Lipids Updated 2 PDF Lecture Notes

Lectures - PHT383 06/10/2024 Lipids Chemistry Essential Information Lipids are heterogonous group of compounds related to fatty...

Lectures - PHT383 06/10/2024 Lipids Chemistry Essential Information Lipids are heterogonous group of compounds related to fatty acids. Classification of Lipids: A. Simple Lipids. B. Compound Lipids. C. Derived Lipids. A) Simple Lipids These are esters of fatty acids with alcohols. According to the alcohol, they are subclassified into: 1. Neutral fats 2 - Waxes 1 Lectures - PHT383 06/10/2024 1. Neutral fats These are esters of fatty acids with glycerol. They carry no charge, therefore called neutral fat. They are also called triglycerides or triacylglycerols. Neutral fats are either fats or oils. Fats are solid triacylglycerols due to high content of saturated fatty acids. Oils are liquid triacylglycerols due to high contents of unsaturated fatty acids. A Typical Neutral Fat Glycerol Head Fatty Acid Tails 2 Lectures - PHT383 06/10/2024 2 - Waxes These are esters of fatty acids with alcohol higher than glycerol in molecular weight. The alcohol is monohydric alcohol containing one OH group. Examples of waxes are beeswax and lanoline. B) Compound lipids These are esters of fatty acids with alcohols and containing other groups. According to these groups they are subclassified into: 1. Phospholipids 2. Glycolipids 3. Lipoproteins 1. Phospholipids Phospholipids contain in addition to fatty acids and alcohol, a phosphate radical (phosphoric acid). They also have nitrogen containing bases. The alcohol of phospholipids is either: Glycerol (glycerophospholipids) or Sphingosine (sphingophospholipids). 2. Glycolipids Glycolipids contain carbohydrate radical in addition to fatty acids and alcohol. 3 Lectures - PHT383 06/10/2024 3. lipoproteins Lipoproteins contain protein radical in addition to fatty acids and alcohols. C) Derived lipids These include substances obtained by the hydrolysis of either simple lipids or compound lipids. They also include substances associated with them in nature and related to them in properties and metabolism. Steroids Cholesterol, cortisol& progesterone Fatty Acids Fatty acids (F.A.) are monocarboxylic acids obtained from the hydrolysis of fats. They are either saturated (containing no double bonds) or unsaturated (containing one or more double bonds). F.A. usually containing an even number of carbon atoms (because they are synthesized from two carbon units). The carbon atoms of fatty acids are numbered from the carboxylic end, which is known as carbon No. 1. The carbon atom adjacent to the carboxyl group is known as carbon No. 2 or α-carbon, and the next carbon as carbon 3 or β- carbon and the next carbon 4 or -carbon, …etc. The methyl carbon at the other end of F.A. is known as omega carbon (ω-carbon). 4 Lectures - PHT383 06/10/2024 Nomenclature of unsaturated fatty acids There are two ways of defining the position of a double bond: by counting from the functional group and by counting from the end opposite to the functional group. A. Counting from the functional group (COOH): Used by chemists, the position of the double bond is represented by the symbol  (delta), followed by a number. For example  9, 12, 18:2, means an 18 carbon fatty acid containing two double bonds between the carbon atoms 9 and 10 and 12 and 13, that is the linoleic acid. B. Counting from the end opposite to the functional group (COOH): Used by biologist and more confusing. The symbol  (omega) is used to depict the end opposite to the functional group. For example,  6, 9, 18:2 is an 18 carbon fatty acid containing two double bonds between the carbon atoms 6 and 7 and 9 and 10, that is also linoleic acid. a. CH3 CH2 CH2 CH2 CH2 CH CH CH2 CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 COOH 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 Functional group b. CH3 CH2 CH2 CH2 CH2 CH CH CH2 CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 COOH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Count from end opposite to the functional group 5 Lectures - PHT383 06/10/2024 There are three major classes of fatty acids: I- Saturated fatty acids (SFAs): SFAs contain no CC double bonds. II- Monounsaturated fatty acids (MUFAs): MUFAs contain one CC double bonds. III- Polyunsaturated fatty acids (PUFAs): PUFAs contain two or more CC double bonds.  Saturated fatty acids do not contain any double bonds. The term "saturated" refers to hydrogen, in that all carbons (apart from the carboxylic acid [COOH] group) contain as many hydrogens as possible.  Acetic acid, palmitic and stearic acids are the most abundant saturated fatty acids.  Oleic acid is the most abundant unsaturated fatty acids. Fatty acids vary in chain length and in the degree of unsaturation. The melting points of fatty acids (and lipids that contain them) increase as the number of carbons increase and decrease as the number of double bonds increase. 6 Lectures - PHT383 06/10/2024 Number of Double Number of Name Bonds Carbons Structure Melting Point (C) Saturated Fatty Acids Lauric 0 12 CH3(CH2)10COOH 44 Myristic 0 14 CH3(CH2)12COOH 54 Palmitic 0 16 CH3(CH2)14COOH 63 Stearic 0 18 CH3(CH2)16COOH 69 Arachidic 0 20 CH3(CH2)18COOH 77 Behenic 0 22 CH3(CH2)20COOH 81 Lignoceric 0 24 CH3(CH2)22COOH 84 Monounsaturated Fatty Acids Palmitoleic 1 16 CH3(CH2)5CHCH(CH2)7COOH 1 Oleic 1 18 CH3(CH2)7CHCH(CH2)7COOH 13 Nervonic 1 24 CH3(CH2)7CHCH(CH2)13COOH 42 Polyunsaturated Fatty Acids (PUFAs) Linoleic 2 18 CH3(CH2)4(CHCHCH2)2(CH2)6 COOH -5 Linolenic 3 18 CH3CH2(CHCHCH2)3(CH2)6 COOH -11 Arachidonic 4 20 CH3(CH2)4(CHCHCH2)4(CH2)2 COOH -49 Eicosapentaenoic 5 20 CH3CH2(CHCHCH2)5(CH2)2 COOH -54 Essential fatty acids (EFAs)  Essential fatty acids (EFAs) are a type of polyunsaturated fatty acids (PUFAs). They are essential in that they are absolutely required for human health but cannot be synthesized by humans due to absence of human enzyme systems that can introduce a double bond except at the ninth carbon atom (9-10 position).  