OBJECTIVE. Lipids are largely hydrocarbon derivatives and thus represent

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1 Paper 4. Biomolecules and their interactions Module 20: Saturated and unsaturated fatty acids, Nomenclature of fatty acids and Essential and non-essential fatty acids OBJECTIVE The main aim of this module is to introduce the students to the basic structure, nomenclature and functions of lipids. To understand as to how the hydrocarbon chain length can affect the properties of lipids. To understand the significance of presence of double bonds in the fatty acid chains. To discuss the significance of cis- and trans bonds in the fatty acid chains. To understand the difference between essential and non-essential fatty acids. To look into the pathways that lead to the synthesis of eicosanoids (prostaglandins, thromboxanes and leukotrienes etc) from arachidonic acid. To understand the biological roles of prostaglandins, thromboxanes and leukotrienes. To understand as to how the non-steroidal anti-inflammatory drugs (NSAIDs) are generated by targeting the pathway that is utilized for the generation of the eicosanoids. 1. INTRODUCTION Lipids, unlike nucleic acids, proteins and carbohydrates do not exist as polymers and a defining feature of these diverse groups of biomolecules is their insolubility in water. They impart structural stability to the biological membranes by forming aggregates. The only similarity amongst various lipids is their hydrophobicity as they can be very different structurally. The biological function of lipids is as varied as their structure. For example: a. Storage lipids: FATS and OILS b. Structural lipids: PHOSPHOLIPIDS and STEROLS (cholesterol and ergosterol) c. Other metabolic lipids: UBIQUINONE (isoprenoids or terpenoids), Vitamin A, K and E Lipids, in general perform the following functions: a. Lipids are important constituents of the biological membranes b. Lipids containing hydrocarbon chains serve as energy storage molecules c. Lipids can function as signaling molecules Lipids are largely hydrocarbon derivatives and thus represent a. most reduced form of carbon AND b. their oxidation, therefore yields high amounts of energy 1

2 2. LIPID CLASSSIFICATION a. Fatty acids b. Triacylglycerols c. Glycerophospholipids/phospholipids d. Sphingolipids e. Steroids such as cholesterol and ergosterol f. Others such as waxes, terpenes, eicosanoids etc 2.1 Properties of fatty acids The hydrocarbon chains in the fatty acid chains are determinants of their various properties, as fatty acids are considered carboxylic acids with long-chain hydrocarbon side groups. They rarely exist as free moieties and are usually found as esterified forms. The length of the hydrocarbon chain can range from 4-36 carbons long (C 4 to C 36 ). The chains in turn can a. be unbranched and saturated (with no double bonds) b. comprise of one or more double bonds and then are referred to as unsaturated fatty acids c. comprise of three carbon rings d. contain hydroxyl groups or e. be branched that are methylated 2.2 Conventions for naming fatty acids Figure 1 A. 18:1(Δ 9 ) cis-9-octadecenoic acid B. 20:5(Δ 5,8,11,14,17 ) Eicosapentaenoic acid (EPA)- an omega-3 fatty acid ω 2

3 The following conventions are followed for naming a fatty acid: 1. Standard nomenclature assigns no. 1 (C-1) to the carboxyl carbon (COOH). 2. The carbon adjacent to the carboxyl carbon is referred to as the α carbon. 3. Δ indicates the position of any double bond followed by a superscript number, which is almost always the lower number carbon in the double bond. 4. For polyunsaturated fatty acids (with one or more double bonds) (PUFA), the carbons are numbered, beginning from the methyl carbon at the opposite end of the chain. This carbon is also referred to as the ω carbon. The double bond positions are indicated then with respect to the ω carbon. For example, palmitic acid is also written as 16:0 while oleic acid as 18:1 (contains single double bond) 2.3. Saturated Versus unsaturated fatty acids The most common occurring fatty acids have even number of carbon atoms (C 12 -C 24 ). The even numbered fatty acids are synthesized by the concatenation of two carbon units (Table 1). Table 1: List common fatty acids 3

