Lecture 4 - LIPIDS

1. Lecture 4 - LIPIDS

2. General Facts about Lipids Lipids (fats and oils): These molecules are essentially all hydrophobic or partially hydrophobic. There is quite a variety of structure among the lipids, but the two best known examples are the steroids and the glycerides. The steroids includesuch substances as cholesterol, testosterone and estrogen. The glycerides include the triglycerides (primary storage lipids) and phospholipids (also called phosphoglycerides, the major component of all biological membranes). Lipids are a diverse group of biomolecules with wide differences in both structure and function: Fats, Oils, Phospholipids, Steroids, Carotenoids. Lipids are a class of biological molecules defined by low solubility in water and high solubility in nonpolar solvents (e.g., ether, benzene, acetone, chloroform). As molecules that are largely hydrocarbon in nature, lipids represent highly reduced forms of carbon and, upon oxidation in metabolism, yield large amounts of energy. Lipids are thus the molecules of choice for metabolic energy storage.

3. Fatty Acids • Fatty acids are monocarboxylic acids that are building blocks for triglycerides and phospholipids. • Each fatty acid consists a chain of carbon and hydrogen atoms with a carboxyl group at the alpha end and a methyl group at the omega end. • 20 different fatty acids with varied length, saturation, and shape exist.

4. Fatty Acids Fatty acidsare building blocks of lipids. They are naturally occurring carboxylic acids with an unbranched carbon chain and an even number of carbon atoms. The pathway by which fatty acids are biosynthesized they almost always contain an even number of carbon atoms. Long-chain fatty acids (12 to 26 carbon atoms) are found in meats and fish; medium-chain fatty acids (6 to 10 carbon atoms) and short-chain fatty acids (fewer than 6 carbon atoms) occur primarily in dairy products. There are saturated and unsaturated Fatty acids. • Fatty acid chains that contain only carbon-carbon single bonds are referred to as saturated. Example: • Palmitic acid:

5. Fatty Acids • Unsaturated Fatty Acid: Those molecules that contain one or more double bonds are said to be unsaturated. • Unsaturated fats have lower melting points (i.e. liquid in lower temperatures). • There are mono- and polyunsaturated(double bonds are usually separated by methylene groups) fatty acids. Oleic acid: A mono-unsaturated fatty acid.

6. Saturated vs Unsaturated Fatty acids • Oils, mostly from plant sources, have some double bonds between some of the carbons in the hydrocarbon tail, causing bends or “kinks” in the shape of the molecules. • The mono unsaturated oleic (18:1 Δ9) and poly unsaturated linoleic (18:2 Δ9,12) are the most abundant fatty acids in living organisms. • The higher the level of unsaturation, the greater the susceptibility to oxidative attack.

7. The Common Fatty Acids

8. Fats vs. Oils • The difference between fats and oils: • fats are solid in room temperature and oils are liquid in room temperature. • Hydrogenation is the process of converting liquid oil to solid fat by adding hydrogen to some of the double bond of the unsaturated carbon chain in presence of nickel as catalyst; e.g., Margarine. • Rancidity: Triglycerides soon become rancid, by either hydrolysis of ester bond or oxidation of double bond, • developing an unpleasant odor and flavor on exposure to moist and warmth air at room temperature.

9. Classification of Lipids Lipids could be in six classes: • Fatty acids and their derivatives • Triacylglycerols • Wax esters • Phospholipids (phosphoglycerides) • Sphingolipids • Isoprenoids (isoprene-containing lipids) Lipids could be classified based on their structure: • Simple: fats, oils, waxes, steroids. • Complex: phospholipids, spingolipids, glygolipids. • Their derivatives: hormones, fat-soluble vitamins Lipids could be classified on the basis of whether they undergo hydrolysis reactions in alkaline solution: 1- Saponifiable lipids: can be hydrolyzed under alkaline conditions to yield salts of fatty acids. 2- Nonsaponifiable lipids: do not undergo hydrolysis reactions in alkaline solution.

