Lipid Reading Module PDF

Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Lipids Introduction Lipids are defined as heterogeneous organic compounds that are in...

Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Lipids Introduction Lipids are defined as heterogeneous organic compounds that are insoluble in water, but soluble in organic solvents such as diethyl ether, chloroform, methylene chloride, and acetone. They include fats, waxes, oils, hormones, and certain components of membranes, and function as energy-storage molecules and chemical messengers. Fats which are a major form of stored energy in biological systems, generate lots of energy compared to carbohydrates when completely oxidized. They occur frequently in nature, and are found in the food we eat, like eggs, butter, meat and dairy products. Learning Objectives The student must be able to: 1. Relate the structures and properties to the functions of lipids. 2. Relate membrane structure to its function. 3. Discuss transport mechanisms in membranes 4. Observe osmotic changes in red blood cells 5. Calculate rate of osmosis using dialysis bags. 6. Perform isolation of lipids from egg yolk 7. Interpret results of lipid qualitative tests 8. Determine acid, ester, and iodine values and saponification number of oils *Objectives 4-8 are Laboratory Objectives Reference Textbooks 1. Campbell, M.K. & Farrell, S.O. (2012). Biochemistry. (7th ed). Belmont, CA, USA: Brooks/Cole Cengage Learning. 2. Voet, D. & Voet, J. (2011). Biochemistry. (4th ed). United States of America: John Wiley & Sons, Inc. 3. Berg, J.M., Tymoczko, J.L., & Stryer, L. (2012). Biochemistry. (7th ed). New York, USA: W.H. Freeman and Company. 4. Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed) Supplemental Readings and Videos 1. Lipids: http://people.uleth.ca/~steven.mosimann/bchm2000/Bchm2000_L11.pdf 2. Lipoproteins: 1. https://www.slideshare.net/LoaloaaBadrawy/lipoproteins-47366441 2. https://www.slideshare.net/namarta28/lipoproteins-structure-classification- metabolism-and-clinical-significance 3. Eicosanoids: https://www.slideshare.net/namarta28/eicosanoids-power-point- presentaion 4. Prostaglandins: https://www.slideshare.net/UDDent/prostaglandinsbioch212pptx 5. Prostaglandins, Leukotrienes and Platelet Activating factor: https://www.slideshare.net/ drdhriti/eicosanoids-5575018446. Page 1 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila I. Classification of Lipids According to their Chemical Nature 1. Lipids consisting of open-chain compounds with polar head groups and long nonpolar tails: fatty acids, triacylglycerols, sphingolipids, phosphoacylglycerols, glycolipids. Lipid-soluble vitamins (A, D, E, K), prostaglandins, thromboxanes, and leukotrienes 2. Lipids consisting of fused- ring compounds: steroids (e.g. cholesterol, steroid hormones, mineralocorticoids, and bile acids) Lipids are also classified into: 1. Simple lipids – esters of fatty acids with various alcohols A. Fats - esters of fatty acids with glycerol B. Waxes – esters of fatty acids with high molecular weight monohydric alcohols 2. Complex lipids – esters of fatty acids containing group in addition to an alcohol and a fatty acid. A. Phospholipids- glycerophospholipids, spingophospholipids B. Glycolipids C. Plasmalogens D. Cardiolipins 3. Derived lipids: fatty acids, glycerol, steroids/sterols, fat soluble vitamins. Fatty Acids Fatty acids are the building blocks of many important lipids. They are carboxylic acids with a long aliphatic chain, which is either saturated (containing no double bond) or unsaturated (containing one or more double bonds). Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, from 4 to 28. They are amphipathic compounds because the carboxyl group is hydrophilic and the hydrocarbon tail is hydrophobic. The carboxyl group is negatively charged at physiological pH. Fatty acids consist of a hydrocarbon chain with a carboxylic acid at one end. A 16-C fatty acid: CH3(CH2)14-COOH Non-polar polar A 16-C fatty acid with one cis double bond between C atoms 9-10 may be represented as 16:1 cis ∆9. The degree of unsaturation refers to the number of double bonds (given by the number after the colon in the notation for fatty acids). The superscript indicates the position of double bonds. For example, ∆9 refers to a double bond at the ninth carbon atom from the carboxyl end of the molecule. The ∆, means the double bond is in the “cis” conformation. Fatty acids occur mainly as esters in natural fats and oils. However, they occur in the unesterified form as free fatty acids, a transport form found in the plasma. Fatty acids are required for the formation of membrane lipids such as phospholipids and glycolipids. Like carbohydrates, they act as fuel molecules and are oxidized to produce energy. Page 2 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Saturated and Unsaturated Fatty Acids: Conformation and Properties Both the length of the hydrocarbon chain and the number of double bonds have an effect on the properties of fatty acids and their compounds. In unsaturated fatty acid, the double bond is usually cis rather than trans, and this affects its overall shape. A cis double bond puts a kink in the long-chain hydrocarbon tail, resulting in the bent shape, different from the fully extended shape of the trans fatty acid and saturated fatty acid. The shape, in turn affects the extent of interaction of fatty acid molecules. The space between the cis unsaturated fatty acids weakens their intermolecular interactions, and thus lower the melting point. The greater the degree of unsaturation, the lower the melting point. The shapes of the saturated and unsaturated fatty acids are compared below: Figure 1. Structures of saturated and unsaturated fatty acids. Unsaturated fatty acids can be cis- or