Medical Biochemistry for Dentistry 2024 PDF

Medical Biochemistry for Dentistry Mansoura National University Contents No. Topic Page I Theoretical (Lectures) Biochemistry 3 1 Enzyme Chemistry...

Medical Biochemistry for Dentistry Mansoura National University Contents No. Topic Page I Theoretical (Lectures) Biochemistry 3 1 Enzyme Chemistry 4 2 ATP & Introduction to Metabolism 9 3 Carbohydrates Chemistry 14 4 Carbohydrates Metabolism Overview 22 5 Lipids Chemistry 31 6 Lipids Metabolism Overview 38 7 Protein Chemistry 45 8 Protein Metabolism Overview 55 9 Minerals Chemistry 63 10 Vitamins Chemistry 69 II Practical Biochemistry 75 1 Detection of Glucose & Diagnosis of Diabetes 76 Mellitus 2 Lipoproteins Metabolism & Lipids Profile 83 3 Detection of Proteins & Liver Function Tests 91 2 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Theoretical BioChemistry 3 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Enzyme Chemistry 4 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Catalytic Proteins, Chemistry of Enzymes Enzymes Def.:  They are organic biological catalysts.  They accelerate or catalyse the chemical reactions inside the biological systems (living cells).  Although they are involved in the reaction, they are not consumed, not changed and not affect the end product at the end point of the reaction. S+E ES E+P Chemical nature of enzymes  All enzymes are protein in nature except ribozymes (RNA in nature). Being protein, they have the properties of proteins Protein enzymes are classified into 2 types: 1- Simple Protein enzymes: They are formed of protein only. 2- Complex (conjugated) Protein enzymes: formed of two parts: 1) Protein part: called apoenzyme 2) Non- protein: called cofactor * The whole enzyme is called holoenzyme - The cofactor may be coenzyme or prosthetic group. Coenzyme Prosthetic group Chemical nature Organic Inorganic Effect of heat Thermo-labile Thermo-stable Binding to enzyme. Loosely bound Firmly bound Examples NAD and FAD Metal ions such as: Cu, Ca, Fe, Mg, Zn Mechanism of Enzyme Action HOW DOES THE ENZYME WORK? 5 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Enzyme action: 1- Enzymes increase the rate of reaction by decreasing the activation energy of reaction. 2- The activation energy is needed to overcome the energy barrier between reactants and products. Factors Affect Rate of Enzyme Action: Concentration of coenzymes Enzyme concentration Concentration of ion activators Substrate concentration Time Temperature Inhibitors pH 1- Effect of enzyme concentration The rate of enzyme action is directly proportional to the concentration of enzyme provided that there are sufficient supply of substrate & constant conditions. 2- Effect of substrate concentration -The rate of reaction increases as the substrate concentration increases up to certain point at which the reaction rate is maximal (Vmax.) At Vmax, the enzyme is completely saturated with the substrate any increase in substrate concentration doesn't affect the reaction rate. Michaelis constant (Km) - It is the substrate concentration that produces half maximum velocity of enzyme. 6 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Enzymes with low Km: they act at maximal velocity at low substrate concentration. E.g. Hexokinase acts on glucose at low concentration (fasting state) Enzymes with high Km: they act at maximal velocity at high substrate concentration. E.g. Glucokinase enzyme acts on glucose at high concentration (fed state) 3- Effects of temperature - Rate of reaction increases gradually with the rise in temperature until reach a maximum at a certain temperature, called optimum temperature - The optimum temperature is 37- 40 °C in humans - After the optimum temperature, the rate of reaction decrease due to denaturation of the enzyme (60-65 °C). 4- Effect of PH - Each enzyme has an optimum PH at which its activity is maximal E.g. Optimum PH of pepsin = 1.5 - 2 Optimum PH of pancreatic lipase = 7.5 - 8 Optimum PH of salivary amylase = 6.8 5- Concentration of coenzymes: the increase in the coenzyme concentration will increase the reaction rate. 6- Concentration of ion activators: The increase in metal ion activator increase reaction rate Enzymes are activated by ions: 1- Chloride ion activate salivary amylase 2- Calcium ion activate thromobokinase enzyme 7- Effect of time: The rate of reaction is decreased by time. 8- Presence of enzymes inhibitor: presence of enzyme inhibitor decreases or stops the enzyme activity. Enzyme Inhibition  Enzyme Inhibition means decreasing or cessation in the enzyme activity.  The inhibitor is the substance that decreases the rate of enzyme action.  According to similarity between the inhibitor and substrate, enzyme inhibition is either:  Competitive inhibition: Reversible.  Non-competitive inhibition: Irreversible. 7 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University I- Competitive Inhibition:  There is structural similarity between the inhibitor and substrate.  The inhibitor and the substrate compete with each other to bind to the same catalytic site of the enzyme.  The inhibition is reversible.  It can be relieved by increasing substrate concentration. II- Non-competitive inhibition There is no structural similarity between the inhibitor and the substrate.  The inhibitor does not bind to the catalytic site as the substrate but it binds to another site.  The inhibition is irreversible.  It cannot be relieved by increasing substrate concentration. Non-competitive inhibition may be caused by:  Inhibition of sulphahydryl (SH) group: e.g. mercury (Hg++) & lead (Pb++).  Inhibition of cofactors: e.g. carbon monoxide (CO) & cyanide  Inhibition of specific ion activator: e.g. Removal of calcium ions by oxalate from blood prevents its coagulation. 8 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University ATP & Introduction to Metabolism 9 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Metabolism Energy: A central theme of Biochemistry Can be produced by oxidation of foods: glucose, FA, aa Energy is essential to: Grow, maintain structure & function of the cell. Metabolism: it is the sum total of all the reactions that take place in a living cell.  Metabolism = Catabolism + Anabolism * Catabolism: larger molecules are broken down into smaller ones in a process that usually releases energy * Anabolism: larger molecules are made from small ones in a process the usually requires energy Energy is produced by oxidation of food stuffs with the production of the end products CO2 and H2O. Biological oxidation is that oxidation which occurs in biological systems to produce energy. ATP - ATP is a high-energy phosphate compound. It is the energy currency of cell. - Structure: adenine + ribose + 3 inorganic phosphates - It contains 2 high energy phosphate bond. Function of ATP: it is a source of energy for: 1. Biosynthetic reactions. 2. Biosynthesis of cAMP. 3. Muscle contraction. 4. Nerve conduction. 5. Active absorption and secretion. 6. Active transport across biological membranes. 7. Activation of monosaccharides, FA and AA. 8. Formation of creatine phosphate: energy store in muscle. 10 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University ATP Production, How do cells make ATP?  By Phosphorylation: i.e. adding a phosphate to ADP ADP + P ------> ATP  2 mechanisms of phosphorylation: 1. Substrate level phosphorylation. 2. Oxidative phosphorylation. (1) Substrate level phosphorylation: * ATP can be formed (during metabolic pathways e.g. glycolysis, Krebs cycle) by transferring the high energy phosphate group from substrates directly to ADP. * A substrate molecule ( X-p ) donates its high energy P to ADP making ATP (2) Oxidative phosphorylation: * The electrons produced by oxidation of foodstuffs are transferred to react finally with oxygen and the produced energy is utilized for ATP synthesis (Electron Transport Chain). Electron Transport Chain (ETC), Respiratory Chain  It is a chain of catalysts that collects hydrogen atoms and electrons from substrates transferring stepwise to be oxidized in a final reaction with oxygen (electrons combine with O2 and protons) to form water and energy (Oxidative Phosphorylation).  Associated with cell breath, also, called respiratory chain. Significance of Electron Transport Chain * Trap the energy released as ATP. * It represents the final stage in the oxidation of carbohydrates, fats, and amino acids. Site: Located in the inner membrane of mitochondria. 11 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Components of the electron Transport Chain 1. Hydrogen and electron carriers. 2. Four membrane-bound enzyme complexes. All are embedded in inner mitochondrial membrane. Oxidative Phosphorylation  It means coupling of the electron transport in respiratory chain with phosphorylation of ADP to form ATP (by Complex V = ATP Synthase).  It is a process by which the energy of biological oxidation is ultimately converted to the chemical energy of ATP.  There are 3 sites (Complex I, III, IV).  