10. Simple Lipids vs Complex Lipids Several levels of complexity: • Simple lipid is a lipid that cannot be broken down to smaller constituents by hydrolysis. • Fatty acids, waxes and cholesterol are examples of simple lipids. • Complex lipid is a lipid composed of different molecules held together mostly by ester linkages and susceptible to cleavage reactions. • acylglycerols - mono, di and triacyl glycerols ( fatty acids and glycerol). • phospholipids(also known as glycerophospholipids) - lipids which are made of fatty acids, glycerol, a phosphoryl group and an alcohol. Many also contain nitrogen. • glycolipids (also known as glycosphingolipids): Lipids which have a spingosine and different backbone than the phospholipids.

11. Biological functions of lipids • Fuel: The most important role of lipids is as а fuel (9.45 kcal/g). • Fat is the most concentrated form in which energy can be stored. Fat is less oxidized, release more energy than carbohydrates (4.1 kcal/g). • Insulation: Since fat is а bad conductor of heat, it provides excellent insulation (of course for mammals!). • Padding: Fat may also provide padding to protect the internal organs. • Some compounds derived from lipids are important building blocks of biologically active materials. • Lipoproteins are constituents of cell walls. • One more important function of dietary lipids is that of supplying the so-called essential fatty acids. • Plants and bacteria can synthesize all the fatty acids they require from acetyl-CoA. Triacylglycerols of plant seeds and fruits are mainly made from unsaturated fatty acids (oleic and linoleic), thus are known as oils. • Mammals can synthesize saturated fatty acids and some unsaturated fatty acids. They can also modify some dietary fatty acids by adding two carbon units and introducing some double bonds. • Fatty acids that can be synthesized are called nonessential fatty acids. • Mammals do not posses the enzymes to synthesize linoleic (18:2 Δ9, 12) and linolenic (18:3 Δ9, 12,15), they are known as essential fatty acids (must be obtained from diet).

12. Glycerides: Triglycerides • Glycerides come in at least two types: Triglycerides and Phospholipids (phosphoglycerides) • All glycerides have as part of their structure the trialcohol glycerol, as well as 2-3 fatty acids (long hydrocarbon chains which terminate with carboxyl groups). • The chemical nature of a glyceride is somewhat contradictory—especially the phospholipids. The glycerol part of the molecule is at least somewhat polar, while the hydrocarbon tails contributed by the fatty acids are highly non-polar. The fatty acids are attached to the glycerol through dehydration synthesis between the hydroxyls of the glycerol and the carboxyls of the fatty acids, creating an ester linkage. The formation of this connecting bond destroys most of the polarity of both the hydroxyl and the carboxyl. The completed glyceride has a polar head with two or three long non-polar tails. • Fatty acids react with alcohols to form esters.

13. Glyceride formation • Fatty acid esters of the trihydric alcohol – glycerol are called acylglycerol or glycerides. Triacylglycerols are esters of glycerol with 3 fatty acids. • Triacylglycerols have no charge, thus they are known as neutral fats. • Reaction of formation of triacylglycerols: • Triacylglycerols may contain a high Proportion of saturated fatty acids, then they are called fat. If the proportion of unsaturated fatty acids is high, then they are called oil. Oils are partially hydrogenated to form oleomargarine.

14. General Structure of glycerides • Since there are three fatty acids attached, these are known as triglycerides. • The longer the fatty acids the higher the melting point of a fat (liquid in higher temperatures). • Decreases in the packing efficiency (double bound-induced kink) decreases the melting point (liquid in lower temperatures). • Animals alter the length and unsaturated level of the fatty acids in lipids (cholesterol too) to deal with the cold temperatures. • Plants could also alter the level of saturation of the fatty acids upon facing low temperature (cold acclimation).

