METABOLIC PATHWAYS



INTRODUCTION TO BIOMOLECULES AND METABOLISM:Bio-molecule Are the biological molecules that particitipates In the physiological functioning of the living things. They are either organic or inorganic in nature. They may be classified as monomeric or polymeric compounds.They include; Carbohydrates, Lipids, Proteins and amino acids, Enzymes, Plasma proteins, nucleic acids, Immunoglobulin, Biological oxidation, Haemoglobin and haemoglobinopathies, Vitamins, Prostaglandins.Metabolism: These are the sequences of the biochemical reaction that degrade, synthesize, or interconvert small molecules inside a cell. This is for the understanding both normal functioning and metabolic basis of human diseases, molecular basis of drug action, drug interaction and many genetic diseases that are caused by the absence of a particular proteins or enzyme. The metabolic pathways include, gluconeogenesis, glycolysis, TCA cycle, β oxidation of fatty acids e.t.cMetabolism is divided into twoCatabolism:The degradation process concerned with breakdown of complex molecules to simpler ones, with a corresponding release of energy in the form of ATP i.e. glycolysis TCA cycle , oxidation of fatty acidsAnabolism:This is the biosynthesis reaction involving the synthesis or the formation of complex molecules from simple precursor i.e. gluconeogenesis,glycogenesisFood and fuels found in the body:There are three kinds of fuels found in the body. That isAmino acids and proteinsCarbohydratesFatty acids and ketonesThese fuels undergoes the inter conversion process to yield or utilize energy in the form of ATP The inter conversion requires ATP as the high energy compound. The process by which the ATP is formed or generated and utilized is known as ATP-ADP cycleThe dietary fuel being the carbohydrates, proteins and fats the can undergoes the digestion and absorbed and the products of digestion undergoes circulation in the blood and enter into various tissues and are eventually taken up by cells and get oxidized to produce energy and carbon dioxide and water molecules and oxygen is required for oxidation process to take place. The fuels are stored in the body as;Fats: triglycerides in the adipose tissuesCarbohydrates: glycogen in muscles, liver and other cellsProtein: in musclesThe oxidation of fuels is also referred to respiration process. It requires the utilization of oxygen to yield carbon dioxide and water. Several pathways are there which involves generation of the ATP from the fuels and the oxidation of glucose, fatty acids, and the amino acids to acetyl CoA a substrate for the TCA cycle. In the TCA cycle they are completely oxidized, electrons are transferred to oxygen by the electron transport chain and the energy is used to generate ATP ( formed conversion of the ADP and Pi to ATP by the process known as Oxidative IC: BIOENERGETICSBioenergetic is the study of the energy transfer and utilization in biochemical reaction. It is also known as biochemical thermodynamics.Bioenergetics is classified into two:Endergonic reaction: this involves energy utilizationExergonic reactions: this involves energy release.Bioenergetics generally deals with the initial and the final state of the reactants and not mechanism of reaction.Free Energy: (G)This is the utilizable or energy available to do work which can be utilized (absorbed) or released and is denoted by (G). A decrease in free energy leads to spontaneous reaction.Enthalpy ( H): is a measure of change in heat between reactants and the products.Entropy( S): represents a change in randomness or disorder of reactants and products which attains its maximum as the reactions approaches equilibrium.Relationship between Free energy, entropy and enthalpy: FREE ENERGY (G) = ENTHALPY ( H) –T ENTROPY ( S) ~ T=absolute temperature in kelvi ( K=273+oC).Standard free energy (G0) is the free energy change