Sample preparation for lipid extraction

• Sampling & Homogenisation
• Storage of extracts

Sampling & Homogenisation

All tissues must be sampled freshly before lipid extraction to prevent any hydrolysis or oxidation before its dispersion in the solvent mixture. The presence of lysophospholipids, phosphatidic acid, monoacylglycerols, diacylglycerols or free fatty acids should indicate a possible degradation before extraction. In some special cases, special precautions are necessary to minimize rapid degradation of lipid constituents. Thus, plant tissues need to be processed immediately after collection by immersion in hot 2-propanol or water. Plant phospholipase D is known to be active even in some solvents !!
Any tissue which cannot be extracted immediately should be frozen as rapidly as possible in dry ice or better in liquid nitrogen and stored in sealed glass containers at -70°C.

Fresh or frozen tissues should be homogenized in the chosen solvent mixture and agitated during a fixed time before elimination of the solid part. Soft tissues may be minced with scissors in the cold and homogenized in solvent with an Ultraturax type device, a Warring blender or a glass-Teflon Potter. Hard tissues (muscle, vessels, bone...) are best pulverized in a mortar filled with liquid nitrogen.

Storage of extracts

Lipid extracts should not be stored in the dry state. Oxidative degradation is slower in solutions even in the absence of added antioxidants. This protection depends on the chemical nature of the solvent and the physical conditions of storage. Air and light should be avoided. The best storage conditions are a super freezer (-70°C) to store for a long time (up to one year) lipid extracts in chloroform-methanol mixtures filling well stoppered glass vials (with Teflon liners), the cap being secured with a wide length of self-sticking tape. For long period of storage, it can be useful to flush vials or tubes with nitrogen before closing to prevent fatty acid oxidation. For the same purpose, a low amount of antioxidant such as BHT (butylated hydroxytoluene or 2,6-di-tert-butyl-4-methoxyphenol) or ethyl gallate (i.e. about 50 µg BHT/ml) may be added in the solvent if the natural antioxidant amount is estimated too low (purified extracts). The antioxidant is used as a concentrated solution in ethanol (10 mg/ml).
After long periods of storage, the cleavage of ester lipids and plasmalogens must be expected with a concomitant rise in free fatty acids, diacylglycerols methyl or ethyl esters. These problems can be minimized by using neutral extracts and 2-propanol instead of methanol or ethanol in the solvent mixture. It must be remembered that best results are obtained with freshly prepared materials.