Starting via NAD, 3 ATP are formed for each substrate molecule oxidized.  Starting via FAD, 2 ATP are formed for each substrate molecule oxidized. P/O Ratio  The relationship between ATP synthesis and O2 consumption.  It is a measure to the efficiency of oxidative phosphorylation.  When NADH is used, P/O = 3/1  When FAD is used, P/O = 2/1 12 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Uncouplers  They are substances that dissociate oxidation from phosphorylation leading to loss of the resulting energy as heat.  There is normal oxygen consumption without ATP generation and P/O ratio becomes zero.  Example of the uncouplers include: 1. Calcium injection. 2. Dicoumarol, used as anticoagulant. 3. Thyroid hormones. 4. Progesterone. 13 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Carbohydrates Chemistry 14 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Carbohydrate Chemistry, Monosaccharides Definition: Carbohydrates are organic compounds composed of C.H.O. Generally, (not always), the hydrogen and oxygen are present in the proportion as in water (H2O), from which the term carbohydrate was derived. The basic formula for carbohydrates is CH2O: 1 carbon atom, 2 hydrogen atoms, 1 oxygen atom. Carbohydrates are defined as polyhydroxy aldehydes or polyhydroxy ketones and all the compounds yielding them on hydrolysis. Function of Carbohydrates: 1- Provide Energy: They serve as energy stores and fuels. 2- Structural Components:  They share in the cell membranes structure.  Pentose sugars (ribose and deoxy-ribose) contributes in the structure of nucleic acids.  They share in the structure of glycoproteins and glycolipids.  Cellulose in plants. 3- Molecules for Recognition: They play important roles in cell recognition. Classification: 1. Monosaccharides (simple sugars): the simplest unit of CHO. Only one sugar molecule. 2. Disaccharides: 2 monosaccharide units. 3. Oligosaccharides: 3-10 units of mono-saccharides and their derivatives. 4. Polysaccharides (glycans): more than 10 monosaccharide units and/or their derivatives. Glyco-conjugates  Also called complex saccharide  Polysaccharide linked to proteins or lipids, including: Glycoproteins Glycolipids Monosaccharides: 15 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Are carbohydrates containing only one sugar unit and cannot be hydrolyzed into smaller units. General formula: CnH2nOn They are further classified according to: 1. Number of carbon atoms. 2. The active sugar (carbonyl) group whether aldehyde or ketone. Monosaccharide Classification:  Monosaccharides can be classified according to the number of carbon atoms they contain,  3 carbons: triose.  4 carbons: tetrose.  5 carbons: pentose.  6 carbons: hexose.  Pentose (5 C): e.g. Ribose.  Hexose (6 C): e.g. Glucose, Galactose, Mannose, Fructose.  Mono-saccharides can be divided into two families: aldoses and ketoses.  The most abundant monosaccharides are Aldo-hexoses.  Glucose is the most important and is used by cells as fuel.  The most abundant monosaccharide in nature is the six-carbon sugar; glucose. 16 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Monosaccharides of Biological Importance 1. Glucose (Dextrose) (Grape Sugar):  The most important sugar representing the major source of energy for humans and animals’ tissues.  Some cells and tissues e.g. brain and erythrocytes depend on glucose because they cannot oxidize alternative fuels.  Therefore, the body maintains a fairly constant blood glucose level of 70–110 mg/dl at all times.  Ingested carbohydrates are absorbed in the form of glucose.  It is converted into other sugars in the liver and other tissues e.g. galactose, fructose, ribose and glycogen. 2. Galactose:  It is synthesized in mammary gland to form disaccharide lactose (milk sugar).  It presents in tissues as a constituent of galactolipid and glycoproteins.  It converts to glucose in liver. 3. Fructose (Fruit Sugar):  It is present in semen and is a constituent of disaccharide sucrose.  Seminal fluid is rich in fructose that is formed from glucose and sperms utilize fructose for energy.  Convert to glucose in liver. 4. Ribose:  Ribose and deoxy-ribose form part of the structural backbone of nucleic acids RNA and DNA respectively.  Ribose enters in the structure of high-energy phosphate compounds (e.g. ATP) and also in the structure of coenzymes such as (e.g. NAD). 17 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Disaccharides Definition:  Disaccharides consist of 2 monosaccharides joined by an O- glycosidic bond. Classification: The disaccharides can be classified into:  Homodisaccharides: formed of the same monosaccharide units e.g. maltose.  Heterodisaccharides: formed of different monosaccharide units e.g. sucrose and lactose.  The most abundant disaccharides are sucrose, lactose and maltose. Clinical correlate:  Low level of lactase enzyme leads to undigested lactose that undergoes bacterial fermentation in the colon with the generation of large amounts of CO2, H2 and irritating organic acids.  These products cause painful digestive upsets known as lactose intolerance. 18 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Oligosaccharides Definition: 3-10 monosaccharide units joined together by glycoside bonds. E.g. Maltotriose Existence and Significance: 1. Oligosaccharides are important constituents of the glycoproteins and glycolipids present in cell membrane. 2. Many secreted proteins, such as antibodies and coagulation factors also contain oligosaccharide units. Polysaccharides Definition:  Polysaccharides, also called glycans, consist of more than 10 monosaccharide units and/or their derivatives joined together by glycosidic linkage.  Polysaccharides are classified into:  Homopolysaccharides (homoglycans): contain only one type of monosaccharide. Include starch, glycogen, dextran, cellulose.  Heteropolysaccharides (heteroglycans) those containing more than one type of monosaccharides. Include glycosaminoglycans (GAGs) and agar.  Polysaccharides can also be classified according to their function into storage and structural polysaccharides.  Storage polysaccharides: include starch, glycogen, dextran.  Structural polysaccharides: include cellulose and agar. Homopolysaccharides 1. Starch Structure: It is formed of α-glucose units (glucosan) consists of 2 layers: Inner linear non branching layer. Outer highly branched layer. The branch points occur about once every 30 linkages. Starch is hydrolyzed by salivary and pancreatic amylase to yield maltose and dextrins. Function: It is the most common storage polysaccharide in plants. 19 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University 2. Glycogen (animal starch): Structure: It is formed of α-glucose units (glucosan). It is highly branched molecule with the branches occurring every 10 glucose units. Function:  It is the major form of storage polysaccharides in animals and human body.  It is found mainly in liver (about 10% of liver mass) and skeletal muscle (about 1 to 2 % of muscle mass).  In fasting: breakdown to glucose to maintain blood glucose level. 3. Dextran: Structure: It is formed of α-glucose units (glucosan). Function: 1. It is a storage polysaccharide in yeasts and bacteria. 2. It is also used as replacement therapy in blood loss. 3. Dental plaque is due to dextran synthesized from sucrose by oral bacteria. 4. Cellulose Structure: It is formed of β-glucose units. Function:  It is the most abundant natural polymer found in the world.  It is the structural component of cell walls of nearly all plants.  It is not digested by the human (due to absence of cellulase enzyme), so its nutritive value is low.  Cellulose is extremely resistant to hydrolysis by acid and by the digestive tract amylases. So, it can stimulate peristaltic movement and prevent constipation.  It can stimulate peristaltic movements of the intestine increasing the bulk of stool so it is used in the treatment of constipation (laxative effect). Hetero-polysaccharides 1- Agar:  A polysaccharide isolated from marine red algae.  Agarose gel is used in gel electrophoresis.  Nutrient agar is used in preparation of culture media used in microbiology. 2- Glycosaminoglycans (GAGs) (Mucopolysaccharides)  Glycosaminoglycans are long linear (unbranched) heteropolysaccharide chains generally composed of a repeating disaccharide unit (acidic sugar-amino sugar)n. 20 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University 21 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Carbohydrates metabolism 22 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Carbohydrates Metabolism CHO Digestion So, the end products of carbohydrate digestion are mainly glucose, galactose and fructose. 23 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University CHO Absorption Absorption of carbohydrates: Sugars are absorbed by 3 mechanisms: 1- Simple diffusion: Absorption of monosaccharides according to concentration gradient e.g. pentoses. 2- Facilitated transport: Glucose is carried by specific protein transporter (GLUT5). Galactose and fructose are absorbed by the same mechanism. 