There are two closely related families of EFAs: omega-3 (ω-3 or n-3) and omega-6 (ω-6 or n-6). The distinction between ω-3 and ω-6 fatty acids is based on the location of the first double bond, counting from the methyl end (opposite end to the carboxylic group) of the fatty acid molecule.  EFAs were originally designated as Vitamin F when they were discovered as essential nutrients in 1923. Around 1930, it was realized that they are better classified with the fats than with the vitamins. 7 Lectures - PHT383 06/10/2024  There are two principal essential fatty acids: alpha-linolenic, ALA (18 carbons) and linoleic, LA (18 carbons) acids, which are considered to be the parent of the ω-3 and ω-6 series, respectively. Principal (parent) essential Fatty Acids (EFAs) No. of No. of  Series Name carbon atoms double bonds  3 Series  3, 6, 9 18 3 alpha-linolenic acid (ALA)  6 Series  6, 9 18 2 Linoleic acid (LA) Branched-chain fatty acids  Almost all fatty acids present in mammalian tissues are aliphatic i.e: straight chain. However, branched chain fatty acids are found in nature.  Some milk products contain branched chain fatty acid called phytanic acid (18 carbons). It contains 4 methyl groups at positions 2,7,11 and 15 carbons.  Refsum’s disease: Is peroxisomal disorder caused by the impaired alph-oxidation of branched chain fatty acids resulting in buildup of phytanic acid and its derivatives in the plasma and tissues. This may be due to deficiencies of phytanoyl-CoA hydroxylase or peroxin-7 activity. In general, Refsum disease is caused by PHYH mutations.  Symptoms also include ataxia, scaly skin, difficulty hearing, and eye problems including retinitis pigmentosa, cataracts, and night blindness Individuals with Refsum disease are commonly placed on a phytanic acid- restricted diet and avoid the consumption of fats from ruminant animals and certain fish, such as tuna, cod, and haddock. Grass feeding animals and their milk are also avoided. 8 Lectures - PHT383 06/10/2024 Digestion of lipids The main lipids in diet are triglycerides (99%). Diet also contains some phospholipids, cholesterol and fat soluble vitamins. Triglycerides are digested by a group of enzymes: 1- Lingual lipase. 2- Gastric lipase. 3- Pancreatic lipase. 4- Intestinal lipase. The presence of emulsifying agent (which helps solubilization) such as bile salts (which are synthesized in the liver and secreted from the gallbladder) is important for the action of pancreatic and intestinal lipase. Emulsification means breakdown of large globules into small ones. This increase the surface area of lipids exposed to lipase enzyme. Cholesterol itself undergoes no digestion and absorbed as such. Cholesteryl esters are digested by cholesterol esterase enzyme into cholesterol and fatty acids. Phospholipids may be absorbed as such or digested by phospholipase enzymes. They act on phospholipids hydrolyzing them into fatty acids, glycerol, phosphate and nitrogenous base. 9 Lectures - PHT383 06/10/2024 Absorption of lipids The end products of lipid digestion are monoacylglycerols, fatty acids [short chains (C4-C11) and long chains (C12-C18)], glycerol, phospholipids and cholesterol. They are absorbed from the intestine. Short chain fatty acids (less than 12 carbons) and glycerol are water-soluble and pass via portal circulation to the liver Other lipids are water insoluble. They combine with bile salts to form water-soluble complex called micelles, which enter the mucosal cell. Bile salts are reabsorbed to the liver again (enterohepatic circulation). Long chain fatty acids are activated in the mucosal cells and combine with mono- and diacylglycerols to form triacylglycerols again. Triacylglycerols, phospholipids and cholesterol are bound to a protein called apolipoprotein to form chylomicrons which enter the circulation through lymphatic vessels. 10 Lectures - PHT383 06/10/2024 Intestinal Wall Lumin Liver Glycerol Glycerol Portal circulation Short chain FA Short chain FA Bile salts Bile salts + Thoracic Duct Monoacylglycerol Monoacylglycerol Micelles Triacylglycerols + Long chain FA Long chain FA + Chylomicrons Cholesterol Cholesterol + Lacteals Phospholipids Phospholipids Protein Systemic Absorption of lipids Circulation Fate of absorbed lipids  After fatty meal, plasma shows a milky appearance. This due to that venous blood contains excess chylomicrons after absorption.  Excess chylomicrons stimulate mast cells to produce heparin that stimulate the lining epithelium of blood vessels to produce an enzyme called lipoprotein lipase (plasma clearing factor).  Lipoprotein lipase enzyme will act on triacylglycerols of chylomicrons, converting them into glycerol and free fatty acids. Lipoprotein lipase Triacylglycerol Glycerol + Fatty acids Glycerol and fatty acids are taken up by different tissues for different functions. 11 Lectures - PHT383 06/10/2024 Lipogenesis  Fatty acids synthesis, or lipogenesis, consists of series of reactions in which a molecule of fatty acid is built up from the two carbon units derived from acetyl CoA.  Any substance can give acetyl CoA (e.g. glucose) is called lipogenic.  There are 3 mechanisms for fatty acids synthesis: Cytoplasmic, mitochondrial and microsomal. Microsome: fragment of the endoplasmic reticulum I- Cytoplasmic system for fatty acid synthesis (de novo synthesis of fatty acids) Also called extramitochondrial system. The main product of this pathway is palmitate (16C). A- Site: Cytoplasm of liver, adipose tissue, lactating mammary gland, lung and kidney. 