ESTIMATION OF TOTAL LIPIDS
Estimation of total lipid amount in
a biological sample may be done by gravimetry or by colorimetry.
Several macro- and micro-gravimetric method have been proposed using various solvent extraction mixtures.
Large samples : For samples from 0.5 to 5 g, the Bligh and Dyer procedure may be used.
Briefly, 5 g aliquots (preferably wet weight) are homogenized (2 min with a polytron homogenizer) with 5ml of chloroform and 5ml of methanol. Five ml of chloroform is added and the mixture homogenized another 30 s. Five ml of water is added to this mixture, and the sample homogenized again for 30 s. The mixture is then allowed to separate, the lower solvent phase removed and passed through a Whatman #1 filter paper, the filtrate is saved in a labeled vial. Another 5ml of chloroform is added to the remaining pellet and aqueous phase, and homogenized another 2 min. The resultant mixture is added to the previous filtrate by passing it through the Whatman #1 filter paper. The filtrate is allowed to separate in graduated cylinder, and the volume of the lower chloroform layer is recorded. The lipids are then gravimetrically determined by placing 0.5 ml aliquots of the chloroform layer into pre-weighed aluminum pans (three pans per sample), allowing the samples to evaporate in a hood overnight, recording the weights, then converting to percent lipids.
The Folch extraction method may also be used before gravimetry measurement, an efficient and simple modification was evaluated for measuring total lipids in small fish (Lu Y et al., Can J Fish Aquat Sci 2008, 65, 2233).
Small samples : If the estimation of total lipids is not a serious problem with large samples, specific method are required for small sample size (up to 100 mg). The method adapted from that of Van Handel (E. Van Handel, J Am Mosq Control Assoc 1985, 1, 302) for single mosquitoes was further miniaturized to minimize the use of reagents. Thus, 10–50 mg are homogenized with 4ml of a chloroform/methanol mixture (1/1, v/v) in a handheld ground glass homogenizing tube. The homogenate was centrifuged at 3000 rpm for 5 min, and the supernatant volume recorded. An aliquot (0.25 ml) of the supernatant was transferred to a glass tube, being careful to deposit the sample directly at the bottom to the tube (Inouye LS et al., Talanta 2006, 70, 584).
Colorimetric methods are mainly used to estimate lipid content in very small samples (some mg) or in small aliquots of large samples.
A micro-colorimetric assay have been developed for a single mosquito, and should be reliable for other small tissue samples (E. Van Handel, J Am Mosq Control Assoc 1985, 1, 302).
Briefly, lipid samples and standards are placed in a heating block set at 100°C to allow the solvent to evaporate. Once the solvent is gone (about 10 min), 0.1 ml of concentrated sulfuric acid is added to each tube, vortexed, and then heated at 100°C for 10 min. Samples are then removed from the heat block and allowed to cool to room temperature before adding 2.4 ml of vanillin reagent (600 mg of vanillin, 100 ml of hot water, 400 ml of 85% phosphoric acid) and vortexed. The pink color is allowed to develop for 5 min, and then 0.2 ml of standards and samples are read at 490 nm. A standard spectrophotometer can be used, but a 96-well spectrophotometer decreases analysis time and increases accuracy by reading of standards at virtually the same time as the samples. Since the color continues to develop over time, the standards should be re-read at 5 min intervals if a standard spectophotometer is used.
An accurate adaptation of the micro-colorimetric method was described using quantification of lipids in a 96-well microplate enabling higher throughput and reduced costs (Cheng YS et al., Lipids 2001, 46, 95).
When compared with two other approaches, the reported procedure was shown to be the most reliable (Lu Y et al., Lu Y et al., Can J Fish Aquat Sci 2008, 65, 2233).
A method for the determination of the total lipid content in fish meat was established using a 2-thiobarbituric acid (TBA) reaction, which had previously been used for the determination of lipid peroxides in animal tissues (Matsushita T et al., JAOCS 2010, 87, 963). In this method, an unspecific peroxidation of fish oils was created by omitting the addition of antioxidant to the reaction mixture during the TBA reaction. The assay procedures in was optimized and a new standard procedure was recommended for determining the total lipid content of fish using a TBA reaction.