3- Active transport (Co-transport): Mainly for glucose and galactose. Use a sodium glucose transporter (SGLUT-1), which binds both Na+ and glucose at separate sites and transport both through the brush border of small intestine. It needs ATP as a source of energy. Fate of absorbed sugars  Fructose and galactose are transported by portal blood to the liver, where they are converted to glucose.  Absorbed glucose is transported to the liver then by systemic circulation to different tissues. Glucose uptake by tissues  Glucose is transported through cell membranes of different tissues by different protein carriers or transporters.  GLUT4 is the major glucose transporter in heart, skeletal muscle & adipose tissue.  GLUT4 is under the control of insulin.  But other glucose transporters are not under the control of insulin. CHO Metabolism 24 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University We eat, we digest, we absorb, then what? Three fates for nutrients 1) Most are used to supply energy for life 2) Some are used to synthesize structural or functional molecules 3) The rest are stored for future use Metabolism of carbohydrates  Glycolysis  Citric acid cycle  Pentose Phosphate Pathway  Glycogen metabolism  Gluconeogenesis  Control of blood glucose level Glucose Oxidation Oxidation of glucose A- The major pathways for oxidation: mainly for energy production 1- Glycolysis. 2- Citric acid cycle (Krebs cycle). B- The minor pathway for oxidation: Not for energy production but mainly for synthesis of other glucose derivatives. Hexose monophosphate pathway (HMP): for production of pentose and NADPH. Glycolysis (Glyco = sugar, lysis = breakdown) Frist stage in cellular respiration Site: Cytosol of all cells.  It is a series of biochemical reactions by which:  Glucose →→→ pyruvate (aerobic condition)  Glucose →→→ lactate (anaerobic condition) Steps: From glucose to pyruvate: 10 steps from glucose to pyruvate, require 2 ATPs. Anaerobic Glycolysis Under anaerobic conditions (e.g. exercising muscles) and in cells lacking mitochondria (RBCs), Glucose →→→→Pyruvic → lactate 25 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Importance of Glycolysis Energy production during aerobic and anaerobic conditions: 1- Under aerobic condition: Glucose →→ 2 pyruvate + 8 ATP 2- Under anaerobic condition: Glucose →→ 2 lactate + 2 ATP Biomedical importance of glycolysis 1. Fluoride: inhibits enolase enzyme → inhibition of glycolysis in bacteria → no production of lactic acid produced by bacteria which cause dental caries. Regulation of Glycolysis Fate of Pyruvate: → → → → (Pyruvate Oxidation)  2 Pyruvate → transported into mitochondria → 2 acetyl CoA for oxidation through the citric acid cycle.  2 pyruvate → 2 acetyl CoA + 2 NADH+H+  So, 6 ATP is formed by oxidation of 2 NADH+H+ by the electron transport chain. 26 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Tricarboxylic acid cycle (TCA) Citric acid cycle / Krebs cycle Definition: It is a series of biochemical reactions that are responsible for complete oxidation of acetyl CoA. Site: Mitochondria of all cells except RBCs which do not contain mitochondria. Steps: consists of eight successive reactions Products:  1 FADH2  2 CO2  1 of ATP  3 NADH+H+ Importance: 1- It is the final common metabolic pathway for oxidation of carbohydrates, fats and proteins (amino acids). 2- Energy production: Every 1 molecule of acetyl CoA produces 12 ATP * 2 acetyl CoA Summary for complete oxidation of glucose Pentose Phosphate Pathway (Pentose shunt) Hexose monophosphate pathway (HMP) Definition: An alternate pathway for oxidation of glucose. Pathway by which glucose is converted into pentose phosphate with production of NADPH. Site: Cytosol of many cells e.g.: liver, mammary gland, adipose tissue, red cells, adrenal cortex, retina,.etc. Regulation: → →→ Insulin:  Produced in response to hyperglycemia.  It stimulates Pentose shunt. 27 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Importance: No ATP is directly consumed or produced 1- Provision of pentose for nucleotide and nucleic acid synthesis. 2- Formation of NADPH: Examples of such reactions where NADPH is used (fates): 1. fatty acid synthesis. 2. Synthesis of cholesterol. 3. Synthesis of steroids. 4. Role as cellular antioxidants: Protect red blood cells from oxidative damage. Glycogen metabolism Glycogen is the main storage form of carbohydrates in animals. Sites: It is present mainly in the liver (8-10% of its net weight) and in muscles (2% of its weight). However, due to the greater mass of muscles, its glycogen store is 3 - 4 times more than that of the liver.  Functions:  Liver glycogen → maintenance of blood glucose especially between meals.  Muscle glycogen → supply glucose within muscles during contraction.  Liver glycogen is depleted after 12-18 hours fasting.  Muscle glycogen is depleted after muscular exercise. Glycogen metabolism includes the following: Glycogenesis: It is the synthesis of glycogen from glucose. Glycogenolysis: It is the breakdown of glycogen. Glycogenesis (Glycogen, genesis = formation) Definition: It is synthesis of glycogen from glucose. Site: In the cytosol of liver cells and muscles. Regulation: Well-fed state: Insulin → increases glycogenesis in liver and muscles. Fasting: Glucagon and epinephrine (anti-insulin hormones) → decreases glycogenesis in liver and muscles 28 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Glycogenolysis (Glycogen, lysis = breakdown) Definition: It is breakdown of glycogen to form glucose 6-phosphate (end product).  In liver: Glucose-6-phosphate is mainly converted to glucose → blood.  In muscles: Glucose-6-phosphate is oxidized by glycolysis to produce energy. Regulation: Well-fed state: Insulin → decreases glycogenolysis in liver and muscles. Fasting: Glucagon & epinephrine (anti-insulin hormones) → increase glycogenolysis in liver and muscles. Gluconeogenesis (Gluco = sugar, neo = new, genesis = formation) Definition: It is synthesis of glucose from non-carbohydrate precursors. Function: to supply blood glucose in case of carbohydrate deficiency (Fasting, starvation, and low carbohydrate diet). Site: It occurs mainly in the liver and to lesser extent in the kidney (cytosol & mitochondria). Regulation: →→ Hormonal regulation of gluconeogenesis: Hyperglycemia (CHO meal) → insulin secreted → decrease gluconeogenesis (& increases glycolysis). Hypoglycemia (CHO deficiency) → anti-insulin hormones secreted → increase gluconeogenesis (& decrease glycolysis). Glucose Homeostasis Def.: Regulation of blood glucose to be kept constant within normal ranges. Maintenance of blood glucose is critical to survival of human being Blood Glucose Normal fasting blood glucose level is about 70-110 mg/dl. It normally fluctuates between 70-140 mg/dl. Reaches 140 mg/dl during the first hour after meal, then returns to fasting level within 2 hours after meal. 29 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University It is kept within relatively narrow range by balance between the rate of addition and rate of withdrawal of glucose from blood 30 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Lipids Chemistry 31 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Lipid Chemistry  Definition of lipids: Lipids are organic compounds which are relatively insoluble in water (hydrophobic), but freely soluble in non-polar organic solvents like benzene, ether and acetone, etc.  Biological importance of lipids: 1. Source of energy: they yield twice the energy produced by same weight of carbohydrates or proteins. 2. Lipids in adipose tissue (as TGs) serve as energy storage. 3. Structural components of membranes (phospholipids and cholesterol). 4. Metabolic regulators (e.g. steroid hormones). 5. Help in absorption of fat-soluble vitamins (A, D, E and K). 6. Lipids (pads of fat) have a role in protection and fixation of internal organs as kidneys. 7. Subcutaneous fat (Lipids under the skin) serve as thermal insulator. 8. Act as electric insulators in myelin sheath of nerve fibers.  General classification of lipids: 1. Simple lipids: They are formed of fatty acids and alcohol. 2. Compound lipids: They are formed of simple lipids (fatty acids + alcohol) and other non-lipid part. 3. Derived lipids: They are substances derived from simple lipids by hydrolysis. They also, include substances related to lipids. Glycerol  It is a polyhydric alcohol containing 3 OH groups.  Importance of Glycerol 1. It is used in pharmaceutical (e.g. glycerin suppositories) and cosmetic preparations. 2. It is used as vasodilator agent in coronary heart disease in form of nitro-glycerine. Chemical properties: Can combine with one or more fatty acids by ester bonds forming >>> mono, di or triacylglycerol (Triglycerides). 32 Prof./ Salwa Abo Elkhair  Medical Biochemistry for Dentistry Mansoura National University Fatty acids  Definition of fatty acids:  Fatty acids are monocarboxylic organic acids, with a methyl group at one end (called the ω-carbon) and a carboxyl group at the other end.  