12 Lectures - PHT383 06/10/2024 Production of malonyl CoA from acetyl CoA This is the irreversible, rate-limiting step of fatty acid synthesis. The carboxylation of acetyl CoA is catalyzed by acetyl CoA carboxylase, which requires the vitamin biotin as a cofactor. Biotin is covalently attached to a lysine residue of the enzyme and takes part in the reaction. The biotin group of the enzyme is first carboxylated, creating an active carboxyl group for further transfer to acetyl CoA. Fatty acid synthase Fatty acid synthesis requires several enzymes. In eukaryotes those enzymes are joined together, forming a multi-enzyme complex called fatty acid synthase. Fatty acid synthase is a dimer of two identical subunits, each with seven different enzymatic activities that catalyze a different reaction of fatty acid synthesis. Each subunit also contains the acyl carrier protein (ACP). Subunits of fatty acid synthase are folded into three domains joined by flexible regions. Both the acyl carrier protein and one of the enzymes, the condensing enzyme (β-ketoacyl synthase), contain important thiol (sulphydryl) groups. 13 Lectures - PHT383 06/10/2024 1 4 6 7 5 2 3 Stages of fatty acid synthesis Formation of saturated fatty acids 1. Addition of acetyl and malonyl groups Acetyl transacylase catalyzes the transfer of the acetyl group from acetyl CoA to the thiol group (SH) of the acyl carrier protein. It is then transferred to the thiol group of the condensing enzyme (β-Ketoacyl synthase). Malonyl transacylase then transfers the malonyl group from malonyl CoA to the acyl carrier protein. 2. Condensation β-Ketoacyl synthase catalyzes the condensation of acetyl (2C) and malonyl (3C) groups to form acetoacetyl-ACP (4C). The reaction is driven by the loss of CO2. 14 Lectures - PHT383 06/10/2024 3. Reduction The keto group at C3 is reduced to an alcohol group by β-ketoacyl reductase. The reducing agent for this reaction is NADPH+H+. 4. Dehydration The removal of water by β-hydroxyacyl dehydratase introduces a double bond. 5. Reduction Enoyl reductase catalyzes the second reduction producing a saturated four-carbon fatty acyl chain. This completes the first elongation cycle. 6. Site-to-site transfer The four-carbon chain is transferred to the thiol group of the cysteine residue of the condensing enzyme (β-ketoacyl synthase). 7. Addition of a second malonyl CoA to the acyl carrier protein The four-carbon chain condenses with malonyl CoA and steps 2-6 are repeated to form a saturated six-carbon acyl chain. The cycle is in fact repeated a further six times until a 16-carbon chain, palmitate, is made, i.e., seven cycles altogether.  The enzyme thioesterase then catalyzes the release of palmitate. Therefore, the synthesis of one molecule of palmitate uses one molecule of acetyl CoA and seven of malonyl CoA.  Remember, all the carbon atoms of fatty acids originally come from acetyl CoA. 15 Lectures - PHT383 06/10/2024 Acetyl transacylase β-Ketoacyl synthase Malonyl transacylase β-ketoacyl reductase β-hydroxyacyl dehydratase Enoyl reductase II- Microsomal pathway for fatty acid synthesis  This is probably the main site for the elongation of existing long chain fatty acid molecules i.e. production of fatty acids longer than 16 carbon atoms.  The elongated molecules (C10-C16) are derived from either: 1- Palmitate: by cytoplasmic pathway. 2- Fatty acids of diet.  The microsomal pathway needs malonyl CoA as acetyl donor and NADPH+H+ as coenzyme.  This system gets active in nervous system in order to provide C22 and C24 fatty acids which are present in sphingophospholipids. 16 Lectures - PHT383 06/10/2024 III- Mitochondrial pathway for fatty acid synthesis  This pathway occurs in mitochondria, by elongation of acyl CoA.  It is important for the elongation of short-chain fatty acids, that is, those containing 14 carbon atoms or less, and it takes place in the mitochondrial matrix.  Two carbon units are added directly from acetyl CoA, not malonyl CoA. Desaturation of fatty acids  This pathway is located in the membrane of the smooth endoplasmic reticulum.  Mammalian systems have four different desaturase enzymes capable of producing double bonds at positions Δ4, Δ5, Δ6, and Δ9.  Unsaturated fatty acids are necessary for the synthesis of important membrane phospholipids and intracellular messengers such as prostaglandins. 17 Lectures - PHT383 06/10/2024 Synthesis of Triacylglycerol Fatty acids are stored as triacylglycerol molecules in the cytosol of adipose cells. Triacylglycerol (Triglycerides) consist of a glycerol backbone esterified with three fatty acids. Synthesis of triacylglycerol molecules can be achieved using fatty acids (acyl CoA) and glycerol (glycerol-3-phosphate). Remember that acyl group is the carboxylic acid part of the compound Acyl group O R C 1- Activation of fatty acids into acyl CoA Acyl group O Thiokinase R-COOH R-C-CoA Fatty acid Acyl CoA ATP + AMP CoASH 2- Synthesis of glycerol-3- phosphate In liver, kidney, intestine, and lactating mammary glands: Glycerol-3-phosphate is formed from glycerol by glycerokinase or from glucose through glycolysis. Glycerokinase Glycerol Glycerol phosphate ATP ADP In muscles and adipose tissues Glycerokinase is deficient. In these tissues, glycerol-3-phosphate is formed from glucose (through glycolysis) as follows: Glucose → Dihydroxyacetone Phosphate → Glycerol phosphate 18 Lectures - PHT383 06/10/2024 CH2OH O HO-C-H O O CH2-O-C-R1 CH2-O-P-O- CoA-C-R1 CoA HO-C-H O O- CH2-O-P-O- Acyltransf earase O- Glycerol phosphate Lysophosphatidic acid CoA-C-R2 Acyltransfearase O O CoA O CH2-O-C-R1 O R2-C-O-C-H O CH2-O-C-R1 CH2OH R2-C-O-C-H O Diacylglycerol CH2-O-P-O- O- Synthesis of triacylglycerol Phosphatidic acid O O CH2-O-C-R1 R2-C-O-C-H O CH2-O-C-R3 Triacylglycerol After meal Insulin is secreted which stimulates glycolysis. Glycolysis supplies dihydroxyacetone phosphate that convert into glycerol-3-phosphate in adipose tissue. This will end up with stimulation of lipogenesis. During fasting Anti-insulin hormones are secreted. These inhibit lipogenesis and stimulate lipolysis. LIPID BREAKDOWN  Triacylglycerol stores in adipose tissue serve as the body's major fuel reserve.  Fatty acid breakdown is the process by which a molecule of fatty acid is degraded by the sequential removal of two carbon units, producing acetyl CoA, which can then be oxidized to CO2 and H2O by the TCA cycle. 