trans-conformation. A B C D Figure 2. Comparison of the conformations of stearic acid and oleic acid. In (A), the saturated chain of stearic acid tends to adopt extended conformations; in (B), the double bond in oleic creates a kink in the chain; in (C) , the saturated fatty acids are closely packed; in (D), the kinked oleic acid molecules are not closely packed. Space-filling models were created using molview.org. Page 3 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila The effect of the presence of double bond on the conformation and extent of interactions among fatty acid chains is further illustrated above using stearic acid (C18:0) and oleic acid (C18:1∆9. The double bond (red) in oleic acid restricts rotation and introduces a rigid bend in the hydrocarbon chain. Because of the different shapes, there is a big difference in the packing of molecules of stearic acid and oleic acid. The oleic acid molecules are not closely packed, and this explains why oleic acid is liquid at room temperature. The liquid state at room temperature is due to the low melting point of oleic acid, as a result of the weak interactions among its molecules. In contrast, fully saturated fatty acids in the extended form pack into nearly crystalline arrays, and stabilized by many hydrophobic interactions. See Figure 2 for clarity. Plant oils are liquid at room temperature because they have higher proportions of unsaturated fatty acids than do animal fats, which tend to be solids. Below is a comparison of the properties of saturated and unsaturated fatty acids: Table 1. Comparison of saturated and unsaturated fatty acids. Properties Saturated Fatty Acid Unsaturated Fatty Acid Packing of molecules Packed in a fairly orderly way Packed less orderly due to kinks Attractions between chains Strong Weak Melting poing High Low Physical state at room temperature Solid Liquid Unsaturated fatty acids are very reactive substances due to the presence of double bonds. They are easily hydrogenated, halogenated and oxidized in cells (in vivo) and in vitro. Some fatty acids can also undergo isomerization and polymerization reactions. Polyunsaturated fatty acids are also susceptible to peroxidation and react readily with molecular oxygen. This results in the development of rancidity or undesirable flavors and color in food, lowering its nutritive value. It may also lead to formation of toxic products. Peroxidation may be catalyzed by visible or UV light as well as metal ions or enzymes. Oxidation, however, can be prevented by the use of antioxidants such as vitamin A, C and E. Conversion of oils to fats by the process of hydrogenation, is a commercially important process. Hydrogenation is a process of adding hydrogen across the double bond of unsaturated fatty acids to produce the saturated counterpart (called trans fatty acid). Fats are preferred than oils in food preparations, not only because of their good taste but also of their stability. However, too much intake of fat is not good for the heart. Usually when oils are used in food, vitamin E, an anti-oxidant, is added to prevent oxidation of double bonds. Two Conventions for Naming Fatty Acids: A. Alpha (⍺) nomenclature: based on position and type of double bonds relative to the carboxylic end of the fatty acids B. Omega (ω) nomenclature: based on where the first double bond is located relative to the methyl (ω) end Page 4 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila In the alpha nomenclature, the number 1 is assigned to the carboxyl carbon (C-1), and ⍺ to the carbon next to it. Each line segment of the zigzag represents a single bond between adjacent carbons. The position of any double bond(s) is indicated by Δ followed by a superscript number indicating the lower-numbered carbon in the double bond. In the omega nomenclature for polyunsaturated fatty acids (PUFAs), carbons are numbered in the opposite direction, assigning the number 1 to the methyl carbon at the other end of the chain, designated as ω (omega; the last letter in the Greek alphabet) end. The positions of the double bonds are indicated relative to the ω carbon. Studies show that imbalance in ω-3 and ω-6 fatty acids in the diet is implicated to the increased risk of cardiovascular diseases. In Eskimo tribes, very little heart disease is diagnosed even though people in these groups eat high-fat diets and have high levels of blood cholesterol. Analysis of their diet showed the presence of highly unsaturated fatty acids found in the oils of fish which they ordinarily eat. One class of these fatty acids is called omega-3 (ω-3), an example of which is eicosapentenoic acid (EPA). The omega-3 fatty acids can also be found in soybean, canola, walnut, salmon, tuna and mackerel; the omega-6 fatty acids in vegetable oils. Learn the conventions for naming fatty acids from the examples below: Figure 3. Comparison of the ⍺- and ω-naming conventions. Omega-3 fatty acids means first double bond is between the 3rd and 4th carbons from the ω-end. Omega-6 fatty acids means first double bond is between the 6th and 7th carbons from the ω-end. Common Biological Fatty Acids Below is a table of the common biological fatty acids, their symbols, names, and melting points. Some of these fatty acids are not synthesize in the body, hence must be provided in the diet. They are classified as essential fatty acids. Examples of these are linoleic, linolenic, arachidonic acids and other polyunsaturated fatty acids. Page 5 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Table 2. Characteristics of the common biological fatty acids. Melting points were adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed) Symbol Common Name Systematic Name Melting Points Saturated Fatty Acids 12:0 Lauric acid Dodecanoic acid 44.2 14:0 Myristic acid Tetradecanoic acid 44.2 16:0 Palmitic acid Hexadecanoic acid 44.2 18:0 Stearic acid Octadecanoic acid 44.2 20:0 Arachidic acid Eicosanoic acid 44.2 Unsaturated Fatty Acids (all double bonds are in cis conformation) 16:1Δ9 Palmitoleic acid 9-Hexadecanoic acid -0.5 18:1Δ9 Oleic acid 9-Octadecanoic acid -0.5 18:2Δ9,12 Linoleic acid 9,12-Octadecanoic acid -0.5 18:3Δ9,12,15 ⍺-Linolenic acid 9.12.15-Octadecanoic acid -0.5 18:3Δ6,9,12 ɣ-Linolenic acid 6,9,12-Octadecanoic acid -0.5 20:4Δ5,8,11,14 Arachidonic acid 5,8,11,14-eicosatetraenoic acid -0.5 20:4Δ5,8,11,14,17 EPA 5,8,11,14,17-eicosapentaenoic acid -0.5 II. Classification of Lipids Based on Function Lipids are classified according to functions: storage lipids and membrane lipids (shown below): Figure 4. Overview of classification of lipids. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed) Page 6 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila A. Triacyglycerols (old name: triglycerides) Triacylglycerols are the most abundant naturally occurring lipids. These organic compounds, composed of three fatty acids and glycerol are commonly known to us as fats (solid triglycerides) and oils (liquid triglycerides). Fats accumulate in adipose tissue and provide a means of storing fatty acids, particularly in animals. The fatty acids are released from the stored fats by the action of lipases during starvation, and oxidized to provide energy. In addition to serving as storage form of energy, triglycerides also provide insulation and protection to body organs. Triacylglycerols serve as concentrated stores of metabolic energy. Moderately obese people with 15 to 20 kg stored TAG could live off of fat stores for 2-3 months In contrast, the human body can store less than a day’s supply of glycogen. Complete oxidation of fats yields about 9 kcal/g, in contrast with 4 kcal/g for carbohydrates and proteins. The ratio of energy derivation per weight basis is (TAG:glycogen)→ 6:1. There are two reasons for such big ratio: one, because fats are more reduced than glycogen; second, one g of fat is pure fat because it does not bind water, and one gram of glycogen is not pure glycogen (has water in it). Below is an example of triglyceride. The pink portion is the glycerol component. It has two saturated fatty acyl chains, and one unsaturated. The triglyceride shown below has three different fatty acids attached to the glycerol backbone. This is classified as mixed triglyceride. A mixed triglyceride is consist of 2 or 3 different fatty acids. In simple triglyceride (e.g. tristearin), the 3 fatty acids are identical (not shown). Figure 5. Structure of a triglyceride: glycerol (pink) + three fatty acids. This tryglyceride is named 1-stearoyl,2-linoleoyl,3-palmitoyl glycerol. Space-filling model was created using molview.org. Saponification Hydrolysis of triglycerides occurs with acids or bases as catalysts. When heated with a strong base such as sodium hydroxide or potassium hydroxide, glycerol and the sodium or potassium salts of the fatty acids are formed. These salts are called soaps, the name derived Page 7 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila from the process called saponification. The other product of saponification, glycerol, is used in creams and lotions as well as in the manufacture of nitroglycerin. Figure 6. Hydrolysis of a tryglyceride with the addition of NaOH to form 1 mol of glycerol and 3 mols of fatty acids for every mol of tryglyceride. When soaps are used with hard water, the calcium and magnesium ions in the water react with the fatty acids to form a precipitate—the characteristic scum left on the insides of sinks and bathtubs. Presence of Double Bond Influences Physical State of Triglycerides Depending on the physical state at room condition, triglycerides are called “fats” if they are solids, and “oils”, if they are liquid. The triglyceride with unsaturated fatty acids tend to remain liquid at room temperature because of their low melting point. Below is a comparison of fats and oils: Table 2. Differences between fats and oils. Fats Oils Remains solid at room temperature Remains liquid at room temperature Relatively more saturated Relatively more unsaturated relatively higher melting point Low melting point More stable Less stable Mainly from animal sources Mainly from plant sources OLESTRA: An Answer to Reduction of Fat Intake According to the dietary guidelines for saturated fatty acid (SFA) consumption, SFA should constitute less than 10% of total energy intake. SFA intake has been related to risks for cardiovascular disease. To reduce fat intake, a synthetic compound called olestra (brand name Olean) is now used as substitute for fats in many foods, including snacks. This tastes like fat, but unlike fat it does not have caloric value. This product of many years of research was prepared from natural compounds, such as sucrose and fatty acids. Although made from natural compounds, it is not digested because of the absence of digestive enzymes for it. It has been used in the preparation of high-fat foods such as potato chips, thereby lowering or eliminating their fat content. Page 8 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 7. General structure of Olestra. R groups are fatty acids. Olestra is a mixture of hexa-, hepta-, and octa-esters of sucrose with various long chain fatty acids. Besides the absence of enzymes, olestra is too large, with irregular structure to move through the intestinal wall and be absorbed into the bloodstream. B. Glycerophospholipids (old name: phospholipids) Glycerophospholipids are lipids made up of glycerol, 2 fatty acids, phosphate and a polar group.They are major components of biological membranes and the second most abundant group of naturally occurring lipids. Moreover, because of their amphipathic nature they can aid