They usually contain an even number of carbon atoms.  They are generally found in ester linkage in different classes of lipids.  The general formula of fatty acid is R-COOH CH3 – CH2 – CH2 – CH2 – CH2 – COOH  Numbering of fatty acid carbon atoms: 1. ω-system: The carbon chain of fatty acids are numbered starting from terminal CH3 group or omega carbon: ώ1 ώ2 ώ3 ώ4 ώ5 ώ6 CH3 – CH2 – CH2 – CH2 – CH2 – COOH ω-9 represent 1st double bond position at C9-C10. ω-6 represent 1st double bond position at C6-C7. ω-3 represent 1st double bond position at C3-C4.  Classification of fatty acids: 1. Depending on length of hydrocarbon chain: a. Short chain: with 2 to 10 carbon atoms. b. Long chain: > 10 carbon atoms. 2. Depending on absence or presence of double bonds: a. Saturated fatty acids. No double bonds. They have the general formula: CH3 – (CH2)n – COOH. b. Unsaturated fatty acids: having double bonds, sub-classified into: Mono-unsaturated: having single double bond. Polyunsaturated: with 2 or more (≥ 2) double bond.  Examples of fatty acids: (a) Saturated fatty acids: Common name No. of carbons Structure Palmitic 16 CH3-(CH2)14-COOH Stearic 18 CH3-(CH2)16-COOH 33 Prof./ Salwa Abo Elkhair  Medical Biochemistry for Dentistry Mansoura National University (b) Mono-unsaturated fatty acids: Common name No. of carbons Structure Palmitoleic (16: 1∆9, W7) CH3(CH2)5-CH=CH-(CH2)7-COOH Oleic (18: 1∆9, W9) CH3(CH2)7-CH=CH-(CH2)7-COOH Common sources: Many vegetable oils: like sunflower, olive, nuts, corn, sesame, soybean and cod liver oils. (c) Poly-unsaturated fatty acids (PUFA) (essential fatty acids): They are polyunsaturated fatty acids i.e. fatty acids which contain more than one double bond. They must be taken in diet because the body cannot synthesize them as the enzymes needed for their synthesis are absent in humans. Common name No. of carbons Linoleic (18: 2, W6) Linolenic (18: 3, W3) Arachidonic (20: 4, W6) Common sources: Many vegetable oils: like sunflower, olive, nuts, corn, soybean and cod liver oils. Importance of essential fatty acids: 1. They are essential for growth. 2. They are essential for phospholipid formation. 3. Arachidonic acid is important for biosynthesis of prostaglandins. Simple lipids  Definition of simple lipids: They are esters of fatty acids with various alcohols. 1- Fats and oils (triglycerides = triacylglycerol):  They are esters of fatty acids with glycerol.  They are called triglycerides because they are triesters formed of glycerol and 3 fatty acids.  Functions of triglycerides: 1- Storage of energy as fat: The triacylglycerols are the storage form of lipids in the adipose tissue. 2- Provide essential fatty acids. 3- Carriers of fat-soluble vitamins (A-D-E-K). 34 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Compound (Conjugated) Lipids Compound lipids contain in addition to esters of fatty acids with alcohols, other groups. According to the type of the group attached, they are classified into: a- Phospholipids:- contain phosphoric acid b- Glycolipids:- contain sugars c- Lipoproteins:- contain proteins 1-Phospholipids - formed of alcohol, fatty acids, phosphoric acid ± nitrogenous base - They are classified according to type of the alcohol present into:- a- Phosphoglycerides: contain glycerol alcohol b- Sphingomyelin: contain sphingol (sphingosine) alcohol They include: 1. Lecithin. 2. Cephalins. 3. Sphingomyelins Phospholipids functions They are having hydrophilic part + hydrophobic part This property helps: A. Formation of lipid bilayer in cell membrane. B. Micelle formation to help T.G absorption in the small intestine C. Structure of plasma lipoproteins that transport lipids in blood. 1- Lecithin (phosphatidyl choline). Nitrogenous base: Choline. Function of lecithin 1. It is the most abundant phospholipids in the cell membrane. 2. It acts as a lipotropic factor: substances that prevent accumulation of lipids in the liver. 3. Dipalmityl lecithin acts as Lung surfactant: substances that line the lung alveoli forming a layer preventing lung collapse. 2- Cephalins They are phosphatidyl ethanolamine & phosphatidyl serine Structure: Nit. Base: Ethanolamine or serine. Function of cephalins They have a role in blood coagulation because they share in the structure of thromboplastin, which is essential for blood clotting. 3. Sphingomyelins Sphingomyelin contains: Sphingosine base (sphingol). Choline base. Function of sphingomelin: It is abundant in the nervous system in the myelin sheath (electrical insulator). 35 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University 2- Glycolipids  Fatty acids + alcohol + sugar.  Glycolipids are classified into:  A- Cerberosides B- Gangliosides Functions of glycolipids 1- Electrical insulator in nervous tissue. 2- Gangliosides are receptors for many hormones. 4- Lipoproteins Def: compound lipids formed of  lipid part (Triglycerides T.G, cholesterol, phospholipids)  protein part (Apolipoprotein) Functions 1- important for lipid transport in the blood.  lipids are insoluble in water so they cannot be transported alone, so, lipids bind to protein make lipoproteins water-soluble and can be transported in the blood Types 1- Chylomicrons (CM) 2- Very Low Density Lipoprotein (VLDL) 3- Low Density Lipoprotein (LDL) 4- High Density Lipoprotein (HDL) Derived Lipids Definition: Derived lipids are compounds derived from simple lipids and compound lipids by hydrolysis (fatty acids, alcohol), also, include substances related to lipids (steroids). Steroids They are large group of biologically important compounds, all of them contain steroid nucleus. Steroid compounds include 1. Cholesterol 2. Steroid hormones 3. Vitamin D 4. Bile acids 36 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University 1- Cholesterol Cholesterol Source Animal source (best known sterol) Absorption from small intestine Easily absorbed Function a) Structure of cell membrane b) Precursor for all steroid hormones, bile acids (liver) c) 7-dehyrocholesterol pro-vitamin D3 ( by U.V rays) 2-Steroid hormones A- Sex hormones: 1- Male sex hormone: testosterone. 2- Female sex hormones: estrogen and progesterone B- Adrenal cortex hormones (corticoids): A- Glucocorticoids: corticosterone, cortisol. B- Mineralocorticoids: 11 deoxy corticosterone, 11 deoxy cortisol, aldosterone. 3- Active form of vitamin D (Steroid vitamin) Structure:  Vitamin D3 is derived from 7-dehydrochoolesteorl by U.V rays. Functions: Have role in calcium metabolism, increase Ca absorption from GIT and increase bone ossification 4- Bile acids A- 1ry bile acids: formed from cholesterol in the liver B- 2ry bile acids: formed by action of intestinal bacteria on 1ry bile acids  Bile salts have role in lipid digestion and absorption. 37 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Lipids Metabolism 38 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Lipid Digestion & Absorption Include digestion of: 1- Triglycerides 2- Phospholipids 3- Cholesterol esters 1- Digestion of Triglycerides include: a- emulsification b- enzymatic hydrolysis by lipase enzymes a- Emulsification:  Breakdown of large fat globules into small ones.  This is done:  in the mouth by chewing,  in the stomach by peristaltic contractions,  in intestine by peristaltic movement and bile salts. b- Lipase enzymes:  lingual lipase, gastric lipase, pancreatic lipase and intestinal lipase.  The most active is pancreatic lipase. 2- Digestion of phospholipids Glycerophospholipids are hydrolyzed by pancreatic enzyme phospholipase A2 to form lysophospholipids. 3- Digestion of cholesterol esters Cholesterol ester is hydrolyzed by cholesterol ester hydrolase (cholesterol esterase) into FA and free cholesterol. Absorption of Lipids  The end products of lipid digestion are: monoglyceride, FA, glycerol, cholesterol and lysophospholipid.  Glycerol & short chain FA are water soluble are carried through portal circulation.  Long chain FA, monoglycerides, cholesterol and lysophospholipids need bile salts to be absorbed.  Bile salts surround these components by their un-polar end while their polar endings directing outward to form water soluble micelles (0.1 –0.5 μ in diameter).  Bile salts are reabsorbed to the liver again by enterohepatic circulation.  In the mucosal cells triglycerides and other lipids are resynthesized once again.  The triglycerides, phospholipids & cholesterol bind with a protein (apolipoprotein B48) to form chylomicrons which enter lacteals and pass with lymphatic drainage to the thoracic duct to reach systemic circulation. 39 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Lipid Transport & Lipoprotein Metabolism Lipoproteins: Functions 1- Lipid transport in the blood: Lipids are insoluble in water. Lipids bind to protein to make lipoproteins water-soluble to be transported in the blood. Types 1- Chylomicrons (CM) 2- Very Low Density Lipoprotein (VLDL) 3- Low Density Lipoprotein (LDL) 4- High Density Lipoprotein (HDL) Summary of lipoprotein metabolism Lipid Storage, Lipogenesis The body undergoes one of the following metabolic states: * The fed state * The fast state * The starved state Fed state: The hormonal profile in the fed state as follow:  There is increase insulin secretion in response to elevated blood glucose level and decrease glucagon and epinephrine.  