19 Lectures - PHT383 06/10/2024  Lipid breakdown occurs in many tissues, especially liver and muscle. Certain tissues are unable to oxidize fatty acids, (examples: brain, and RBCs) because they lack the necessary enzymes. Stages of lipid breakdown 1) Lipolysis: Hydrolysis of triacylglycerol by lipase enzyme. 2) Activation of fatty acids. 3) Transport of fatty acids into mitochondria. 4) -Oxidation. 1) Lipolysis  Lipolysis is the hydrolysis of triacylglycerols in adipose tissue into glycerol and fatty acids.  Glycerol travels to the liver, where it is phosphorylated and oxidized to dihydroxyacetone phosphate, which in turn is isomerized to glyceraldehyde-3-phosphate. This intermediate is on both the glycolytic and gluconeogenic pathways. It can therefore be converted into pyruvate or glucose in the liver.  The free fatty acids travel in the blood bound to albumin and are taken up by muscle or liver cells for oxidation. 20 Lectures - PHT383 06/10/2024 Lipolysis is carried out by a number of lipase enzymes, which are present in adipose tissue. These are: 1. Hormone sensitive triacylglycerol lipase. 2. Diacylglycerol lipase. 3. Monoacylglycerol lipase. Hormone-sensitive triacylglycerol lipase Triacylglycerol diacylglycerol + free fatty acid Monoacylglycerol Diacylglycerol lipase Glycerol + lipase free fatty acid monoacylglycerol + free fatty acid The key enzyme controlling lipolysis is the hormone sensitive triacylglycerol lipase enzyme. It exists in 2 forms: active (phosphorylated) and inactive (dephosphoryalted). 21 Lectures - PHT383 06/10/2024  Many hormones (epinephrine, norepinephrine, glucagon, ACTH and TSH) are stimulating adenylate cyclase enzyme → formation of cAMP → activation of protein kinase enzyme → phosphorylation of hormone sensitive triacylglycerol lipase → stimulation of lipolysis.  Insulin is stimulating both of phosphodiesterse enzyme and lipase phosphatase enzyme → dephosphorylation and inactivation of hormone sensitive triacylglycerol lipase enzyme → inhibition of lipolysis. Caffeine is a substance present in coffee and tea. It inhibits phosphodiesterase enzyme → stimulation of lipolysis. Epinephrine Nor-Epinephrine Glucagon Thyroid H. Glucocorticoids H. Insulin (+) (+) Adenylate cyclase Phosphodiesterase ATP cAMP 5/-AMP (-) (-) (+) Prostaglandin E PPi Caffeine Nicotinic acid Protein Kinase phosphorylase HS Lipase (b) ATP ADP P (Inactive) Insulin HS Lipase (a) (Active) (+) Lipase phosphatase Stimulate Lipolysis Pi H 2O Rregulation of Lipolysis 22 Lectures - PHT383 06/10/2024 2) Activation of fatty acids  Before they can be oxidized, fatty acids are activated by attachment to CoA to form acyl CoA molecules; this takes place in the cell cytosol.  Fatty acyl CoA synthetase (thiokinase) activates fatty acids and this requires ATP, which is hydrolyzed to AMP and pyrophosphate (PPi), breaking a high-energy phosphate bond. The reaction is made irreversible by the rapid hydrolysis of the pyrophosphate to two free inorganic phosphates by pyrophosphatase, consuming a second high-energy phosphate bond.  Therefore, the activation of a fatty acid consumes two ATP equivalents. 3) Transport of fatty acyl CoA molecules into mitochondria  The activation of fatty acids occurs in the cytosol, but the enzymes for β oxidation are in the mitochondrial matrix. The inner mitochondrial membrane is relatively impermeable to long-chain acyl CoA molecules, so a special transporter system is required to carry the fatty acid across.  The carnitine shuttle consists of three enzymes: a translocase and two carnitine acyl transferases, CAT I and II. 23 Lectures - PHT383 06/10/2024 1) The acyl group is transferred from CoA to carnitine by carnitine acyl transferase I (CAT I), an enzyme found on the cytosolic side of the inner mitochondrial membrane. 2) Acylcarnitine is transported across the membrane by the translocase to the mitochondrial matrix. 3) The acyl group is transferred back to CoA by carnitine acyl transferase II (CAT II), located on the inner surface of the inner mitochondrial membrane. 4) Carnitine is returned to the cytosolic side in exchange for another molecule of acylcarnitine. -Oxidation Acyl CoA molecules inside the mitochondrial matrix undergo β oxidation in a cyclical sequence of four reactions: 1) Oxidation. 2) Hydration. 3) Oxidation. 4) Thiolytic cleavage. 24 Lectures - PHT383 06/10/2024 1) Oxidation (first oxidation – unsaturation of fatty acids) The oxidation of acyl CoA introduces a double bond between the C2 and C3 atoms (α and  carbons). FADH2 is produced and enters the electron transport chain. 2) Hydration Hydration is the addition of water across the double bond between α and  carbons by Δ2 enoyl-CoA hydratase. 25 Lectures - PHT383 06/10/2024 3) Oxidation (second oxidation) β-Hydroxyacyl CoA dehydrogenase converts the OH group at C3 (the β carbon) to a keto group. NADH+H+ is produced and enters the electron transport chain. 4) Thiolytic cleavage Thiolase cleaves the molecule to release acetyl CoA, and acyl CoA is shortened by two carbon atoms. The shortened acyl CoA is ready to undergo another sequence of β oxidation. 26 Lectures - PHT383 06/10/2024 The four steps are repeated until the fatty acid is oxidized completely to acetyl CoA. The last round of oxidation produces two molecules of acetyl CoA. Ch3-co-ch2-co-scoA For even-numbered, saturated fatty acids, (n/2)-1 cycles are required for complete oxidation, where n = the number of carbons of the fatty acid. For example, C16 palmitate requires (16/2)-1 = 7 cycles. ATP yield from the -oxidation of palmitic acid (16 carbons)  β-Oxidation of palmitic acid will be repeated 7 times (turns) to produce 8 acetyl CoA. Oxidation of one molecule of acetyl CoA in citric acid cycle gives 12 ATP. ˙. 8 Acetyl CoA x 12 ATP = 96 ATP.  In each turn, one molecule of reduced FADH2 and one molecule of reduced NADH+H+ are produced. They are oxidized in respiratory chain to give 5 ATP. FADH2 → 2 ATP, NADH+H+ → 3 ATP.