in the digestion, absorption, and transport of dietary fats. General formula of glycerophospholipids: glycerol (black); 2 fatty acids (blue); phosphate (red); and a polar group (green). In most glycerophospholipids (phosphoglycerides), Pi (red) is esterified to OH of a polar head group (X, green): e.g., serine, choline, ethanolamine, glycerol, or inositol. The 2 fatty acids (blue) tend to be non-identical. They may differ in length and/or the presence/absence of double bonds. Unsaturated fatty acid is commonly attached to C2 of glycerol (black). Figure 8. General structure of a glycerophospholid where the X group can be serine, choline, ethanolamine, glycerol, or inositol. An example is phosphatidylcholine. Each glycerophospholipid includes a polar region: glycerol, carbonyl O of fatty acids, phosphate (Pi), and the polar head group; and a nonpolar region: hydrocarbon tails of fatty Page 9 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila acids (R1 and R2). Phosphatidylcholine, a phospholipid, is the major component of most eukaryotic cell membranes. Other important glycerophospholipids (phospholipids) are phosphatidyl ethanolamine (cephalin), phosphatidyl serine, phosphatidyl choline (lecithin), phosphatidyl inositol, and phosphatidyl glycerol. In an aqueous solution, the phospholipids arrange themselves to form bilayer (lipid vesicles). In this spherical structure, the polar groups of phospholipids interact with water, while the fatty acid chains interact with each other away from the aqueous environment. Optimum van der Waals interactions are achieved in a side-by-side arrangement of the fatty acid chains found in a bilayer. Cross-sectional view of the structures that can be formed by phospholipids in aqueous solution. Micelles are usually formed by single-chain lipids; the 2 fatty acid chains in the phospholipids exhibit steric effect – reason why phospholipids prefer the bilayer (liposomes) Figure 9. Cross-section view of phospholipids in aqueous solution. Although bilayers can be quite extensive, the bilayer can achieve a lower energy configuration if it wraps around upon itself to form a continuous surface. This results in a physical enclosure called liposome. Synthetic lipid vesicles are often used to carry substances into the cells. Micelles, on the other hand are single layered spherical structure usually formed by single- chain lipids, like fatty acids. Monolayers are formed with dilute solutions of fatty acids, but at higher concentrations (i.e. at the critical micelle concentration, or CMC), fatty acids will associate to form micelle structures. This concentration is typically 0.1 - 10mM. Some glycerophospholipids have ether-linked fatty acids: Plasmalogen: - have an ether-linked alkenyl chain - vinyl ether analog of phosphatidylethanolamine - head-group alcohol is ethanolamine - Found in large quantities in heart tissue Platelet-activating factor: - aliphatic ether analog of phosphatidylcholine - with acetic acid esterified at C2 of glycerol - stimulates aggregation of blood platelets - Important in inflammation and allergic reactions Page 10 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 10. Structures of plasmalogen and platelet-activating factor. C. Sphingolipids Sphingolipids are fatty acid derivatives of sphingosine that serve as membrane component, and participates in many signaling pathways in the cell. The structures of sphingosine, its fatty acid derivative (ceramide) and sphingomyelin are shown below: Figure 11. Structures sphingosine and its derivatives. The amino group of sphingosine can form an amide bond with fatty acid carboxyl, to yield ceramide. In the more complex sphingolipids, a polar “head group” is esterified to the terminal hydroxyl of the sphingosine moiety of the ceramide. Below is a structure of sphingomyelin, a sphingolipid. Figure 12. Structure of sphingomyelin. Page 11 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Sphingomyelin, an abundant sphingolipid found in the myelin sheath of some nerve cells in animals, has phosphocholine or phosphoethanolamine as its polar head group. Sphingosine may be reversibly phosphorylated to produce the signal molecule sphingosine-1-phosphate. The molecular structures of the two types of membrane lipid classes: phospholipids and sphingolipids are similar. Sphingomyelin (a sphingolipid), with a phosphocholine head group, is similar in size and shape to phosphatidylcholine (a glycerophospholipid); with similar dimensions and physical properties (both are amphipathic), but play different roles. Figure 13. Structure of phosphatidylcholine and sphingomyelin. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed). D. Glycolipids Glycolipids are lipids with bound carbohydrate. In most cases, sugar is either glucose or galactose. Many glycolipids are derived from ceramides, and are called glycosphingolipids. These glycolipids are commonly found in the outer leaflet of the plasma membrane bilayer, with their sugar chains extending out from the cell membrane. They are often found as markers on cell membranes and play a large role in tissue and organ specificity Types of glycosphingolipids 1. Gangliosides - sphingolipids with complex carbohydrate moiety that contains more than 3 sugars, one of them is always a sialic acid; present in large quantities in nerve tissues. The structure of a ganglioside is shown in Figure 14. The most common ganglioside is composed of the ceramide backbone, the typical GalNAc-Gal-Glc trisaccharide and the NANA (sialic acid or N-acetylneuaramidic acid) attached via an - glycosidic linkage to the galactose residue. In the ganglioside trisaccharide core, glucose is attached to the ceramide backbone via a β1- glycosidic linkage, the galactose is attached to the glucose via a β(1,4) glycosidic linkage and the GalNAc is attached to the galactose via a β(1,4) glycosidic linkage. 