So, Insulin/Anti-insulin ratio is increased.  So, there are plenty of fats and in high energy levels, body will start lipid storage. 40 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Adipose tissue metabolism in fed state, Storage of FAT  Most fat is stored (as TG) in adipose tissue, which is composed of specialized cells known as adipocytes.  Adipocytes are specialized storage cells for triglycerides. Lipogenesis  Def: It is the biosynthesis of triglycerides (TGs) principally from glucose.  Site: It occurs in most tissues especially adipose tissue, liver, lactating mammary gland.  Function: storage of excess glucose after a carbohydrate rich meal. Include 3 processes: A. Biosynthesis of glycerol 3- phosphate. B. Biosynthesis of fatty acids. C. Biosynthesis of the triglycerides (TGs). A- Biosynthesis of glycerol 3-phosphate: Glucose  glycolysis  DHAP  glycerol-3 phosphate dehydrogenase  glycerol-3 phosphate B- Biosynthesis of fatty acids: Site: Fatty acid synthesis occurs in the Cytosol. Fatty acids biosynthesis has many steps: 1st step is Formation of acetyl-CoA from Glucose: - Glucose  glycolysis  pyruvic acid  oxidative decarboxylation  acetyl-CoA (building block of fatty acid synthesis). - fatty acid synthesis are catalyzed by a multifunctional enzyme called fatty acid synthase. 41 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University End product: palmitic acid (16-C) which can be elongated/shortened with/without desaturation to synthesize different types of fatty acids according to body need. Regulation of FA synthesis 1. Dietary factors: carbohydrate promotes synthesis 2. Hormone factors  Insulin， “store hormone”: increase FA synthesis.  Glucagon，“release hormone”: inhibit FA synthesis. C- Biosynthesis of the triglycerides (TGs): Glycerol 3-phosphate + 3 FA    Triacylglycerol (TAG) Adipocyte Lipolysis Fasting state:  There is decreased circulating levels of glucose, amino acids, and TAG.  There is decline in insulin secretion and increase in glucagon release.  The decreased insulin/anti-insulin and the substrates availability leads to increase catabolism. 42 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Fat metabolism in adipocytes in fasting 1) Increased TAG degradation (Lipolysis).  Due to activation of hormone sensitive lipase enzyme by the elevated level of catecholamines. 2) Increased release of FA:  FA obtained from hydrolysis of stored TAG are released into the blood, then transported to a variety of tissues for use as a fuel.  Glycerol produced following TAG hydrolysis is used for synthesis of glucose (gluconeogenesis) in the liver. Mobilization and Transport of fatty acids:  Short chain fatty acids are freely transported in blood as Free Fatty Acids (FFA),  Long chain fatty acids are transported bound to serum albumin. Fatty Acids Oxidation (β oxidation)  It occurs in the form of repeated cycles, in each cycle, 2 carbon fragment are removed from the carboxyl end of fatty acid producing: acetyl CoA (2C), FADH2 & NADH+H.  It occurs in many tissues including liver, kidney and heart.  The mitochondria is the principle site of fatty acids oxidation.  Fatty acids oxidation doesn’t occur in RBCs (no mitochondria) and not occur in the brain (fatty acid can’t be taken up by the brain).  Acetyl CoA is entered Krebs's cycle.  FADH2 and NADH+H transport in the respiratory chain of electrons will lead to the synthesis of ATP. Energy out-put of β oxidation: Oxidation of Fatty Acids Produces a Large Quantity of ATP  eg. Palmitic acid (16 C): by β oxidation, it will produce 129 ATP Ketogenesis  During high rates of fatty acid oxidation, primarily in the liver, large amounts of acetyl- CoA are generated.  These exceed the capacity of the TCA cycle, and result in synthesis of ketone bodies (ketogenesis).  The ketone bodies are acetoacetate, β-hydroxybutyrate, and acetone.  Mitochondrial in location.  Site: liver  ketone bodies are produced in the liver to be used in peripheral tissues.  Ketone body synthesis and utilization: occur during starvation or prolonged exercise 43 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Liver & lipid metabolism 44 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Protein Chemistry 45 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Protein Chemistry Definition:  Proteins are organic nitrogenous compounds of high molecular weight formed of C, H, O, N … [N = 16%], (+/- S).  They are formed of amino acids linked together by peptide linkage [-CO-NH-]. Biological importance of proteins: 1- Antibodies (immunoglobulin) are proteins 2- Buffer system of blood (plasma proteins) 3- Carry hormones and minerals in blood (plasma proteins), Carry oxygen (Hemoglobin), Carry lipids (lipoproteins) 4- Structure of cell membrane. 5- Support bone, skin, nails, and hair. 6- Hormones as insulin, Enzymes are proteins. 7- Provide body with Nitrogen, Sulfur, & some Vitamins. Amino Acids Amino acids are the structural units (Building block) of proteins. They are obtained from protein by acid, alkali or enzymatic hydrolysis. There are 20 known Amino acids. General Structure of amino acids: Each amino acid (except proline and hydroxy proline) has: a- COOH group. b- NH2 group attached to α-carbon (α-amino acid). c- Side chain (R) which is characteristic for each amino acid. 46 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Classification of Amino Acids: Chemical Classification of AAs According to the number of amino group and carboxylic groups. (1) Neutral Amino Acids They are further classified into the following categories: 1) Aliphatic: [ Glycine - Alanine - Valine - Leucine - Isoleucine ] 2) Aromatic: [ Phenylalanine - Tyrosine – Tryptophan ]. 3) Hydroxy amino acids: [ Serine-Threonine ]. 4) Sulphur containing: [ Cysteine - Methionine - Cystine ] N.B.  Imino acids (heterocylic) [contain an imino group; NH]: proline , hydroxy proline. 47 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University (2) Basic Amino Acids These amino acids contain ( -NH2 ) group ˃ (-COOH) group Positively (ve+) charged e.g.: Arginine - Histidine – Lysine (3) Acidic Amino Acids These amino acids contain (-COOH ) group ˃ ( - NH2 ) group Negatively (ve-) charged e.g.: Glutamic acid - Aspartic acid Nutritional Classification of Amino Acids Based on the requirement of amino acids in the diet, and synthesis of them in the body: (1) Essential amino acids (2) Non-essential amino acids 48 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Metabolic classification of AAs Protein classification Simple proteins Definition: They are proteins that on hydrolysis gives only amino acids Types: Albumin, Globulin, Histone, Albuminoids 49 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University 5- Scleroproteins (Albuminoids)  Fibrous proteins  Supportive & protective functions.  Not coagulated by heat  Insoluble in water, acids, alkali and all neutral solvents.  Not digested by proteolytic enzymes.  Not in plants. Gelatin  Obtained from collagen by boiling  Forming gel by cooling  Easily digested  It is not adequate diet (deficient in some essential AA as tryptophan). Conjugated (Compound) protein Definition: They are protein conjugate with non protein part (other groups) 50 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Derived protein Definition: These are denaturated or hydrolytic products of simple or conjugated proteins. e.g. coagulated albumin (cooked egg albumin), coagulated globulin (cooked egg globulin) Protein structure Protein structure & function The functions of proteins depend on the ability to recognize and bind with a variety of molecules. This ability depends on 3D- structure of proteins. Protein structure  They are a number of amino acids linked together by peptide linkage [-CO-NH-].  The carboxylic group of the first amino acid unites with the amino group of the second amino acid and so on.  At one end of the polypeptide chain, there is free COOH group called C-terminal, while at the other end free NH2 group called N-terminal There are four orders of protein structures  Primary structure  Secondary structure  Tertiary structure  Quaternary structure 51 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University 1- Primary structure  It is the number, type and sequence of AAs in the polypeptide chain.  Main bond: Peptide bond (-CO-HN-)  Any change in one AA  physiological (functional) defect.  It is not affected by denaturation. 2. Secondary structure * 1ry structure (The polypeptide chain) will be folded by hydrogen bonds between specific atoms to give a specific shape / form which may be: α-Helix or β-Sheets 3. Tertiary structure  Definition: It is three-dimensional (3D) shape of a protein (polypeptide chain)  Formed of: Secondary structures (α and β) are arranged to form final functional 3D structure of protein called domain. The function of a protein depend on its tertiary structure If it is disrupted  protein loses its activity It is affected by denaturation. 