4 cis- Versus trans- configuration The location of double bonds also follows a common pattern: a. the double bond is between C-9 and C-10 in most monounsaturated fatty acids, while b. the bonds are generally between C-12 and C-15 in PUFAs except for arachidonic acid. c. The double bonds of PUFAs are never conjugated meaning that the double bond always alternates with a single bond such as CH=CH-CH=CH- and are separated by a methylene group such as CH=CH-CH 2 -CH=CH- All the double bonds invariably are always in cis configuration. Fermentation in the rumen of dairy animals results in the generation of trans fatty acids. Additionally, they are also obtained from dairy products and meat (Figure 2). Figure 2 cis-oleic acid Elaidic acid (trans-oleic acid) 4

5 Advantages of cis configuration are the following: the cis bond generates a kink in the fatty acid because of which the unsaturated fatty acids do not pack as tightly as the saturated fatty acids (imagine a box of chalks!). This provides the fluidity and flexibility to the biological membranes. the cis bond prevents free rotation around the C-C bond, thereby introducing a rigid bend in the hydrocarbon chain. Due to the above reasons, fatty acids with cis bonds have low melting points thereby keeping them as liquids at room temperature Disadvantages of trans configuration: trans bonds provide more linearity to the fatty acid structure because of which they stack together, have high melting points and remain solid at room temperature these are artificially created fatty acids and are generated through a process referred to as hydrogenation unsaturated fats are hydrogenated to increase their stability, thus increasing their shelf-lives. Hydrogenation adds hydrogen to a fatty acid and hence can convert a unsaturated fatty acid to a saturated one. This though is not the case with trans fatty acids as these are unsaturated fatty acids, which means that they still have double bonds in them. trans fats therefore are considered to be produced as a by-product of partial hydrogenation, which means that not all double bonds have been converted to single bonds by addition of hydrogen. Thus, any food item that mentions contains partially hydrogenated oils on its label actually contains trans fats as these are risk factors for cardiac diseases! Characteristics of saturated and unsaturated fatty acids More than half of the lipids in plants and animals contain unsaturated fatty acid residues. On the contrary, presence of unsaturated fatty acids is a rarity in bacteria. Instead they are branched, hydroxylated or have cyclopropane rings Saturated fatty acids Saturated fatty acids are highly flexible molecules as they are free to rotate around the C-C single bonds and assume a range of conformations. There is minimum steric interference 5

6 between neighboring methylene groups in these fatty acids as they are able to attain an extended, minimum energy conformation. The melting point of these fatty acids is directly proportional to their molecular mass or in other words it is directly proportional to the length of the hydrocarbon chain length (Table 1) Unsaturated fatty acids The length and degree of unsaturation are the major determinants of the physical properties of these fatty acids. The longer the fatty acyl chain and the lesser the number of double bonds, the lesser is the solubility in water. Carboxyl group comprises of the only polar group in these fatty acids, which actually accounts for their slight solubility in water. For example, lauric acid (12:0) has a solubility of 0.063mg/g, compared to a highly polar glucose molecule with a solubility of 1100mg/g. Melting points are influenced by a. length of the hydrocarbon chain AND b. degree of unsaturation Thus fatty acids with same number of carbon atoms but with different degrees of unsaturation will differ in their melting points. Lower the degree of saturation= lower the melting point Free fatty acids circulate in humans bound to serum albumin, which serves as the protein carrier for them. However, in the blood plasma these fatty acids majorly occur as derivatives of carboxylic acid or as amides. The absence of a free carboxyl group therefore accounts for the lower solubility of these esterified fatty acids. Amongst the family of PUFAs exist another class of fatty acids that have double bonds between the 3 rd and 4 th carbon from the methyl end of the chain. These fatty acids are important in humans as these cannot be synthesized by the humans. These fatty acids are referred to as Essential fatty acids (Figure 3). Therefore, ω-3 and ω-6 fatty acids are designated essential in humans. α-linolenic acid (ALA) is an ω-3 fatty acid which is essential and hence should be obtained from the diet. ALA then serves as a precursor for the synthesis of two other essential fatty acids: eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6). Figure 3 α-linoleic acid is a precursor for arachidonic acid (Figure 4). Figure 4 6

