Thursday, December 8, 2011

Measurement of Fatty Acids

Erythrocyte fatty acid concentrations were identified using gas-liquid chromatography. Samples were analyzed at the University of Pittsburgh's Heinz Laboratory Total lipids (500pl of packed red blood cells) were extracted according to the general technique of Bligh and Dyer. Briefly, the samples were homogenized in 4 ml of methanol, 2 ml of chloroform and 1.1 ml of water. Two ml of chloroform and 2 ml of water were added to the samples after 15 min. The tubes were then centrifuged at 1200 g for 30 min at 16°C and the upper phase discarded. The lower phase was dried under nitrogen and resuspended in 1.5 ml 14% boron trifluoride methanol. The samples were heated at 90°C for 40 min and after cooling extracted with 4.0 ml pentane and 1.5 ml water. The mixtures were vonexed and the organic phase recovered. The extracts were dried under nitrogen, resuspended in 50 pi heptane and 2 ml injected into a capillary column (SP-2380, 105 m x 53 mm ID. 0.20 um film thickness). Gas chromatographic analyses were carried out on a Perkin Elmer Clarus 500 equipped with a (lame ionization detector Operating conditions were as follows the oven temperatures were 140C for 35 min; 8°C/min to 220°C, held for 12 min; injector and detector temperatures were both at 260°C; and helium, the carrier gas, was at 15 psi. Identification of fatty acids was by comparison of retention times with those of authentic standards (Sigma). A random subset of 27 samples was analyzed for reproducibility; laboratory personnel were blinded to duplicate samples and subject identification. The inter-assay coefficients of variation (CV) for the fatty acid measures reported ranged between 1.7-15.2%. CVs's were 4.6% for LA, 3.4% for AA, and 1.7% for total n-6 fatty acids. CV's were higher for the n-3 fatty acids, with CV's of 15.2% for ALA. 5.3% for EPA, 7.5% for DHA and 5.3% for total n-3 fatty acids. The CVs for the total n-6:n-3, LAALA. AA:EPA, and AAEPA+DIIA ratios were 5.2%, 111%, -4.5% and 5.7% respectively. The individual and total n-6 and n-3 fatty acids are expressed as a percentage by weight of the total erythrocyte fatty acid content.

Fats and Oils Are Hydrophobic

Fats and oils are hydrophobic Chemically, fats and oils are triglycerides, also known as simple lipids. Triglycerides that are solid at room temperature (around 20'C) are called fata; those that are liquid at room temperature are called oils. Triglycerides are composed of two types of building blocks: fatty acids and glycerol. Glycerol is a small molecule with three hydroxyl (—OH) groups (thus it is an alcohol). A fatty add is made up of a long nonpolar hydrocarbon chain and a polar carboxyl group (—COOH). These chains are very hydrophobic, with their abundant C—H and C—C bonds, which have low electronegativity and are nonpolar. A triglyceride contains three fatty acid molecules and one molecule of glycerol. Synthesis of a triglyceride involves three condensation (dehydration) reactions. In each reaction, the carboxyl group of a fatty acid bonds with a hydroxyl group of glycerol, resulting in a covalent bond called an ester linkage and the release of a water molecule. The three fatty acids in a triglyceride molecule need not all have the same hydrocarbon chain length or structure; some may be saturated fatty acids, while others may be unsaturated:

  • In saturated fatty acids, all the bonds between the carbon atoms in the hydrocarbon chain and single bonds—there are no double Kinds. That is, all the bonds are saturated with hydrogen atoms. These fatty add molecules an? relatively rigid and straight, and they pack together tightly, like pencils in a box.
  • In unsaturated fatty acids, the hydrocarbon chain contains one or more double bonds, Linoleic acid is an example of a polyunsaturated fatty acid that has two double bonds near the middle of the hydrocarbon chain, which causes kinks in the molecule. Such kinks prevent the unsaturated fat molecules from packing together tightly. The kinks in fatty acid molecules are important in determining the fluidity and melting point of a lipid. The triglycerides of animal fats tend to have many long-chain saturated fatty acids, packed tightly together; these fats are usually solids at room temperature and have a high melting point. The triglycerides of plants, such as corn oil, tend to have short or unsaturated fatty acids. Because of their kinks, these fatty acids pack together poorly and have a low melting point, and these triglycerides are usually liquids at room temperature. Fats arc excellent storehouses for chemical energy. When the C—H bond is broken, it releases significant energy that an organism can use for its own purposes, such as movement or building up complex molecules. On a per weight basis, broken-down fats yield more than twice as much energy as do degraded carbohydrates.