4. Quaternary Structure  Definition: it is the association (arrangement) of several polypeptide chains or subunits into a closely packed arrangement, (subunits = monomers). Each of the subunits has its own primary, secondary, and tertiary structure.  Examples: 1- Haemoglobin: is tetramers (formed of 4 subunits) 52 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Protein Folding Definition:  Protein folding is a physical process by which a polypeptide chain folds into a biologically active protein in its unique stable 3D structure. Importance  Protein structure is crucial to its function. Protein misfolding Definition Proteins that are not able to form stable 3D structure recognized as misfolded The misfolded protein generate aggregates (large size, insoluble)  loss its structure and function Example of diseases related to misfolded protein: 1. Alzheimer's disease 2. Type II diabetes 3. Parkinson's disease 53 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Protein Denaturation Def: Change in the native state (physical, chemical, and biological properties) of proteins without destruction of their peptide linkages but destruction of secondary (non- covalent) bonds leading to unfolding. Denaturating agents: Heat, UV rays, Shaking, Acid, alkali, organic solvents. Results: Physical: Decrease solubility & mobility Chemical: Unfolding of the protein molecule. Biological: Loss of activity, Easily digested 54 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Protein Metabolism 55 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Protein Digestion & Absorption Protein Digestion:  In general, animal protein is more efficiently digested than plant protein.  The dietary proteins are hydrolyzed to amino acids by proteolytic enzymes, which can be easily absorbed.  Proteolytic enzymes responsible for degrading proteins are produced by three different organs; stomach, pancreas and small intestine I- Digestion in Stomach: 1- Role of gastric HCL  It causes denaturation of proteins  It activates pepsinogen to pepsin  It makes PH of stomach suitable for the action of pepsin. 2- Pepsin:  It is secreted in an inactive form (zymogen) called pepsinogen.  Its optimum PH: 1.5-2.2  It is activated by HCL then by autoactivation.  It is an endopeptidase, which acts on central peptide bond. 3- Rennin  It is a milk-clotting enzyme.  It is present in the stomach of infants and young animals.  Its optimum pH = 4.  It acts on casein (milk protein). * The end products of protein digestion in the stomach are:  Proteoses  Peptones  Large polypeptides 56 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University II- Digestion in Small Intestine: A. Pancreatic Juice 1. Trypsin  It is secreted in an inactive form (zymogen) called trypsinogen.  Its optimum PH = 8.  It is activated by enterokinase enzyme then by auto-activation.  It is an endopeptidase, which acts on central peptide bonds. 2. Chymotrypsin  It is secreted in an inactive form called chymotrypsinogen.  It is activated by trypsin.  Its optimum PH = 8.  It is an endopeptidase, which acts on central peptide bonds. * Trypsin & chymotrypsin convert proteoses & peptones into large polypeptides 3. Carboxypeptidase  It secreted in an inactive form called procarboxypeptidase.  It is activated by trypsin.  Its optimum PH = 7.4  It is an exopeptidase that hydrolyzes the terminal (peripheral) peptide bond at the carboxyl terminus (end) of the polypeptide chain. B. Intestinal Juice 1. Aminopeptidase  It is an exopeptidase that acts on the terminal peptide bond at the amino terminus of the polypeptide chain.  Both carboxypeptidase & aminopeptidase convert large polypeptides into tripeptide. 2. Tripeptidase  It acts on tripeptides.  It releases a single amino acid and dipeptide. 3. Dipeptidase  It acts on dipeptides.  It releases 2 amino acids. N.B. The end products of protein digestion in the small intestine are amino acids. 57 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Amino Acids Absorption  It is an active process that needs energy derived from ATP.  It occurs in small intestine.  There are two mechanisms for amino acids absorption. - Carrier proteins transport system. - Glutathione transport system (γ-Glutamyl cycle). 1- Carrier proteins transport system  The main system for amino acid absorption.  An active process needs energy derived from ATP.  Absorption of one amino acid needs one ATP molecule.  There are 7 carrier proteins, one for each group of amino acids. 2- Glutathione transport system(-Glutamyl cycle)  Glutathione (tripeptide) is used to transport AAs from intestinal lumen to cytosol of intestinal mucosa cells.  An active process which needs energy derived from ATP.  Absorption of one amino acid needs 3 ATP molecule. Glutathione Protein Metabolism Introduction Turnover of Proteins: Continuous degradation and re-synthesis of protein. In adults, the nitrogen balance is generally in equilibrium i. e., the quantities of protein nitrogen taken in and excreted per day are approximately equal. Positive nitrogen balance: synthesis > degradation Growth, pregnancy,.. Negative nitrogen balance: degradation > synthesis Fever, chronic diseases, cancer. 58 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Amino acids pool:  The amount of free amino acids distributed throughout the body is called amino acids pool.  Plasma level for most amino acids varies widely throughout the day.  It ranges between 4-8 mg/dl.  It tends to increase in the fed state. Sources of amino acid pool: 1. Dietary protein. 2. Breakdown of tissue proteins. 3. Biosynthesis of nonessential amino acids. Fate of amino acid pool: 1. Biosynthesis of structural proteins e.g. tissue proteins. 2. Biosynthesis of functional proteins e.g. hemoglobin, myoglobin, protein hormones and enzymes. 3. Biosynthesis of small peptides of biological importance e.g. glutathione, endorphins, and encephalin. 4. Biosynthesis of non-protein nitrogenous compounds (NPN) as urea, uric acid, creatine, creatinine and ammonia. 5. Individual specific pathway for each amino acid 6. Catabolism of amino acids to give ammonia and α-keto acids (carbon skeleton). 59 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University General structure of amino acids: Each amino acid has:  COOH group.  NH2 group.  Side chain R Catabolic pathways of amino acids: 1- Transamination: Def: It is transfer of amino group (NH2) from α-amino acid to α-keto acid with formation of a new α-amino acid and a new α-keto acid. Site: The liver is the main site for transamination. Enzyme: aminotransferases (transaminases). Coenzyme: pyridoxal phosphate (PLP). Properties: All transamination reactions are reversible Examples of transaminases 1. Alanine transaminase (ALT = GPT) , Liver Enzymes (Liver Function Tests) 2. Aspartate transaminase (AST = GOT), Liver Enzymes (Liver Function Tests) 3. Glutamate transaminase 2- Deamination: Def.: Deamination means the removal of amino group (NH2) from α-amino acid in the form of ammonia (NH3) with formation of α-keto acid. Site: The liver and kidney are the main sites for deamination. Types: Oxidative deamination and Non-oxidative deamination 3- Decarboxylation: (Amines Formation): - Def. Decarboxylation means removal of the carboxylic group in the form of CO2 from amino acid with formation of corresponding amines. - It is catalyzed by decarboxylase enzyme. - It needs pyridoxal phosphate as a coenzyme.  E.g.: Histamine synthesis. 60 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Histamine synthesis:  Histamine produced by decarboxylation of histidine amino acid.  Histamine has many important biological functions: - 1) In stomach it stimulates secretion of HCl. 2) It is a potent vasodilator released locally in sites of inflammation & allergic reactions. 3) It is mediator of allergic and hypersensitivity reactions. So, antihistamines are used to treat allergy. The nitrogen has to go!  If amino acids are not required for synthetic reactions, they can be used for energy, or converted to energy storage compounds  To do this, the amino group MUST be removed, and the nitrogen excreted in the form of ammonia AMMONIA  Blood ammonia level is 10-60 µg/dl  Ammonia is toxic to central nervous system and its accumulation in body is fatal.  Hyperammonaemia results in tremor, vomiting, cerebral oedema, coma and death.  Once formed in the body, ammonia must be removed from blood.  The liver is the organ that converts it to urea, which is:  less toxic,  water soluble &  easily excreted in urine. Sources of ammonia: 1- Deamination of amino acid with formation of α-keto acids and ammonia. 2- Glutamine in the kidney by glutaminase enzyme gives glutamic acid and ammonia, which is used by kidney to regulate the acid base balance. 3- Ammonia produced by action of intestinal bacteria on non-absorbed dietary amino acids. 4- Ammonia is released during purine and pyrimindine catabolism. Fate of ammonia: 1- Biosynthesis of nonessential amino acids 2- Biosynthesis of glutamine by glutamine synthetase enzyme. 