˙. 7 turns x 5 ATP → 35 ATP. 27 Lectures - PHT383 06/10/2024  Tow high energy phosphate bonds are utilized in the first reaction (catalyzed by acyl CoA synthetase) which occurs for one time only. Net energy gain = (35 ATP + 96 ATP) – 2 ATP= 129 ATP Calculation of energy production of oxidation of any fatty acid = {(N/2 – 1) X 5 ATP} + {N/2 X 12 ATP} – 2 ATP Where N= Number of carbons of fatty acid e.g. palmitic acid = 16 carbons, so energy production = {(16/2 – 1) x 5 ATP} + {16/2 X 12 ATP} – 2 ATP ={(8 – 1} X 5 ATP}+{8 X 12 ATP} - 2 ATP = 129 ATP Oxidation of odd- Margaric acid (C17) numbered fatty acids  The oxidation of odd- numbered fatty acids is essentially the same as for even-numbered fatty acids, except that the last round of β oxidation produces one molecule of acetyl CoA and one of propionyl CoA (3C) instead of two molecules of acetyl CoA. (n-1/2)-1=(17- 1/2)-1  Propionyl CoA is metabolized to succinyl CoA, which can then enter the TCA cycle. 28 Lectures - PHT383 06/10/2024 Cholesterol Metabolism Role of cholesterol in the body  An essential component of cell membranes.  A precursor of the major classes of steroid hormones (eg: estrogens, androgens, glucocorticoids, and mineralocorticoids).  A precursor of bile acids and vitamin D. The body therefore requires a continuous supply of cholesterol. Cholesterol can either be obtained from the diet or be synthesized endogenously by the body.  Regulatory mechanisms try to balance the amount of cholesterol made by the body daily with both the dietary intake and the amount excreted.  Cholesterol is a 27-carbon steroid molecule. All 27 carbon atoms of cholesterol come from acetyl CoA. Structure of cholesterol 29 Lectures - PHT383 06/10/2024 Cholesterol Synthesis  Cholesterol is made by most tissues (except RBCs) but the main site of synthesis is the liver.  Cholesterol synthesis occurs in cell cytosol, although certain reaction occurs in the endoplasmic reticulum.  The easiest way to view cholesterol synthesis is to divide it into two stages. Stage I: The formation of the isoprene unit [isopentenyl pyrophosphate (IPP)]. This is formed by the condensation of three molecules of acetyl CoA to 3-hydroxy-3- methylglutaryl CoA (HMG-CoA), followed by the loss of CO2. 30 Lectures - PHT383 06/10/2024 Stage II: The progressive condensation of isoprene units to form cholesterol. Six five- carbon isoprene units link up to form squalene (30C atoms) which cyclizes to lanosterol, from which Squalene synthase catalyzes the reductive condensation of two cholesterol arises. molecules of FPP, forming the 30-carbon molecule, squalene Cyclization of squalene to lanosterol (30C) by squalene monoxygenase Conversion of lanosterol to cholesterol: The exact pathway is not known, but it is thought to consist of about 20 steps. Regulation of cholesterol synthesis  Regulation is necessary in order to prevent high plasma cholesterol levels, which may lead to cholesterol deposition in arterial walls and the formation of atherosclerotic plaques.  The primary control site is the rate-limiting enzyme HMG-CoA reductase. HMG-CoA reductase is inhibited by cholesterol (product inhibition). Glucagon inhibits HMG-CoA reductase and therefore decreasing the rate of cholesterol synthesis 31 Lectures - PHT383 06/10/2024  Insulin activates HMG-CoA reductase and therefore increasing the rate of cholesterol synthesis.  HMG-CoA reductase inhibition is a mechanism of action of drugs inhibiting cholesterol synthesis (statins).  Long-term regulation of HMG-CoA reductase. This is the most important control mechanism. The amount of cholesterol, both dietary and endogenous, taken up by cells affects the amount of HMG-CoA reductase synthesized. A high cholesterol level in cells causes a decrease in the rate of transcription of the HMG-CoA reductase gene, inhibiting it and leading to a reduction in cholesterol synthesis. 32 Lectures - PHT383 06/10/2024 Packaging of cholesterol  Most of the cholesterol in the blood is in the form of cholesterol esters, formed by the addition of a fatty acid to the C3-OH group (Recall cholesterol structure). Esterification makes the cholesterol more hydrophobic, enabling it to be packaged, stored, and transported more easily.  Two enzyme systems are responsible for the esterification of cholesterol. One in cells and the other in high density lipoproteins. 1- In cells:  If the cholesterol taken up or synthesized by cells is not immediately required, then it is esterified by acyl CoA : cholesterol acyl transferase (ACAT).  ACAT transfers a fatty acid from a fatty acyl CoA to cholesterol, forming a cholesterol ester that can be stored in the cell. 33 Lectures - PHT383 06/10/2024 2- In high-density lipoproteins:  A similar enzyme, known as lecithin : cholesterol acyl transferase (LCAT), is found associated with high- density lipoproteins (HDLs). HDL is responsible for picking up free cholesterol from peripheral tissues and transporting it to the liver; that is, it acts as a cholesterol scavenger.  LCAT catalyzes the transfer of a fatty acid from the phospholipid, phosphatidylcholine, to cholesterol. HDL then carries cholesterol esters to the liver, either to be reused or excreted. 34 Lectures - PHT383 06/10/2024 Lipids transport  Lipids are insoluble in aqueous solution and therefore are transported in the plasma in association with proteins in the form of lipoproteins.  Lipoproteins consist of a lipid core of mainly non polar triglycerides and cholesterol esters surrounded by a surface layer of more polar phospholipids, free cholesterol and protein fraction known as apoprotein or apolipoprotein.  Lipoproteins function both to solubilize the lipids and to provide an efficient transport system for them. If the system fails, the plasma lipid concentration will increase with an increased risk of different diseases.  