2. Cerebrosides - sphingolipids (ceramide) with a monosaccharide such as glucose or galactose as polar head group; found in nerve and brain cells, primarily in cell membranes surface. The structure of galactocerebroside is shown in Figure 15. Page 12 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 14. Structure of the ganglioside GM1. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed). Figure 15. Structure of galactocerobroside. E. Waxes Waxes are esters of long-chain (C14-C36) fatty acids with long-chain(C16-C30) alcohols. They are insoluble in water, and with highmelting points. In plants, waxes are used to prevent excessive water loss and defense against parasites. They are also used as water repellent to protect hair, skin, feathers; and are used in lotions, ointments, and polishes. An example of a wax produced by bees is shown in Figure 16. Page 13 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 16. Structure of wax produced by bees. F. Steroids They are a group of lipids that have fused-ring structure of 3 six-membered rings, and 1 five-membered ring, called cyclopentanoperhydrophenanthrene (steroid nucleus). Examples of these steroids are steroid hormones, bile acids and cholesterol. A. Cholesterol Cholesterol is the precursor of all other animal steroids. It is an important component of animal cell plasma membranes; a component of lipoprotein complexes in the blood that helps transport lipids; and a membrane plasticizer. It modulates fluidity and permeability of cell membranes. Figure 17. Structure of cholesterol. Although cholesterol plays important roles in humans, it exhibits harmful effects when present in excess amount in the blood. Excess cholesterol causes the development of atherosclerosis, a condition in which lipid deposits block the blood vessels and lead to heart disease. Cholesterol in “low density lipoproteins”, LDL, also known as bad cholesterol, tends to deposit and clog arteries. B. Bile acids and their salts Bile acids and their salts are detergents that emulsify fats in the gut during digestion. They aid in the absorption of fats and fat-soluble vitamins in the small intestine. Like other steroids, they are synthesized from cholesterol. Because they contain both hydrophobic and hydrophilic groups, bile salts are highly effective detergents and emulsifying agents; they aid in the digestion of fats by breaking down large fat globules into smaller ones and keep those smaller globules suspended in the aqueous digestive environment. In this way, enzymes can access the ester linkages in fat, thus, hydrolyzing them more efficiently. To aid in the digestion of fats is the major function of bile salts. Page 14 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 18. Structure of two bile salts, cholic acid and sodium glycocholate. C. Steroid hormones Steroid hormones are lipids derived from cholesterol. They have the sterol nucleus, but lack the alkyl chain found in cholesterol, and are more polar than cholesterol. Below are examples of steroid hormones: Figure 19. Structures of steroid hormones which are all derived from cholesterol. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed). Testosterone - male sex hormone; produced in the testes. Estradiol, one of the female sex hormones; produced in the ovaries and placenta. Cortisol and aldosterone – regulate glucose metabolism and salt excretion, respectively; synthesized in the cortex of the adrenal gland Prednisone and prednisolone are synthetic steroids used as anti-inflammatory agents. Page 15 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Brassinolide is a growth regulator found in vascular plants. Mode of Action of Steroid Hormones In general, steroids effect changes in the cell by mediating DNA/RNA synthesis. When a steroid hormone binds to a receptor site in the cell membrane, both the hormone and the receptor enter the cell as receptor-hormone complex. Inside the cell, the mobile receptor breaks up and the released hormone binds to the cytoplasmic receptor. The cytoplasmic receptor-hormone complex goes through the nuclear envelope and in the nucleus, binds to the specific binding site (regulatory region of a gene) in the DNA to mobilize transcription of DNA to RNA, and synthesis of specific protein. Unlike peptide hormones (e.g. insulin), the action of which is mediated by a second messenger (e.g. cAMP), the steroid hormones effect changes directly at the DNA level. G. Eicosanoids Eicosanoids are local hormones produced as a response to injury and inflammation. These compounds are fatty acid derivatives, derived from arachidonic acid (20:4Δ5,8,11,14). Fiigure 20. Types of eicosanoids derived from arachidonic acid. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed). Classes of Eicosanoids 1. Prostaglandins (PG) Prostaglandins are lipids that contain a five-carbon ring originating from the chain of arachidonic acid. The name “prostaglandin” was derived from the prostate gland, the tissue from which they were first isolated. There are two groups of prostaglandins (original definition): PGE, for ether-soluble, and PGF, for phosphate (fosfat in Swedish) buffer– soluble. Each group contains numerous subtypes, named PGE1, PGE2, and so forth. Prostaglandins act in many tissues by regulating the synthesis of the intracellular messenger 3,5-cyclic AMP (cAMP). Some prostaglandins stimulate contraction of the smooth muscle of the uterus during menstruation and labor; some types of prostaglandins elevate body temperature (producing fever) and cause inflammation and pain. PGE – increases FA levels by increasing cAMP synthesis; mediates inflammation Page 16 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila PGE2 -causes dilation of blood vessels; activates cellular immune cells and attracts macrophages towards the site of injury macrophages – with hydrolytic enzymes that destroy tissue structure (site of injury) → entry of water → inflammation Prostaglandins are synthesized from arachidonic acid by the enzyme, PGH synthase, an enzyme with two functions. One is cyclooxygenase, that forms the ring and the hydroperoxyl group at C-15 by introducing molecular oxygen. The other activity mediates the 2 electron reduction of peroxide to give PGH2. The early step in the pathway of synthesis of prostaglandin, catalyzed by cyclooxygenase or COX is inhibited by aspirin. This inhibition is the basis of anti-inflammatory action of aspirin. 