3- Small amounts of ammonia are excreted in urine in the form of ammonium ions (NH4+). 4- Urea biosynthesis. 61 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Urea Biosynthesis  Ammonia is highly toxic to the central nervous system.  The liver is the site of Urea biosynthesis.  Urea biosynthesis occurs by: Urea cycle Urea cycle  It occurs in five steps.  The first 2 steps occur in the mitochondria & the last 3 steps occur in cytoplasm.  It utilizes 3 ATP and 4 high energy bonds.  It is catalyzed by five enzymes.  Any defect in one of these enzymes leads to ammonia intoxication. Ammonia Intoxication:  Ammonia is highly toxic to the central nervous system.  Blood ammonia level 10-60 ug/dl  Accumulation of ammonia in the body leads to nervous manifestations and may be fatal (due to inhibition of Krebs cycle in brain, which is the main source of energy in the brain). Causes of hyperammonemia: 1- Acquired hyperammonemia a. Liver cell failure. 2- Congenital hyperammonemia * Defect in any one of the five enzymes urea cycle. 62 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Minerals Chemistry 63 Prof./ Salwa Abo Elkhair  Medical Biochemistry for Dentistry Mansoura National University Minerals Metabolism According to body needs, minerals are divided into: 1- Major minerals [Macronutrients]:  They are required in amounts > 100 mg / day.  They include: calcium, phosphorus, magnesium, sodium, potassium, chloride and sulphur. 2- Trace elements [Micronutrients]:  They are required in amounts < 100 mg / day.  They include: iron, copper, zinc, selenium, chromium, cobalt, iodine, fluoride, manganese and molybdenum. Calcium, Phosphorus Ca Distribution:  1200 g of Ca in adult body.  99 % in skeleton:  1 % (ionized and non-ionized) in body fluids, serum and CSF. P Distribution:  800 g distributed in all body cells.  80 % in bone and teeth combined with Ca.  20 % in all body cells: in (chemical compounds) as nucleic acids, nucleotides, coenzymes & creatine phosphate Ca P Distribution  1200 g of Ca in adult body.  800 g distributed in all body cells.  99 % in skeleton:  80 % in bone and teeth combined with Ca.  1 % (ionized and non-ionized) in body fluids, serum and CSF.  20 % in all body cells: in (chemical compounds) as nucleic acids, nucleotides, coenzymes & creatine phosphate Function A. Bone mineralization: 1. Bone and teeth mineralization.  Ca is an essential element in bone and teeth 2. Phosphate ester compounds for transfer  Ca is captured into bones by binding osteocalcin and storage of energy. B. Ionized Ca: 3. Nucleic acids formation. 1. Blood coagulation and milk clotting. 2. Membrane permeability. 4. Coenzymes [TPP, NADP]. 3. Muscle and Nerve excitability: Generation 5. Phosphate buffers system in blood. and conduction of nerve impulse & muscle contraction 4. Second messenger of many hormones. 5. For activity of some enzymes: lipase, amylase & phosphorylase. 64 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Factors affecting absorption: Calcitriol (1,25 dihydroxycholecalciferol): PTH: through activation of vit D Both Vit D and PTH ….increase absorption. pH of intestinal contents: - Alkaline pH leads to less soluble Ca salts - Acidic pH  Ca solubility and  absorption. Dietary factors: A- Vit D, lactose and high protein diet  the absorption. B- Phytate, oxalate (insoluble Ca salts) & fatty acids  Ca absorption. C- (antacid)  P absorption. Control of blood calcium & phosphorus: o The precise control is regulated by action of PTH, calcitriol and calcitonin on intestine, kidney and bone. 1- Calcitriol: produced in kidney in response to low calcium & phosphorus level. It increases their levels by:  Increasing their intestinal absorption.  Decreasing renal excretion of Ca.  Stimulate bone resorption. 2- PTH: secreted in response to low plasma calcium level. It  calcium &  phosphorus levels through;   renal tubular reabsorption of Ca++ &  phosphate excretion.  Activation of vit. D. 3-Calcitonin: lowers both plasma calcium and phosphorus by: 1. Inhibiting calcium mobilization from bone. 2. Decreasing renal tubular reabsorption of calcium & phosphorus 3.  Ca and P excretion. 65 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Normal Blood Calcium level: 9 – 11 mg/dl Disorders of blood calcium level: Hypocalcemia:  Blood level < 8.8 mg/dl.  < 7.5 mg/dl→ tetany [ neuro-muscular irritability] → muscle & Laryngeal spasm.  Chronic ↓→ bone deformity (Rickets and osteomalacia). Hypercalcemia:  Blood level > 11 mg/dl  Calcification of soft tissue: Ca deposit in:  Lungs, heart, Kidney, blood vessels  Hardening of arteries, stone formation in kidneys Magnesium Mg Distribution: 21 g.  70% with calcium & phosphrus in bones.  30% in all tissues and body fluids (intracellular and extracellular). Mg ions are activators for: 1. phosphate transferase enzymes e.g. Kinases, phosphorylase. 2. Co factor in RNA & DNA synthesis 3. Important in Nerve Impulse conduction Sodium, Potassium and Chloride 66 Prof./ Salwa Abo Elkhair  Medical Biochemistry for Dentistry Mansoura National University Sulphur Distribution & Functions:  In all body tissues and cells as: Mucopolysaccharieds Lipids [sulpholipid and bile salt]. Proteins [sulphur a.a] Hormones as insulin Co-enzymes; CoA-SH, PAPS and SAM (s adenosyl methionine). Detoxification mechanism. Vitamins as thiamine and biotin. Trace Elements Trace element Function Deficiency Iron (Fe) Iron enters in the structure of: Decrease serum iron: - Hemoglobin & myoglobin. Iron deficiency anemia. - Respiratory cytochromes. - Cytochrome P450. - Peroxidases and catalases. - Thyroperoxidase Zinc Essential for; 1. Growth retardation  Reproduction, tissue repair and wound healing. 2. Delayed wound healing  insulin storage & release 3. Impaired sexual development Copper (Cu)  Essential for: 1- Hb synthesis, 2- bone formation, 3-maintenance of myelin. Manganese  Essential for normal bone structure (Mn)  Function of CNS and spermatogenesis.  Involved in activation of many enzymes Iodine Formation of thyroid hormones (if prolonged) Goitre 67 Prof./ Salwa Abo Elkhair  Medical Biochemistry for Dentistry Mansoura National University Fluoride Distribution: Bone, teeth and very low amount in soft tissues Functions: - protective against dental caries - protective against osteoporosis & osteomalacia. Selenium (Se) -Component of: glutathione peroxidase, - Essential for: Antioxidant activity Cobalt (Co) as a constituent of vitamin B12, it is involved in erythropoiesis 68 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Vitamins Chemistry 69 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Vitamins Vitamins are organic nutrients that are: 1- Required in small amounts 2- Cannot be synthesized by the body and must therefore be supplied by the diet 3- They are essential for a variety of biochemical functions which are required for the normal growth, development and maintenance of life. 4- Vitamins are not utilized as structural unit of the cell or tissue and do not provide energy 5- They act as a cofactors for enzymes in biochemical reactions. 6- Absence or relative deficiency of vitamins in the diet leads to characteristic deficiency states and disease. Classification of vitamins: The solubility of vitamins was used as a basis for their classification, Vitamins are divided into two main groups: 1. Fat soluble vitamins: Vitamins A, D, E, K. 2. Water soluble vitamins: Vitamins B group and C. Fat soluble vitamins: 1- can only be absorbed efficiently with normal fat absorption 2- Conditions such as mal-absorption syndrome and biliary system disorders can lead to fat soluble vitamins deficiencies 3- Fat soluble vitamins are transported in the blood in lipoproteins or attached to specific binding proteins 4- They are adequately stored in the body. 5- When taken in excessive amount, they may lead to toxic effects (hyper-vitaminosis). Water soluble vitamins: 1- The storage of water soluble vitamins in the body is limited (except vit. B12). 2- Excesses of water soluble vitamins are excreted in urine because of their water solubility 3- Rarely accumulate to toxic level in the body. 4- They must be provided regularly. 70 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Fat soluble vitamins Vitamin A (Anti-xerophthalmic vitamin) Vitamin A (Anti-xerophthalmic vitamin) Function 1- in vision: Colored vision at night 2. normal growth important for normal growth 3. Maintenance of integrity of epithelial tissues as skin, mucus membranes, bone tissues, and spermatogenic epithelium. 4. Necessary for healthy skin & hair growth 5. Reproduction 6. antioxidant 7. anticancer activity Deficiency 1. Defective night vision. 2. Failure of growth. 3. Xerosis and keratinization of mucus membranes 4. Certain forms of skin disease. 5. Impaired bone growth with nerve lesions. 