous solution) add 5 ml of Dole reagent (40 ml isopropanol, 10 ml heptane and 1 ml 1M H2SO4), 3 ml heptane and 2 ml water.
After vortexing and centrifugation, the upper phase is collected. Residual phospholipids may be removed by the addition of 200 mg silica gel (vortex and centrifuge) before evaporation under nitrogen.