2. Thromboxanes Thromboxanes are eicosanoids that have a six-membered ring containing an ether. These lipids are produced by platelets, also called thrombocytes. They cause platelet coagulation (formation of blood clot). 3. Leukotrienes Leukotrienes are a type of eicosanoids first found in the leukocytes. These lipids induce contraction of the muscle lining the airways to the lung. Its overproduction causes asthmatic attacks, making the leukotriene synthesis one of the targets of anti-asthmatic drugs such as prednisone. Leukotrienes were also observed to help regulate blood pressure and blot clot formation. H. Terpenes Terpenes are lipids built from 5 carbon isoprene units. Unlike other common lipids, they do not contain fatty acids. They are generally assembled by a 'head to tail' linkage of isoprene units. Examples: fat-soluble vitamins (A, D, E, K) and electron carriers (ubiquinone, plastoquinone), etc. Figure 21. Structure of terpenes and some of its derivatives. The presence of terpenes in many plants such as cannabis, pine, and lavender, as well as fresh orange peel gives them characteristic aroma. Terpenes found in the cannabis plants were observed to have health benefits from treating pain to relieving anxiety. Secreted from the same glands that produce THC and CBD in cannabis, terpenes are potent and carry the potential to affect animal and human behavior when inhaled (myrceneterpeneeffects). Page 17 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Myrcene, a monoterpene is the most abundant terpene in many cannabis strains. https:// straingenie.com/cannabis-terpenes/myrcene/. It is also found in very high concentrations in sweet basil, hops, mangoes. http://www.internationalhempassociation.org/jiha/jiha4208.html. It has powerful antibiotic, antimutagenic, analgesic, anti-inflammatory, and sedative effects. Working in synergy with THC, the terpene is ideal for patients suffering from: sleep disorders like insomnia, pain and bodily discomfort (myrceneterpeneeffects ; https://labeffects.com/ terpene-glossary-myrcene/) Figure 22. Other types of terpenes. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed). Other Common Terpenes A. Vitamin E (tocopherol) - antioxidant; free radical scavenger; traps HOOᐧ and ROOᐧ radicals formed as a result of oxidation by O2 of unsaturated hydrocarbon chains in membrane phospholipids B. Vitamin K - essential cofactor of carboxylase, an enzyme that catalyzes the carboxylation of glutamic acid residues in certain proteins (prothrombin, factor VII, factor IX, factor X); essential in the blood clotting process; deficiency leads to excessive bleeding. C. Ubiquinone - a mitochondrial electron carrier (coenzyme Q), n ranges from 4 to 8 D. Plastoquinone - a chloroplast electron carrier, n ranges from 4 to 8 E. Vitamin A: plays important role in vision, bone growth, reproduction, cell division, etc. F. Vitamin D: regulates calcium and phosphorous metabolism The active form of vitamin D, 1,25-dihydroxycholecalciferol which regulates metabolism of Ca2+ in kidney, intestine and bone, is synthesized from 7-dehydrocholesterol in the following steps: Page 18 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Figure 23. Metabolism of Vitamin D3.. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed). Polyketides (biologically active lipids) with medicinal uses: Figure 24. Polyketide natural products used in human medicine. Adapted from Nelson, D.L. & Cox, M.M. (2004). Lehninger Principles of Biochemistry (4th ed). Laboratory Activity: Isolation of Lipids from Egg Yolk III. The Biological Membrane Every cell has a cell membrane which separates it from the external environment. It plays important roles in regulating the passage of molecules in and out of the cell, and providing location for hormone receptors and some important enzymes, e.g. adenyl cyclase. It also provides cell shape (in animal cells) e.g. the characteristic shape of red blood cells, nerve cells and bone cells. In addition to the cell membrane, eukaryotic cells also have membrane- enclosed organelles, such as nuclei and mitochondria. Page 19 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila An understanding of membrane structure requires knowledge of how the protein and lipid components contribute to the properties of the membrane. The current model of biological membrane is the Fluid-Mosaic model, shown below: Figure 25. Depiction of the Fluid Mosaic Model. Adapted from Voet, D. & Voet, J. (2011). Biochemistry. (4th ed). United States of America: John Wiley & Sons, Inc. Phospholipids are the principal lipid components of membranes. There are also proteins found at the top and bottom of the bilayer, and some even span the whole membrane. The protein composition varies from 20-80% of the weight of membrane, depending on its function, e.g. mitochondrial membrane where the electron transport chain can be found, is rich in proteins. In addition, there are glycolipids, and in animal membranes, there are cholesterol molecules. Figure 26. Lipid bilayers. Adapted from Campbell, M.K. & Farrell, S.O. (2012). Biochemistry. (7th