when reactants or products are at concentration of 1 mol/l at pH 7.0.Negative and Positive free Energy (G):Negative free energy (-G): is known as exergonic reaction referring to loss of energy leading to spontaneous reaction.Example:ATP +H2O ADP +Pi (G0 =-7.3 Cal/Mol)Positive free energy (+G): is known as endergonic reaction referring to utilization or supply of energy hence non-spontaneous reaction.ADP +Pi ATP(G0 =7.3 Cal/Mol)Free energy change is zero when the reaction is at equilibrium hence at a constant temperature and pressure, change in free energy (G) depends on the concentration of product and concentration of reactant. Example; In the conversion of A to B,Mathematically;G =G0 +RT ln [B] [A]Whereby;G0 =Standard free energy changeR=gas constant (1.987 Cal/mol)T=Absolute temperature (273+0C)Ln =Natural Logarithm[B]=concentration of products[A]= concentration of reactantsG0 Is related to equilibrium constant in case of reactions; example; conversion of A B while free energy is 0.Hence;.G =0 =G0 +RT ln [B]Eq [A]EqHence =G0 =-RTln KeqHIGH ENERGY COMPOUND:These are the substances which posses sufficient free energy to liberate at least 7 cal/mol at PH of 7.0 i.e ATP.High energy compounds can classified as;Pyrophosphates i.e ATPAcylphosphates i.e. 1,3 bisphosphoglyceratesEnol phosphates i.e. phosphoenolpyrophosphateThioether e.g acetyl COAPhosphoguanidines e.g. phosphocreatine.High energy compounds: are compounds that possess acid anhydride bonds (phosphoanhydride bonds) whish are formed by the condensation of two acidic groups or related compounds.Lipmann suggested the symbol ~ to represent high energy bond hence ATP can be written as AMP~P~PSNOCOMPOUNDG0 (Cal/Mol)HIGH ENERGY PHOSPHATESPhosphoenol pyruvate-14.8Carbamoyl phosphate-12.3Cyclic AMP-12.01,3 bisphosphoglycerate-11.8Phosphocreatine-10.3Acetyl phosphate--10.3S-Adenosylmethionine*-10.0Pyrophosphates-8.0Acetyl COA**-7.7ATP-7.3LOW ENERGY COMPOUNDSADP-6.6Glucose 1-phosphate-5.0Fructose 1-phosphate-3.8Glucose 6 phosphate-3.3Glycerol 3 phosphate-2.2*Sulfonium compounds** thioether ATP-ADP CYCLE:The hydrolysis of ATP is associated with the release of large amount of energy.ATP +H2O ADP +Pi (G0 =-7.3 Cal/Mol)The energy which is liberated can be utilized by various process like muscle contraction, active transport, e.t.c ATP can also act as donor of high energy phosphates to low energy compounds making them rich and on the other hand ADP can accept high energy phosphates from compounds possessing higher free energy contents to form ATP.ATP serves as an immediately available energy currency of a cell which is constantly utilized and regenerated bearing a high turn over.ATP acts as an energy link between the catabolism (degradation of molecules) and anabolism (synthesis) in the biological system.SYNTHESIS OF ATP:ATP can be synthesized in two ways;Oxidative phosphorylationIs an aerobic major source of energy which is linked with mitochondrial ETCSubstrate level phosphorylationATP synthesis occurring directlysubstrate oxidation bearing high energy compounds like phosphoenolpyruvate and 1,3 bisphosphoglycerate (intermediate in glycolysis), succinyl COA in TCA cycle which are involed in the transfer of phosphate energy to produce ATPHigh energy compounds are stored in vertebrates as phosphocreatine or creatine phosphate in muscles and brains and in inverterbrates is stored as phosphoarginine or arginine ANIZATION OF ELECTRON TRANSPORT CHAIN: METABOLIC PATHWAYSTOPIC: CARBOHYDRATE METABOLISMGLYCOLYSISCBA5443KEY:A,B,C are allosteric regulation steps of glycolysis3- bromohydroxyacetone phosphate inhibitor4- iodoacetate and arsenate inhibitor5-fluoride inhibitorGlylycosis pathway is also known as Embden- Meyerhof pathway and it is a catabolic reactionGlycolysis pathway Occur in two ways:1. Aerobic: in the presence of oxygen and the end product is pyruvate and later it is oxidized