Extraction and Measurement of Total Lipids

In addition, to release lipids from source material, such as those in starch, fish meal, or milk, it might be necessary to treat the sample with an acid prior to lipid extraction (see Basic Protocol 4). In the case of milk, addition of ammonium hydroxide is necessary to dissolve casein prior to lipid extraction, which will release the lipids from its surrounding matrix (e.g., from the film surrounding the fat globules in milk). Furthermore, in certain cases, it is necessary to predry the sample in order to allow efficient and complete extraction of lipids. Particle size reduction is another factor that may improve lipid extraction efficacy. 

Accurate determination of lipids in foods is required for nutritional labeling, certification, or for evaluation of standard of identity and uniformity, as well as examination of their effects on functional and nutritional properties of foods. Following lipid extraction and precise quantitative analysis, lipids so obtained may be used for analysis of other lipid characteristics and properties provided that nondestructive and mild extraction procedures are employed that retain the integrity of lipids. Thus, determination of lipid classes, fatty acid composition (unit du), and oxidative state of lipids (Chapter D2), amongst others, may be pursued following the extraction process. 

CAUTION The solvents used in these protocols are flammable, treat accordingly. See appendix 2B for more information. 

IMPORTANT NOTE Use only ACS grade reagents and deionized water. 

SOLVENT EXTRACTION OF OILSEEDS, NUTMEG, AND OTHER FOODS USING THE SOXHLET METHOD 

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Dl.l Extraction and Measurement of Total Lipids Basic Protocol 1 Solvent Extraction of Oilseeds, Nutmeg, and Other Foods Dl.1.1... 

Takeuchi, Y. et al. Effects of oleic acid/propylene glycol on rat abdominal stratum comeum lipid extraction and appearance of propylene glycol in the dermis measured by Fourier transform infrared/attenuated total reflectance (FT-IR/ATR) spectroscopy. Chemical and Pharmaceutical Bulletin 4/(8) 1434-1437, 1993. 

Studying Angola green and roasted coffees (eight arabicas, 32 robustas, after dry or wet processing), Derbesy et al. (1969) and Roffi et al. (1973) measured for total lipids (ether extraction, 24h) 11.7-14% (d.b.) of fatty acids with 1.6-2.3% unsaponifiable in the arabicas, and 7.6-9.5% acids with 1.0-2.4%... 

To quantitate total lipids, defined as the sum of the free and bound lipids, both polar and nonpolar, acid hydrolysis may be necessary to release the bound lipids by dissociating lipid-starch and lipid-protein intermolecular forces. The resultant lipids may then be removed and measured however, the nonlipid components so obtained are not usable for further analysis. Removal of some of the polar lipids may hinder the use of the extracted material for further analysis. 

The enzyme DGAT has not been purified to date, probably because it is a hydrophobic and integral membrane protein. Therefore, DGAT activity was measured using rat liver microsomes as an enzyme source and radiolabeled palmitate as a substrate by the method of Mayorek and Bar-Tana [52] with some modifications [53], The reaction mixture contains microsomal protein, BSA, [14C]palmi-toyl-CoA, MgCl2, diisopropyl fluorophosphate, 1,2-dioleoyl-vw-glyccrol, and a test sample in a total volume of 0.2 ml. After a 15-min incubation at 23°C, lipids are extracted and separated by thin-layer chromatography (TLC). The distribution of radioactivity on TLC is analyzed with a radioscanner to determine the amount of [14C]TG. 

To investigate the specificity of DGAT inhibitors, synthesis of lipids including TG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) was measured in an intact cell assay using Raji cells by our established method [54], Raji cells are incubated in the presence of [14C]oleic acid with or without a test sample in a total volume of 0.2 ml. After a 20-min incubation at 37°C, [14C]oleic acid is incorporated mainly into PC, PE, and TG, which are extracted and separated by TLC. The distribution of radioactivity in these lipids on TLC is analyzed. 

Another approach for the direct measurement of SFs from total lipid extracts was presented by Lilitchan et ah (2008), who developed a method applying partial extraction for the analysis of SFs and total lipids from rice bran. In this method an adsorption coefficient (X j) is... 

Quantitative measurement of phospholipids is rare in routine clinical practice but more common in research (e.g., in studies of dietary influences). The choline-containing phospholipids lecithin, lysolecithin, and sphingomyelin, which account for at least 95% of total phospholipids in serum, are readily measured by an enzymatic reaction sequence using phospholipase D, choline-oxidase, and horseradish peroxidase. Kit methods with this enzymatic sequence are available commercially. Before the availability of enzymatic reagents, the common quantitative method involved extraction and acid digestion with analysis of the total lipid-bound phosphorus. ... 

Antonis (A3) has estimated phospholipids by a procedure for determining fatty acids. This technique requires a total serum extract and a phospholipid-free extract for the measurement of both total and free fatty acids, the difference between them being a measure of the phospholipid content. Free fatty acids (A4) are determined on a phospholipid-free extract by a procedure based on partitioning the fatty acids as copper soaps into chloroform, and subsequent photometric determination of the copper with diethyldithiocarbamate. Phospholipids, as well as the free fatty acids present in the total lipid-extract, are measured by the same method, since they also form a complex with copper that is soluble in chloroform. A criticism of this technique is that equal response is not given by dipalmitoyl lecithin, dipalmitoyl cephalin, or beef brain sphingomyelin. 

Although representing complex mixtures of neutral, polar, free, and bound lipid forms, the total lipid content of a sample is usually measured by simple gravimetric techniques whereby residues are extracted with organic solvents and the solvent-free extracts determined by weighing. ... 

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