Apolipoproteins are only weakly associated with lipoprotein complexes and can be transferred easily between them. They did not only serve for solubility purposes, but rather function as recognition molecules for the membrane receptors and enzymes that are involved in lipid exchange. 35 Lectures - PHT383 06/10/2024 Classes of lipoproteins There are five main classes of lipoproteins: 1) Chylomicrons (CMs). 2) Very-low-density lipoproteins (VLDLs). 3) Intermediate-density lipoproteins (IDLs). 4) Low-density lipoproteins (LDLs). 5) High-density lipoproteins (HDLs). They are classified according to increasing density, with CMs having the lowest density and HDLs the highest. Remember, protein is more dense than lipid and so HDLs, with the highest density, must contain the most protein. Each one of the 5 fractions of plasma lipoproteins contains almost all types of lipids but they differ in: 1- The main lipid content of each fraction. 2- Source of each fraction. 3- Types & amounts of associated proteins (apolipoproteins). Briefly Chylomicrons: Transport exogenous (dietary) triglycerides. VLDL: Transport endogenous triglycerides from liver to peripheral tissues. LDL: Transport cholesterol from liver to peripheral tissues. HDL: Reverse cholesterol transport. It transport cholesterol from tissues to liver (cholesterol scavenger). 36 Lectures - PHT383 06/10/2024 Pathways of lipid transport Lipids can either be obtained from the diet (exogenous lipids) or synthesized by the body (endogenous lipids). There are two different pathways for lipid transport in the body: I. Exogenous pathway: CMs transport dietary lipid absorbed from the intestine to the tissues. II. Endogenous pathway: VLDLs, IDLs and LDLs form a continuous cascade. They transport endogenously synthesized triacylglycerol and cholesterol from the liver to the tissues. Exogenous pathway  CMs are assembled in intestinal mucosal cells from dietary fat. They contain mainly triacylglycerol.  These newly formed chylomicrons are referred to as nascent CMs.  Nascent CMs enter the blood and carried to tissues. As they pass through the capillaries of tissues, the enzyme lipoprotein lipase (LPL), found on the luminal surface of the capillary endothelium, is activated.  LPL hydrolyzes CMs content of triacylglycerol to glycerol and free fatty acids. The fatty acids are taken up by cells, either for oxidation or resynthesis of triacylglycerol to be stored.  The removal of triacylglycerol leaves behind a much smaller CM remnant particle. The CM remnants are taken up by the liver and degraded. 37 Lectures - PHT383 06/10/2024 Endogenous pathway  VLDLs are synthesized in the liver, mostly from triacylglycerol. VLDL transports endogenous triacylglycerol to peripheral tissues.  Some triacylglycerol are transferred to HDL, converting VLDL to the denser lipoprotein, IDL.  Cholesterol esters are, then, transferred from HDL to IDL in exchange for triacylglycerol by cholesterol ester transfer protein (CETP). Some of the IDLs are taken up by the liver; but the rest forms LDL. LDL provides cholesterol for peripheral tissues  LDL binds to LDL receptors on cell membranes. Lysosomal enzymes hydrolyze LDL, releasing free cholesterol into the cell. HDL metabolism  HDL is made in the liver. It accepts the free cholesterol from peripheral tissues and esterifies it by the action of LCAT. The cholesterol esters formed are either transferred to IDL to form LDL, or are carried back to the liver by "reverse cholesterol transport."  Whereas an increase in LDL-cholesterol is harmful, an increase in HDL-cholesterol has a protective effect because it removes cholesterol from tissues and takes it to the liver for degradation and excretion. 38 Lectures - PHT383 06/10/2024 Ketone bodies Ketone bodies are 3 compounds formed by the liver and include: O OH CH 3 - C - CH 2 - COOH CH 3 - CH - CH2 - COOH 1- Acetoacetate. 2- -Hydroxybutyrate. O CH3 - C - CH 3 3- Acetone. Ketogenesis (Synthesis of ketone bodies) Site: Organ location: Liver Intracellular location: Mitochondria. Precursor: Acetyl CoA (derived from fatty acids oxidation and ketogenic amino acids). 39 Lectures - PHT383 06/10/2024 O 2 CH3C - CoA Fatty acyl CoA 2 Acetyl CoA O O CH3C - CH2 - C - CoA Acetoacetyl CoA H2O O CH3C - CoA HMG CoA Acetyl CoA synthase CoASH O OH O HO - C - CH2 - C - CH2 - C CoA CH3 HMG CoA HMG CoA O HMG CoA lyase CH3C - CoA O O Acetyl CoA CH3C - CH2 - C OH Acetoacetate Spontaneous NADH+H+ -Hydtoxybutyrate dehydrogenase CO2 NAD+ O H O CH3 - C - CH3 CH3 - C - CH2 - C - OH Acetone OH -Hydroxybutyrate 40 Lectures - PHT383 06/10/2024 Regulation of Ketogenesis Adipose tissue Liver Blood Lipolysis Esterificateion TG FFA FFA→ Acyl CoA TG -Oxidation Insulin Glucagon Acetyl CoA Citric acid cycle Ketogenesis Ketone bodies Ketolysis (Oxidation of ketone bodies) Site: Organ location: Extrahepatic tissue because liver does not contain enzymes for ketolysis. Intracellular location: Mitochondria. 41 Lectures - PHT383 06/10/2024 Steps: 1) Acetone is volatile and removed in the expired air. 2) β-hydroxybutyrate is converted into acetoacetate by hydroxybutyrate dehydrogenase enzyme. 3) Acetoacetate is then converted into acetoacetyl CoA by thiophorase enzyme in the presence of succinyl CoA. 4) Acetoacetyl CoA is splitted into 2 molecules of acetyl CoA which are oxidized in citric acid cycle. Liver Amino acid Fatty acid Glycolysis Catabolism Oxidation 2 Acetyl CoA CoA Peripheral Tissues Acetoacetyl CoA Blood (e.g. muscle) Acetyl CoA CoA Acetoacetyl CoA 2 Acetyl CoA HMG CoA Succinate CO2 Acetyl CoA Thiophorase TCA Cycle Acetoacetate Acetoacetate Acetoacetate Succinyl CoA Acetone NADH + H+ NADH + H+ NAD+ NAD+ -Hydroxybutyrate -Hydroxybutyrate -Hydroxybutyrate Ketogenesis Ketolysis 42 Lectures - PHT383 06/10/2024 Blood ketone bodies: Less than 3 mg/dl. Urine ketone bodies: Less than 40 mg/day. Ketonemia: is the increase of blood ketone bodies above normal concentration. Ketonurea: is the increase of urine ketone bodies above normal concentration.  