ed). Belmont, CA, USA: Brooks/Cole Cengage Learning. In the lipid bilayer part of the membrane, the polar head groups are in contact with water, and the nonpolar tails lie in the interior of the membrane. The surface of the bilayer is polar and contains charged groups. The nonpolar hydrocarbon interior of the bilayer consists of the Page 20 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila saturated and unsaturated chains of fatty acids and the fused-ring system of cholesterol held together by van der Waals and hydrophobic interactions. Membranes are flexible, self-sealing and selectively permeable to solutes. Because membranes are self-sealing, membranes can undergo fusion and fission without leaking cellular components. Asymmetry of Biological Membrane The biological membrane exhibits asymmetry. Asymmetry refers to differences in composition of the outer half and inner half of the membrane bilayers; also on the types of localization of proteins and on localization of carbohydrates. Figure 27. Asymmetry of the lipid bilayer. Adapted from Campbell, M.K. & Farrell, S.O. (2012). Biochemistry. (7th ed). Belmont, CA, USA: Brooks/Cole Cengage Learning. and Voet, D. & Voet, J. (2011). Biochemistry. (4th ed). United States of America: John Wiley & Sons, Inc.. Both the inner and outer portion of the bilayer contain mixtures of lipids and the compositions on inside and outside of lipid bilayer are different. Bulkier molecules tend to occur in the outer layer, and smaller molecules in the inner layer. Because the bilayer is curved, the molecules of the inner layer are more tightly packed. Cerebrosides, the bulky lipids tend to be located in the outer layer. Glycolipids are found only on the outer half of the membrane, with their carbohydrate components located outside the bilayer, exposed to the aqueous environment. Phospholipids that contain bulky nitrogenous base, e.g. phosphatidyl choline, are found in the outer half of the bilayer. Phosphatidic acids which are smaller phospholipids are found in the inner half. Cholesterol occupies both bilayers of the membrane in variable concentrations and frequency. Page 21 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Fluidity of Membrane Depends on Its Composition The interior of a lipid bilayer is normally highly fluid. The fluidity of membrane is due to the presence of unsaturated fatty acids that are loosely packed, and disordered due to the kinks in their hydrocarbon chains. The linear conformation of hydrocarbon chains of saturated fatty acids, on the other hand, leads to close packing of the molecules in the bilayer. Thus, whether the membrane will be fluid or not, depends on component fatty acids. Fluidity also depends on the temperature. Figure 28. Adapted from Voet, D. & Voet, J. (2011). Biochemistry. (4th ed). United States of America: John Wiley & Sons, Inc. In the liquid crystal state (Figure 28a), hydrocarbon chains of phospholipids are disordered and in constant motion. At lower temperature, a membrane containing a single phospholipid type undergoes transition to a crystalline state (Figure 28b) in which fatty acid tails are fully extended; packing is highly ordered, and van der Waals interactions between adjacent chains are maximal. With heat, mobility of the lipid chains increases dramatically. Ordered bilayers become less ordered, and bilayers that are comparatively disordered become even more disordered. This transition from ordered to less ordered state takes place at a characteristic temperature, called transition (melting) temperature or Tm. The transition temperature is higher for more rigid and ordered membranes than it is for relatively fluid and disordered membranes. The transition temperature (Tm) of the bilayer increases with chain length and degree of saturation of fatty acids. Bacteria and cold-blooded animals can modify the fatty acid composition of their membranes in order to adjust fluidity. As seen in the table below, as the growth temperature of E. coli is increase, the more saturated fatty acids are incorporated in its membrane and less unsaturated fatty acids. The exact fatty acid composition depends not only on growth temperature but on growth stage and growth medium composition. See the table on the next page for more details. Role of Cholesterol in Membrane Fluidity Cholesterol plays important role in adjusting the fluidity of cell membranes. Above the transition temperature (Tm), when phospholipids are in rapid motion, cholesterol decreases membrane fluidity by increasing hydrophobic interactions with the fatty acid chains. However, Page 22 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Table adapted from Marr, A. G., & Ingraham, J. L. (1962). Effect of temperature on the composition of fatty acids in Escherichia coli. Journal of Bacteriology, 84(6), 1260-1267. below the transition temperature, cholesterol increases membrane fluidity because of its bulky structure. Figure 29. Role of cholesterol in membraned fluidity. Adapted from Campbell, M.K. & Farrell, S.O. (2012). Biochemistry. (7th ed). Belmont, CA, USA: Brooks/Cole Cengage Learning. The hydrophobic portion of cholesterol interacts with the nonpolar fatty acyl chain via van der Waals interaction. This role of cholesterol in reducing fluidity is important at higher temperature when the membrane is highly fluid. At lower temperature (lower than the Tm), cholesterol serves a different role. It helps increase fluidity of the cell membrane by interfering with the close packing of fatty acid tails in the crystalline state, and hence inhibits transition to the crystal state. Phospholipid membranes Page 23 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila with a high concentration of cholesterol have a fluidity intermediate between the liquid crystal and crystal states. The inner