to carbon dioxide and water2. Anaerobic: in the absence of oxygen and the end product is Lactate and it is summarized asGlucose +2ADP +2Pi 2 Lactate + 2ATPIt occurs in the tissue which are lacking mitochondria i.e erythrocytes, cornea, lens and it is essential source of energy for brains.REACTIONS OF GLYCOLYSIS:A. Energy investment phase or priming phaseB. Splitting phaseC. Energy generation phaseEnergy investment phase:Glucose gets phosphorylated to glucose 6 phosphate by hexokinase or glucokinase depending on ATP ang Mg2+NB: glucose 6-phosphate is impermeable into cell membrane hence its metabolic fates are glycolysis, glycogenesis, gluconeogenesis, and pentose phosphate pathway (Hexose Monophosphate Shunt pathway-HMP)Isomerization of glucose 6 phosphate to fructose 6 phosphate in the presence of phosphohexose isomerase and Mg2+Fructose 6 phosphate gets phosphorylated tofructose 1-6-bisphosphate by phosphofructokinase (PFK). PFK is an allosteric and regulatory step in glycolysisSplitting Phase:Fructose 1,6 biphosphate gets split into glyceraldehydes 3 phosphate and dehydroxyacetone phosphate by the enzyme aldolase (Fructose 1,6 biphosphate aldolase).Glyceraldehydes 3 phosphate and dehydroxyacetone phosphate get interconverted by phosphotriose isomerase enzyme. It gets inhibited by bromohydroxyacetone phosphate.Energy generation phase:Oxidative phosphorylation occurs when Glyceraldehydes 3 phosphate get converted into 1,3 bisphosphoglycerate in the presence of Glyceraldehydes 3 phosphate dehydrogenase in the precence of NAD+ which undergoes reduction by accepting Hydrogen ions to NADH and addition of phosphate to Glyceraldehydes 3 phosphate. 1,3 bisphosphoglycerate is a high energy compound formed. (NADH has 3 ATP hence 3X2= 6 ATPs formed). The reaction is Inhibited by iodoacetate and arsenateSubstrate level phosphorylation occurs when1,3 bisphosphoglycerate is converted into3-phosphoglycerate hence synthesis of ATP in the presence of phosphoglycerate kinase.3 phosphoglycerate is converted into 2 phosphoglycerate in the presence of phosphoglycerate mutase and Mg2+.Phosphoenol pyruvate is formed from 2-phosphoglycerate in the presence enolase enzyme and Mg2+ or Mn2+ which is inhibited by fluoride.Pyruvate is formed from phosphoenol pyruvate in the presence of pyruvate kinase which requires K+and Mg2+ or Mn2+ with release of ATP.NB: Total number of ATP Generated: 8 ATPs -2ATP Utilized= 6 ATPsREGULATION OF GLYCOLYSIS:Regulation of glycolysis is done by three enzymes, hexokinase or glucokinase, phosphofructokinase and pyruvate kinase due to its irreversible reactions.Hexokinase is inhibited by glucose 6 phosphatase accumulation due to product inhibition.Phosphofructokinase (PFK) which is allosteric enzyme and is regulated by allosteric effectors, ATP, citrate and H+ ions (low PH). Fructose 2,6 bisphosphate,AMPand Pi are allosteric activators.Pyruvate kinase is inhibited by ATP and activated by F16BP. Pyruvate kinase is active in dephosphorylated state and inactive in phosphorylated state which is brought about by cAMP dependent protein i.e. hepatic glucagonROLE OF HORMONES IN METABOLISMINSULIN:Secreted in the pancreas by islets of langerhansIt is made up of amino acids, cystein,proline, isoleucineThe hormone requires zinc (metal)for its crystallizationFunctions:Increases glucose uptake ,glycolysis, conversion of pyruvate to acetyl CoA and fatty acid synthesisIt stimulates glycogenesisIt decreases lipolysisGLUCAGON:Secreted in the pancreas by islets of langerhansIt is made of amino acids like tyrosine methionine and tryptophan.It does not require metal ions for its crystaizationFunctions:Mobilization of hepatic glycogen to give glucose by glycogenolysisIt increases glycogenolysis, gluconeogenesis and lipolysis hence regulating decreased amount of blood glucoseHormones of hypophylysisThyrotropin i.e. thyroid stimulating