Ketonemia occurs when the rate of formation of ketone bodies (ketogenesis) is grater then the rate of their oxidation (ketolysis). The concentration of blood ketone bodies may reach up to 90 mg/dl.  Ketonurea occurs with ketonemia. The concentration of urine ketone bodies may reach up to 5000 mg/day.  Severe ketonemia may lead to acidosis (ketosis). 43 Lectures - PHT383 06/10/2024 Causes of ketonemia and ketonurea: 1- Starvation. 2- Severe diabetes mellitus. Ketosis (= Ketoacidosis): It is a condition of metabolic acidosis results from ketonemia. Increase ketone bodies in blood is neutralized by blood buffers [mainly bicarbonate (HCO3-)]. Bicarbonate will be depleted and this leads to decreased blood pH (acidosis). Phospholipids A class of lipids that are a major component of all cell membranes. They can form lipid bilayers because of their amphipathic characteristic. Fatty acids + Alcohol + Phosphoric acid + Nitrogenous base. Classification of phospholipids I- Glycerophospholipids or phosphoglyceride:- Alcohol is Glycerol II- Sphingophospholipids or sphingomyelins: Alcohol is Sphingosine They are Amphipathic molecules i.e. contain a hydrophilic part (phosphate , nitrogenous base) and a hydrophobic part (fatty acid tails) 44 Lectures - PHT383 06/10/2024 Glycerophospholipids The major lipid in biological membranes Glycerol -3-p is esterified at c1& c2. C1---saturated fatty acid C2 ---unsaturated fatty acid And has a phosphate and a variable (X) group at C3 Phosphatidic acid is an intermediate in synthesis of Triglyceride & Phospholipids Phosphatidic acid 45 Lectures - PHT383 06/10/2024 Types of Glycero-phospholipids 1. Phosphotidyl Choline (Lecithin)  Present in membrane, plasma, & bile  Forms component of cell membrane  Decrease surface tension of aqueous later of lung  Detergent property solubilize cholesterol in bile (Gall stone)  LCAT esterify cholesterol in HDL 2. Phosphotidyl Ethanol amine.(Cephalin) Cephalins are found in most cell membranes, particularly in brain tissues. They also important in the blood clotting process as they are found in blood platelets 3. Phosphotidyl Serine. Apoptosis 4. Phosphotidyl Inositol Precursor of Second Messengers 5. Cardiolipin. Mitochondrial Membranes 6. Phosphatidyl Glycerol. 46 Lectures - PHT383 06/10/2024 Sphingophospholipids Sphingomyelin is found in myelin, a bilayer that wraps around nerve cell axons. 47 Lectures - PHT383 06/10/2024 Functions of phospholipids 1- structural component of membranes regulating membrane permeability 2- lecithin, cephalin and cardiolipin assist cell respiration and are part of the electron transport chain 3- their amphipathic nature allowed them to interact with polar and non polar molecules 4- help in fat absorption from the intestine 5- part of lipoproteins for lipid transport (LDL/VLDL/HDL) 6- protect against fatty liver (lipotropic factors) 7. Take part in synthesis of prostaglandins, prostacyclins and thromboxanes 8. Signal transmission across membranes 9. Component of bile to aid fat emulsification 10. Involved in reverse cholesterol transport 11. Lowers surface tension (surfactant) e.g dipalmitoyl phosphatidyl choline (a lung surfactant that causes respiratory distress syndrome if deficient) 48 Lectures - PHT383 06/10/2024 Types of Glycolipids Glycolipids are glycoconjugates of lipids. The term glycolipid Glycoglycerolipids designates any compound containing one or more monosaccharide residues bound Glycosphingolipids by a glycosidic linkage Function Carbohydrates on glycolipids are the most exposed structures on the extracellular surface of cells and are flexible with numerous binding sites which make them optimal for cell signaling.  Signal Transduction  Cell Proliferation  Calcium Signaling  Help in determine blood group of an individual  Acts as surface receptors for some toxins and viruses Disease associated with Glycolipids metabolism Genetic defects in glycolipid metabolism are the cause of lysosomal storage diseases, a group of progressive, and often fatal disorders, and are implicated in some neurological conditions like  Niemann-Pick disease which can cause pain and damage to neural networks, and is usually fatal in early infancy.  Tay–Sachs disease is a genetic disorder that results in the destruction of nerve cells in the brain and spinal cord 49 Lectures - PHT383 06/10/2024 Phosphololipids (PS) and phosphatidylcholine (PC) Brain cell receptors that catch the neurotransmitter signals like dopamine (attention and motivation), serotonin (happiness) and acetylcholine (memory) are located inside the fatty cell membrane. For cell communication to function properly, it should have enough supply of healthy membrane building blocks. Human brain contains very high amounts of PS in cell membranes which regulate nutrients and toxins movement. PS are important for neurotransmitter flow and effective communication between brain cells. Supplements with PS helps brains cells to work better improving attention, learning, memory, mood and stress reduction. PC help neurogenesis; a process that help making new brain cells and neural connections. PC maintains memory, concentration and mood 50 Lectures - PHT383 06/10/2024 Fatty liver This is an accumulation of abnormal amount of fat in the liver for a long time with subsequent compression of liver cells. This result in liver fibrosis and impairment of liver function. Causes: 1- Over mobilization of fat from extrahepatic tissue to the liver. 2- High carbohydrate diet. 3- Under mobilization of fat from the liver to the plasma. Over mobilization of fat from extrahepatic tissue to the liver: 1- During high fat diet. 2- Due to excessive lipolysis as in carbohydrate low diet, starvation and diabetes mellitus. High carbohydrate diet: On high carbohydrate diet, liver is first saturated with glycogen, then any further amount of carbohydrates will be directed into lipogenesis. Under mobilization of fat from liver to the plasma: This is due to deficiency of any factor essential for plasma lipoproteins formation. 