15. Phospholipids • Phospholipids (phosphoglycerides) are very much like triglycerides, except that one of the fatty acids is replaced by a phosphate functional group. The phosphate group affects the behavior of the molecule in two significant ways. First, it decreases the number of hydrophobic tails from three to two, thus weakening the overall force for hydrophobicity in the molecule. Second, since the phosphate functional group is small (and thus functionally part of the head of the molecule) and negatively charged, it is a very powerful force for hydrophilicity. Because of this arrangement, phospholipids spontaneously form bilayers in water, with the hydrophobic tails sequestered between the two layers of hydrophilic heads (polar head). The tails create a water-free environment between the two layers. This (bimolecular layer) is the structural basis for all biological membranes. The membrane is a fluid mosaic composed of proteins of various sorts “floating” in a phospholipid bilayer.

16. Phospholipids are formed when two fatty acids are covalently linked to a glycerol, which is linked to a phosphate. • The phosphate component of a phospholipid is attached to a “head group”, such as choline (which is an alcohol). • Head group is polar, i.e. hydrophilic. • Tail of the phospholipid is non-polar, so it is hydrophobic. • The tail of a phospholipid varies in length from 14 to 28 carbons. • Phospholipids are two types, i.e. phosphoglycerides and sphingomyelins. • Sphingomyelins differ from phosphoglycerides in that they contain sphingosine instead of glycerol. Sphingolipids (found in animal and plant membranes) contain a long-chain amino alcohol. • Phosphoglycerides are the foremost phospholipids in cell membranes. • Phosphoglycerides often contain 16-20-carbon fatty acids.

17. Types of Lipids: a comparison

18. Trans and Cis Isomers • In unsaturated fatty acids, because double bonds are rigid structures, molecules that contain them can occur in two isomeric forms. In fact, there are two ways the pieces of the hydrocarbon tail can be arranged around a C=C double bond. • TRANS-isomer • The two pieces (in fact two identical groups) of the molecule are on opposite sides of the double bond, that is, one “up” and one “down” across from each other. • CIS-isomer • The two pieces of the carbon chain on either side of the double bond are either both “up” or both “down,” such that both are on the same side of the molecule. The presence of a Cis double bond causes an inflexible kink in a fatty acid chain.

19. Trans and Cis • Naturally-occurring unsaturated vegetable oils have almost all Cis bonds. Because of this feature, unsaturated fatty acids do not pack as closely as saturated fatty acids. Thus: unsaturated fatty acids are liquid in the room temperature (i.e. lower melting point). • But using oil for frying causes some of the Cis bonds to convert to Trans bonds. • If oil is used only once like when you fry an egg, only a few of the bonds convert to Trans. • However, if oil is constantly reused, like in fast food French fry machines, more and more of the Cis bonds are changed to Trans until significant numbers of fatty acids with Trans bonds build up. • The reason this is of concern is that fatty acids with Trans bonds are carcinogenic!

20. Waxes А waxis а monoester formed from the reaction of а long-chain monohydroxy alcohol (having 16-30 carbon atoms) with long-chain saturated and unsaturated fatty acids(having 14-36 carbon atoms) molecule. Example: Bees wax or Brazilian wax palm which produces carnauba wax. Biological role:Waxes serve as protective coatings on leaves, stems, and fruit of plants and the skin and fur of animals. Waxes are low- melting, stable solids which appear in nature in both plants and animals. A wax coat protects surface of many plant leaves from water loss and attack by microorganisms.

21. Plant Waxes • Waxes comprise the outermost layer of leaves, fruits, and herbaceous stems and are called epicuticular waxes. • Waxes embedded in the cuticle of the plant are cuticular waxes. • Cutin is another wax in the cuticle and it makes up most of the cuticle. • Suberin is a similar wax that is found in cork cells in bark and in plant roots. Both cutin and suberin help prevent water loss by the plant. • Structures of waxes vary depending on which plant produced them. • Waxes are usually harder and more water repellant than other fats.