6. Reproductive disorders including: Degeneration of the testes, abortion, production of malformed offspring Vitamin D (Anti-rachetic vitamin) Formation of Vitamin D: * Skin (UV light): 7- dehydro cholesterol vitamin D3 * Liver: OH-group added 25- hydroxy vitamin D3, Storage form of vitamin D * Kidney: OH-group added 1, 25- dihydroxy vitamin D3 (or 1,25-dihydroxy cholecalciferol), Active form of vitamin D Functions of vitamin D: 1- It is to maintain adequate serum level of calcium. This function is performed by: A - Increasing uptake of calcium by intestine. B - Minimize losses of calcium by the kidney. C - Stimulate resorption of bone and mobilization of Ca and PO4 from bone when PTH is released in response to low serum calcium. 2- Bone formation: Stimulate calcium uptake for deposition as calcium phosphate (Osteoblasts: bone-forming cells). 71 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Vitamin D deficiency In Children: Rickets * Failure of bones to grow properly * Delay setting, walking & teething. * Deformed bones (skull, chest and legs) * Decrease of Ca & P in the blood In Adult: Osteomalacia. Bone deformation, fracture, Decrease of Ca & P in the blood. Osteoporosis (porous bones): Loss of vitamin D activity with advancing age. Associated with fractures, very serious for elderly. Toxicity of vitamin D * It is the most toxic of all vitamins. * Loss of appetite, nausea, thirst. * Calcification of soft tissue: Hypercalcemia may lead to calcium deposit in Lungs, heart, blood vessels * Hardening of arteries, stone formation in kidneys * Does not occur from sunlight or dietary sources * Does occur with supplementation 72 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Vitamin E (Tocopherol) Functions 1- Vit. E is the most important natural anti-oxidant 2- The first defensive mechanism against cell damage, it protect cells against oxidative damage by free radicals, e.g. oxidation of the lipids in the cell membranes 3- Protect the RBCs from toxic haemolysis. 4- plays a role in aging, sexual performance, or prevention of cancer and/or heart disease Vitamin E – Deficiency: Rare 1- Erythrocyte hemolysis and hemolytic anemia: disruption of red blood cell membranes, perhaps due to polyunsaturated fatty acids (PUFAs) oxidation 2- Sterility: reproductive failure in rats Vitamin K (Anti-haemorrhagic vitamin) Functions of Vitamin K: 1- Essential for clotting of blood.  Vitamin K is essential for synthesis of blood clotting factors II, VII, IX and X in liver.  Clotting factors are synthesized in the liver as inactive precursors - vitamin K converts them to their active forms - Conversion of prothrombin to thrombin, an active enzyme - Formation of fibrinogen to fibrin, leading to clot formation 2- Stimulates bone formation and decreases bone resorption. Vit K deficiency: 1- Increase bleeding and Hemorrhage 2- Prolonged prothrombin time * Rare, may occur when antibiotic kill intestinal bacteria that produce the vitamin. 73 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Water Soluble Vitamins Vitamin C ( L-Ascorbic acid) (Anti-scurvy) Functions: 1- Vitamin C is required for the activity of many hydroxylases: important in the biosynthesis of collagen 2- Required in the biosynthesis of steroid hormones in adrenal cortex, 3- Important for absorption, transport and storage of iron. 4- Required for activation of folic acid to tetra-hydrofolic acid (active form) 5- Act as a general water soluble antioxidant. Vitamin C deficiency: Scurvy: * It is characterized by defective collagen synthesis which is indicated by: 1- Soft swollen gums and loose teeth. 2- Bleeding tendency and subcutaneous hemorrhage. 3- Swollen joints and muscle weakness 4- Delayed wound healing. 5. Iron deficiency anemia due to impaired utilization of iron. B Complex  Found in the same foods: single deficiency rare  B complex vitamin are necessary for healthy skin, hair, eyes and liver, also help the nervous system function properly.  Co-enzymes formation (activate enzymes)  Important and Act together in metabolism  Metabolic pathways of protein, carbohydrate, & fat  B vitamins help the body to produce energy.  Energy metabolism Thiamin (B-1), Riboflavin (B-2), Niacin (B-3), Pyridoxine (B-6), Biotin (B-7), Pantothenic Acid (B-5)  Red blood cell synthesis Folate (B-9), B-12 74 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University practical biochemistry 75 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Detection of Glucose in Body Fluids & Diagnosis of Diabetes Mellitus 76 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Detection of Glucose in Body Fluids Agenda Estimation of glucose in body fluids. Different methods for glucose identification in blood and urine. Diagnosis of diabetes mellitus. Blood glucose or blood sugar?!  Glucose is referred to as “blood sugar” because it circulates in bloodstream as a source of readily available energy.  Measurement of glucose level in body fluids (e.g. serum, urine) is important to diagnose many diseases e.g. Diabetes Mellitus. Principle of Glucose Measurement: “Glucose Oxidase Method”  This method is highly specific for glucose only.  It is a double sequential enzymatic reaction (glucose oxidase and peroxidase) in the presence of an indicator that changes in color in presence of glucose. Lab Techniques based on Glucose Oxidase Method Urine Glucometer Colorimeter Dipstick Test 77 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University 1. Glucometer (Glucose meter) It is a simple method of monitoring blood sugar at home. Most glucometers use an electrochemical method. They analyze a drop of capillary blood. The glucose in the blood reacts with an enzyme electrode containing glucose oxidase. Requirements: Glucometer (Glucose Meter) Test strip Lancet device Needle (Lancet) Alcohol swab Dry swab Steps of measurement: Wash your hand with soap and water (before and after). Take one finger with an alcohol swab. Press the lancet device against the side of your finger. Squeeze your finger to get a drop of blood. Touch the end of the glucometer strip to the drop of blood. Over the puncture site, apply a dry swab. The level of your blood glucose level is displayed. Fasting blood glucose level: A normal range: 70–110 mg/dl Hypoglycemia: blood glucose 140 mg/dl. 2. Urine Dipstick Test (Urine Test Strips) The urine dipstick test is the quickest way to test urine. Plastic strips have pads impregnated with chemicals that react with the compounds present in urine producing a characteristic color. The chemicals in the pads indicate the presence of specific substances in the urine such as glucose, protein, blood, etc. The strips can also indicate the pH and specific gravity of the urine. In case of glucose detection, Active reagents are Glucose oxidase and peroxidase. The color chart value is mg/dl for glucose 78 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Steps: 1- Fill a sterile specimen container with urine. 2- Dip the test strip into the urine at the thick end. 3- Once saturated, Remove it from the container. Drag the strip along the edge of the container. then Use an absorbent material e.g Filter paper, to soak up the excess urine. 4- Wait 1-2 minutes to read the test strip results. 5- Compare the test squares to the color chart and interpret the results (The color chart values are mg/dl for glucose). The color chart value is mg/dl for glucose. Normally, Test result is negative. No glucose should be detected in urine (trace amount). If test is positive, this means urine contains glucose (glucosuria) and this occurs when blood glucose level exceeds the renal threshold for glucose ( > 180 mg/dl) or in case of diseased kidney. 3. Colorimeter A colorimeter is used to determine the concentration of compounds in solution. Certain reagents are added on the biological samples to produce colored products when certain compounds are present in the sample. Then by measuring the absorbance of the colored products at a specific wavelength of light, the concentration can be calculated. N.B: colored solutions can absorb lights at certain wavelengths. The amount of light absorbed is proportional to the solute concentration present in solution. Based on Glucose Oxidase method, the intensity of the colored product is proportional to the glucose concentration and is measured at wave length between 490 and 540 nm. 79 Prof./ Salwa Abo Elkhair Medical Biochemistry for Dentistry Mansoura National University Diabetes Mellitus Def: It is a state of chronic hyperglycemia due to disturbances in carbohydrate, Lipid and protein metabolism caused by  Lack of insulin secretion  Decreased sensitivity of the tissues to insulin Blood Glucose: Normal fasting blood glucose (8-12 hours after CHO meal) level is 70-110 mg/dl. It normally fluctuates between 70-140 mg/dl. Reaches 140 mg/dl during the first hour after meal (post prandial), then returns to fasting level within 2 hours after meal. * Kidney can reabsorb glucose from glomerular filtrate up to the level of 180 mg/dl in blood (renal threshold). Diagnosis of DM: 1. Measurement of blood Glucose level. 2. The Glucose Tolerance Test. 3. Glycated Haemoglobin. 4. Glycated plasma proteins. 5. Microalbuminuria. 1- Measurement of blood Glucose level: Increas