7 All eicosanoids (C 20 compounds) are derived from arachidonic acid. Arachidonic acid consists of four double bonds and therefore is classified as a polyunsaturated fatty acid. It is stored in the cell membranes in an ester form of phosphatidylinositol and other phospholipids. Phospholipase A 2 action results in the release of the fatty acid residue. Prostaglandins and related compounds like prostacyclins, thromboxanes, leukotrienes and lipoxins are collectively referred to as eicosanoids. Prostaglandins are compounds that contain a five-carbon ring, which comes from arachidonic acid. Prostaglandins can be divided into two groups (a) ether-soluble (PGE) and fosfat; phosphate-buffer soluble (PGF). Each group is further divided into many subtypes such as PGE 1, PGE 2, PGF 1 etc. Prostaglandins have diverse functions. Such as some of them are required for stimulating the contraction of the smooth muscle of the uterus during menstruation and labor. Some others have an effect on the flow of the blood to specific organs, while some of them elevate body temperature and cause inflammation and pain. Thromboxanes have a six-membered ring that contains ether. Thrombocytes/platelets are the main source of thromboxanes and function during clotting of blood and reduce the flow of blood to the clot. Leukotrienes are produced by the leukocytes, which contain three conjugated double bonds. Leukotrienes serve as potent biological signals. Such as leukotriene A4 is responsible for the contraction of the muscle that lines the airways to the lungs and overproduction of leukotrienes causes asthmatic attacks. Hence the anti-asthamatic drugs target the synthesis of leukotrienes. During anaphylactic shock, the strong contraction of the muscle lining the lungs is in fact an allergic reaction that individuals stung with bees or hypersensitive to penicillin undergo. Eicosanoids function at very low concentrations and have a role in generating fever and pain, in regulating blood pressure, blood coagulation and reproduction. They act locally unlike true hormones and are not transported through the blood stream Enzymes involved in the synthesis of the C 20 compounds are major drug targets of the non-steroidal anti-inflammatory drugs (NSAIDS) like aspirin, ibuprofen etc (Figure 5). 7

8 PGH 2 synthase has two isoforms: COX-1 and COX-2 of which aspirin and ibuprofen target COX-1 Figure 5 Aspirin acetylates a Ser residue of the prostaglandin H2 synthase, which prevents prostaglandin from binding to the active site of cyclooxygenase, thus inhibiting this enzyme. COX-1 and COX2- share a high degree of sequence identity. The expression of COX-1 is constitutive that helps in maintaining the basal levels of prostaglandins in all the tissues as opposed to COX2, which is expressed in response to inflammation in some tissues. Both aspirin and ibuprofen can cause side effects due to their non-specific mode of action. Therefore, drugs were designed to target the COX-2 active site as the active site of this enzyme is larger than COX-1. The size of the active site is the sole difference between COX_1 and COX-2. Inhibitors that target COX-2 are called coxibs such as rofecoxib and celecoxib. These drugs are used to treat inflammatory diseases like arthritis. The advantage of the coxibs is that they lack the side effects of the non-specific NSAIDs. Later though rofecoxib was found to have an effect on cardiac functioning and hence was withdrawn from the market. Daniel Simmons recently found that acetaminophen binds to the third COX isozyme, COX-3, while it binds poorly to the other two COXs. COX-3 is expressed largely in the central nervous system. It is targeted by drugs that are known to reduce pain and fever. 8

9 Summary In terms of fats you are what you eat! Lipids are soluble in organic solvents and compared to other biomolecules cannot form polymers, though they function by forming aggregates. Fatty acids consist of long hydrocarbon chains that could either be saturated (without double bonds) or unsaturated (with double bonds). Melting points of fatty acids depends on the hydrocarbon chain length and the degree of unsaturation. Essential fatty acids (omega-3 and -6) are PUFAs that cannot be synthesized by humans and hence should be obtained from the diet. ALA is an essential fatty acid, which is obtained from the diet and serves as a precursor for EPA and DHA, two other essential fatty acids. Arachidonic acid, a PUFA that serves as precursor for a class of C 20 compounds called eicosanoids. Eicosanoids serve as precursors for prostaglandins, leukotrienes, thromboxanes etc. 9