22. Characterization of fats • Fats could be characterized based on: • Acid number: It is the number of milligrams of potassium hydroxide required to neutralize the free fatty acids in 1 g of the oil or fat. • Saponification number:It is number of milligrams of potassium hydroxide required tо completely saponify l00 g of the oil or fat. • Soap can be made by heating beef tallow or coconut oil with potash (potassium hydroxide, alone or mixed with potassium carbonate), thus triacylglycerol is hydrolyzed to give glycerol and the potassium salt of fatty acid, i.e. saponification. Fatty acid salts are called soap. Soap can act as an emulsifying agent. Mixing soap and grease forms emulsion, thus it is a cleansing agent. • Iodine number:It is the number of grams of iodine that combine with 100 g of oil or fat. It is а measure of the degree of unsaturation of а fat or oil; а high iodine number indicates а high degree of unsaturation of the fatty acids of the fat. • Reichert-Meissl number. (R. M. number): It is the number of milliliters of potassium hydroxide required toneutralize the distillate (obtained by saponification, acidification and steam distillation of the fat) оf 5 g of the fat.

23. Nonsaponifiable Lipids • Lipids do not undergo hydrolysis in alkaline solution. • Nonsaponifiable Lipids: steroids, eicosanoids, terpenes, pheromones, fat-soluble vitamins • A steroid is a lipid whose structure is based on the tetracyclic (four-ring) system shown in the following examples. Three of the rings are six-membered, while the fourth is five-membered. Steroids have many diverse roles throughout both the plant and animal kingdoms. • All plant steroid molecules are sterol, mainly involved in membrane structure and function. β–sitostrol and stigmastrolare the most abundant algal and plant sterols. Plant sterols have toxic and medicinal properties (e.g. overdoses • of digitoxin from Digitalis purpurea may inhibit Na-K ATPase and thus cause heart failure).

24. Isoprenoids (Steroids and Terpenes) • Theisoprenoids (terpenes, steroids) are a vast array of biomolecules containing repeating 5-carbon structural units known as isoprene units. • Steroids (each composed of four fused rings and found in all eukaryotes) are complex derivatives of triterpenes. Cholesterol is a steroid specific to animals which is an essential component of animal cell membrane and a precursor of biosynthesis of steroid hormones.Ergosterol ( plant sterol) occurs in small amounts in grains, vegetables, fruits, legumes, nuts and has powerful cholesterol-lowering properties. • Terpenesare an enormous group of molecules that are found largely in the “essential oils” of plants. • Terpens are classified based on the number of their isoprene residues: monoterpenes (composed of two isoprene units), diterpenes (composed of four isoprene units), sesquiterpenes (composed of three isoprenes), tetraterpenes (composed of eight isoprene units). • Carotenoidsare the only tetraterpenes. Carotenes are the hydrocarbon member of the group. Xanthophyll is oxygenated derivative of carotene. • Polyterpenes are composed of 100’s to 1000’s of isoprene units. Natural rubber is a polyterpene composed of 3000-6000 isoprene units. • Mixed terpenoids are important biomolecules composed of nonterpene components attached to isoprenoid groups. Vitamine E (α-tocopherol), ubiquinone, vitamin K (phylloquinone in plant), cytokinin phytohormones.

25. Biological Membranes • All biological membranes have the same general structure: they contain lipid and protein molecules. • Membrane is a bimolecular lipid layer (i.e. lipid bilayer). According to the fluid mosaic model of membrane structure, proteins are mainly floated within the lipid bilayer and determine a membranes function. • The fluid mosaic model of membrane structure proposes that lipids of the bilayer are in constant motion, gliding from one part of their bilayer to another at high speed.

26. Membrane Structure • Each type of living cell has its own functions, thus structure of its membranes is also unique. Hence, the proportion of lipid and protein varies among cell types and among organelles within each cell. The types of lipid and protein in each membrane also vary. • Amphipathic molecule is a molecule containing both polar and nonpolar domains. This type of molecules rearrange into ordered structures, upon being suspended in water. Then, hydrophobic groups become buried in the interior and exclude water and hydrophyllic groups become oriented so that they are exposed to water. Phospholipids form into bimolecular layers when sufficiently concentrated. This property of phospholipids (and other amphipathic lipid molecules) is the basis of membrane structure.