mitochondrial membrane lacks cholesterol, but includes phospholipids whose fatty acids have one or more double bonds to lower its Tm to below physiological temperature. Membrane Proteins Proteins in the membrane may be integral or peripheral. Integral proteins traverse the entire lipid bilayer while peripheral proteins are found in only one half of the bilayer, either outer or inner. Proteins that function as channels or gates are usually integral proteins. Peripheral proteins include receptors, enzymes, and some regulatory proteins, e.g. G-proteins. Integral proteins are often in the form of an -helix or -sheet. In this form, the polar parts of the peptide backbone are kept inside, and the nonpolar parts are directed towards the nonpolar lipids in the interior of the bilayer. Proteins can be anchored to the lipids via covalent bonds from cysteines or free amino groups on the protein to one of several lipid anchors, e.g. myristoyl and palmitoyl groups Integral proteins are bound tightly to the interior of the membrane, and can be removed by treatment with detergents or ultrasonication. However, removal generally denatures them. The hydrophobic domains of detergents substitute for lipids, coating hydrophobic surfaces of proteins and the polar domains interact with water. If detergents are removed, purified integral proteins tend to aggregate and come out of solution. Peripheral protein Integral protein Figure 30. Proteins can be anchored in biological membranes. Adapted from Campbell, M.K. & Farrell, S.O. (2012). Biochemistry. (7th ed). Belmont, CA, USA: Brooks/Cole Cengage Learning. Peripheral proteins are usually bound to the charged head groups of the lipid bilayer by polar interactions or electrostatic interactions, or both. They can be removed by such mild treatment as raising the ionic strength of the medium. The relatively numerous charged particles present in a medium of higher ionic strength offer more electrostatic interactions with Page 24 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila the lipid, hence removing the protein which has comparatively fewer electrostatic interactions with the lipid. Mobility of Membrane Lipids Phospholipids contribute to membrane fluidity. Fluidity is maintained through continuous motions of phospholipids, which include: A. Rotational – motion due to electrostatic repulsion between negatively charged groups, e.g. phosphate groups. B. Segmental – motion attributed to the turning and twisting of single C-C bonds in fatty acids C. Transformational – motion of single C-C bonds adjacent to the double bond; results in trans to gauche, trans to trans, and gauche to trans transformations. D. Lateral diffusion – motion of the entire phospholipid molecule from one point to another in the same leaflet of the membrane (horizontal motion) E. Transverse or flip-flop – motion of entire phospholipid molecule from the outer half to the inner half of the bilayer, or vice versa. Lateral motion of lipid molecules within one of the two layers frequently takes place, however, especially in more fluid bilayers. High speed tracking of individual lipid molecules has shown that lateral movements are constrained within small membrane domains. The apparent constraint on lateral movement of lipids (and proteins) has been attributed to integral membrane proteins anchored to the cytoskeleton, functioning as a picket fence. Figure 31. Lateral diffusion and transverse diffusion (flip-flop) of phospholipids in the lipid bilayer. Hopping from one domain to another occurs less frequently than rapid movements within the domain. Flip-flop of lipids from one half of a bilayer to the other is thermodynamically not feasible because it would require the polar head group of a lipid to traverse the hydrophobic core of the membrane. However, some membranes contain enzymes (flippases) that actively transport particular lipids from one monolayer to the other. Page 25 of 30 Module on Lipids CHEM41 and 43 Lecture Course Guide Department of Physical Sciences and Mathematics College of Arts and Sciences, University of the Philippines Manila Transport Processes There are three general classes of transport system in the cell membrane: uniport, symport and antiport. Uniport Antiport Symport Figure 32. Three general classes of transport system: uniport (blue protein), antiport (green protein), and symport (orange protein). Both antiport and symport are collectively called cotransport. Membrane Transport 1. Passive transport - driven by a concentration gradient A. Simple diffusion: a molecule or ion moves through an opening (e.g. oxygen, short chain fatty acids) B. Facilitated diffusion: a molecule or ion is carried across the membrane by a carrier/ channel protein (e.g. glucose) 2. Active transport – a substance is moved against a concentration gradient A. Primary active transport: transport is linked to the hydrolysis of ATP or other high- energy molecule (e.g. Na+/K+ ion pump). B. Secondary active transport: against electrochemical gradient, driven by ion moving down its gradient (e.g. transport of lactose). In simple diffusion, a substance that is present in greater concentration outside the cell diffuses rapidly through the phospholipid bilayer. This is the simplest type of transport mechanism where a substance flows down a concentration gradient. Example of this transport is the transport of molecular oxygen. Since oxygen is a nonpolar molecule, it is soluble in the nonpolar lipid bilayer, and allows it to readily enter the cell. In facilitated diffusion, transport is aided by a transport protein (e.g. protein pores, carrier molecules as well as membrane vesicles). The transporter proteins have 2 important features: (a). they facilitate net movement of solutes only in a thermodynamically favored direction, that is G