51 Lectures - PHT383 06/10/2024 Fatty liver Fatty liver or hepatic steatosis describes the buildup of fat in the liver large vacuoles of triglyceride fat accumulate in liver cells via the process of steatosis i.e., abnormal retention of lipids within a cell Fatty liver develops when the body creates too much fat or cannot metabolize fat efficiently enough. So that The excess fat is stored in liver cells where it accumulates causing fatty liver disease Liver is the main metabolic organ and too much fat in the liver cells can lead to long-term liver damage mild fatty liver can be reversed by lifestyle modifications such as diet changes, weight loss, and increased physical activity. Causes of fatty liver Nutritional causes Metabolic causes obesity glycogen storage diseases Malnutrition Weber–Christian disease Diet rich in processed sugar acute fatty liver of pregnancy and triglycerides Lipodystrophy Heavy alcohol drinking Diabetes total parenteral nutrition hyperlipidemia, especially high severe and rapid weight loss triglycerides Re-feeding syndrome Jejuno-ileal bypass gastric bypass 52 Lectures - PHT383 06/10/2024 Other causes include inflammatory bowel disease, Viral infections like HIV and hepatitis C Some drugs like methotrexate, tamoxifen, amiodarone, and valproic acid low physical activity Genetics celiac disease The Risk Factors for fatty liver include obesity high triglycerides diabetes Diagnosis: Physical examination, blood tests, imaging and biobsy In many cases, fatty liver has no symptoms, but the Amount of fat in liver can be estimated by Ultrasounds, CT scans, and MRI scans. Fatty liver Normal Laboratory Abnormalities 2 - 4 fold  AST and ALT AST: ALT Ratio < 1 High serum triglycerides Normal serum urea and creatinine Normal Albumin and prothrombin time High Serum Ferritin High Iron saturation SGOT: SGPT Ratio > 1 if Cirrhosis starts SGOT: serum glutamate oxaloacetate transaminase (AST) SGPT: serum glutamate pyruvate transaminase (ALT) 53 Lectures - PHT383 06/10/2024 Types of Fatty liver There are two basic types of fatty liver: nonalcoholic and alcoholic and both are basically chronic disorders. a- Nonalcoholic fatty liver disease (NAFLD) It develops when the liver has difficulty breaking down fats, which causes a buildup in the liver tissue. The cause is not related to alcohol. NAFLD is diagnosed when more than 5 percent of the liver is fat. B- Nonalcoholic steatohepatitis (NASH) Nonalcoholic steatohepatitis (NASH) is a type of NAFLD. As fat builds up, it can cause inflammation. Once more than 5 percent of the liver is fat and inflammation is also present, the condition is known as NASH. C- Alcoholic fatty liver Alcoholic fatty liver is the earliest stage of alcohol-related liver disease. Heavy drinking damages the liver so it cannot break down fats. Stopping alcohol drinking will likely reverse the fatty liver within six weeks. if excessive alcohol use continues, inflammation known as alcoholic steatohepatitis may develop, leading to cirrhosis. The Two Hit Concept Saturated > Unsaturated 1st Hit Fats Burnt Diet high in Fatty Liver VLDL-TG FFA Lipid 1st Susceptibility Accumulation HIT Oxidative Stress Oxidative 2nd Hit Stress Toxins 2nd Cytokine HIT Inflammatory Activation Molecules Damaged Liver Apoptosis Donnelly et al. J. Clin. Invest. 113: 1343, 2005; Day and James. Gastroenterol. 114: 842, 1998 54 Lectures - PHT383 06/10/2024 Acute fatty liver is a rare and potentially life threatening complication of pregnancy. Signs and symptoms begin in the third trimester. These include: persistent nausea and vomiting pain in the upper-right abdomen headache jaundice general malaise fatigue decreased appetite Treatment includes managing any complications and prompt delivery. Most women improve within several weeks after delivery and have no lasting effects. If the patient is diagnosed by NASH, it is very common to find that the patients suffers also from diabetes (70%), insulin resistance (98%) or metabolic syndrome (85%) The opposite is correct too, if diabetes, insulin resistance or metabolic syndrome are confirmed, it is very common to find a condition of NASH IR 98% NAFLD DM, MS, NASH CVD NASH DM MS 70% 85% IR= insulin resistance, MS= metabolic syndrome, DM= diabetes mellitus, CVD=cardiovascular disease 55 Lectures - PHT383 06/10/2024 The New Definition of Metabolic syndrome the patient is diagnosed to suffer from metabolic syndrome if he shows 2 of the following 5 signs: Waist Circumference  90 cm (Males), 80 cm (Females) Triglycerides >150 mg/dl HDL100 or diabetes Hypertension >130/85 Prognosis of FLD fatty liver NAFL steatohepatitis NASH steatohepatitis + fibrosis steatohepatitis + cirrhosis cryptogenic cirrhosis The liver produces new liver cells when the old ones are damaged. But repeated damage causes permanent scarring or “cirrhosis”. Fatty liver causes Liver inflammation (steatohepatitis) when it progresses, leading to liver scarring, liver cancer, and end- stage liver disease. 56 Lectures - PHT383 06/10/2024 Prognosis of Fatty Liver Simple Steatosis or Fat Deposition of > 5% Benign course 3% develop cirrhosis NASH – Ballooning, Inflammation,  Fibrosis Worse prognosis 30% develop cirrhosis Severe NASH with fibrosis – 75% go in for cirrhosis 5 yr survival 67% 10 yr survival 45% The first-line of treatment is to reduce risk factors including: limiting or avoiding alcoholic beverages AFLD managing cholesterol and reducing the intake of sugars and saturated fats losing weight controlling blood sugar 57 Lectures - PHT383 06/10/2024 Potential Drugs for treatment of NAFLD Insulin Sensitizing Agents Membrane-Stabilizing Agents  Glitazones; Metformin Urso deoxy cholic Acid Lipid-Lowering Agents Betaine  Clofibrate; Gemfibrozil Anti-Oxidants Future Potential Treatments Vitamin E; Vitamin C  Anti-fibrotics; Probiotics Lecithin; beta -Carotene  Silymarin; Selenium Vitamin B Complex 58