27. An Amphipathic Molecule Two-tailed amphipathic molecules are cylindrical (a), and tend to naturally form a bilayer structure (b).

28. Lipid Bilayer and Water Molecules • Model of a lipid bilayer surrounded by water:

29. Lipids in biomolecules

30. Membrane Features • Features of biological membranes • Membrane fluidity: resistant of membrane components to movement. A membrane fluidity is largely determined by the percentage of unsaturated fatty acids in its phospholipid molecules. A high proportion of unsaturated chains results in a more fluid membrane. Rapid lateral movement is responsible for the proper functioning of many membrane proteins. In animals cholesterol moderates membrane stability without great compromising fluidity, because it contains both rigid (ring system) and flexible (hydrocarbon tail) structural elements. • Selective permeability: impenetrable barrier (i.e. to ionic and polar substances) created by the hydrophobic hydrocarbon chains. Crossing a lipid bilayer (i.e. a polar substance) requires the shedding of some or all of the hydration sphere and binding to a carrier protein or otherwise passing through an aqueous channel. In both methods the hydrophyllic molecule is shielded from the hydrophobic core of the membrane. Nonpolar substances simply diffuse through the lipid bilayer down their concentration gradients. Each membrane exhibits its own transport capability or selectivity based on its protein component.

31. Membrane Features • Self-sealing capability: when lipid bilayers are disrupted (it is lethal) they are immediately and spontaneously reseal, i.e. it is critical to do so. • Asymmetry: lipid composition of each half of a bilayer in the membrane is different, because each side of a membrane is exposed to a different environment. • Membrane proteins can be classified based on their function: structural, enzymes, hormone receptors, transport proteins. • Membrane proteins can be classified according to their structural relationship to membrane: • Integral proteins: those that are embedded in and/or extend through a membrane. Such molecules can be extracted only by disrupting the membrane with organic solvents or detergents. • Peripheral proteins: can be released from membrane by relatively gentle methods, e.g. salt solutions or pH changes.

32. Membrane Functions • Membranes play a number of functions, two functions are more critical: • Transport of molecules and ions, binding of hormones and biomolecules. • Membrane transport must be carefully regulated to meet cell’s metabolic needs. Due to the inherent impermeability, specific transport components must be inserted into membranes. Transport proteins or permeases are harnessed to deal with the membrane impermeability. • Passive and active transport could be involved. • Passive transport (e.g. oxygen and carbon dioxide and small organic molecules) could be via simple diffusion (moving down a concentration gradient until an equilibrium is reached), facilitated diffusion through carriers or channels of tunnel-like proteins. Each type of carrier or channel is designed for the transport of a specific molecule (certain large or charged molecules). Many channels are chemically or voltage-regulated. • Carrier proteins (i.e. passive transporters) undergo conformational changes upon binding, i.e. to a specific solute on one side of the membrane. This type of diffusion increases the rate of diffusion but cannot cause a net increase in solute concentration in one side.

33. Membrane Functions • Active transport is inevitable, as often cells must accumulate molecules against a concentration gradient. • Primary active transport: energy is provided by ATP via transmembrane ATP-hydrolyzing enzymes, e.g. Na-K ATPase pump is an active transporter. • Secondary active transport: concentration gradients generated by primary active transport are harnessed to move substances across membranes. Example: the Na gradient created by Na-K ATPase pump is indirectly used for transport of some negatively charged substances. • Membrane receptors provide mechanisms by which cells monitor and respond to changes in their environment. E.g. binding of chemical signals such as hormones to membrane receptors is a vital tool in intracellular communication. Or binding of a ligand to a membrane receptor results in a conformational change which then causes a specific programmed response.