hormone (TSH)Functions:Stimulates the synthesis of thyroid hormonesIncreases the DNA contentsStimulates glycolysisCONVERSION OF PYRUVATE TO ACETYL COA.Pyruvate is converted to Acetyl CoA by the process of Oxidative decarboxylation.This reaction is catalyzed by the enzyme pyruvate dehydrogenase complex (PDH) found in the mitochondria, cardiac muscles and kidneyThe co-factor (co-enzymes) required are:TPP, lipoamide, FAD, Coenzyme A and NAD+The overall reaction of PDH is:Pyruvate +NAD+ + CoA PDH Acetyl Coa + CO2 +NADH +H+Reaction:TCA CYCLETRI CARBOXYLIC ACID is also known as KREBS CYCLE or CIRTIC ACID CYCLE It involve the oxidation of Acetyl Coa to caron dioxide and water and it is the common oxidative pathway to carbohydrates,fats, and amino acids.It intermediates include:amino acids, heme, glucose.Occurance: mitochondriaIn triarboxylic acid cycle it involves combination of 2 carbon atom ACETYL CoA and 4 carbon atom OXALOACETATE to form a 6 carbon atom tricarboxilic acids CITRATEOXALOACETATE plays a major catalytic role in TCA cycleFunction: To burn the acetyl-CoA made from fat, glucose, or protein in order to make ATP in cooperation with oxidative phosphorylation.Location: All cells with mitochondria.Connections: From glycolysis through acetyl-CoA. Pyruvate makes oxaloacetate and malate through the anaplerotic oxidation reactions through acetyl-CoA. Amino acid degradation through acetyl-CoA and various intermediates of the cycle.Regulation: Supply and demand of TCA cycle. Availability of NAD- and FAD as substrates.Inhibition by NADH High-energy signals turn off. Low-energy signals turn on. ATP yield: Pyruvate 15ATP Acetyl-CoA 12ATP.Equations:COMPONENTS OF ELECTRON TRANSPORT CHAINSOURCES OF CARBOHYDRATES:GLYCOGENOLYSIS: This is the process of the breakdown of glycogen in liver and tissues to replenish glucose in blood. The process of glycogenolysis requires enzyme Glycogen Phosphorylase. The first step in glycogenolysis is catalyzed by glycogen phosphorylase, commonly called phosphorylase. This enzyme cleaves the a-1,4 glycosidic bonds of glycogen Glycogen + HPO4 2- glycogen phosphorylase glucose 1 -phosphate + glycogen (glucose residues) (- 1 glucose residues) The reaction catalyzed by glycogen phosphorylase is physiologically irreversible and is the regulated step in glycogen mobilization. Different isozymes of glycogen phosphorylase are present in muscle and liver, permitting organ-specific regulation of glycogenolysis. The debranching enzyme:α- 1,6-glucosidase catalytic site of the debranching enzyme then hydrolyzes the a- 1,6 glycosidic bond of the remaining glucose unit, releasing free glucose and producing a long, unbranched a-1,4 glycosidic chain that is a suitable substrate for continued glycogenolysis by glycogen phosphorylaseFate of glucose 6 phosphate obtained from glycogenolysis:The glucose molecules obtained from glycogenolysis is either utilized in the liver to regulate blood glucose or it undergoes Glycolysis to yield energy that can be utilized in Brain and musclesBlood sugar level and its significance:Normal values: the normal fasting or absorptive glucose taken atleast three hours after the last meal is:As per glucose oxidase method (“true glucose”) 60 mg to 100mg %As per “ folin and wu’s method” is 80mg to 120 mg %Abnormalities in blood glucose level:Increased in blood glucose level above the normal is called “ hyperglycemia”Decreased in blood glucose below the normal values is called “ hypoglycemia”A: HYPERGLYCEMIACauses of hyperglycemia:Diabetes meritus ?fasting blood glucose varying from the normal to more than 500 mg % depending on severity of the disease.Hyperactivity of the thyroid, pituitary and adrenal glandsEmotional stress Diffuse diseases of the pancreas i.e. pancreatitis and carcinoma of the pancreas readily increases blood glucose.Sepsis Intracranial diseases like meningitis, encephalitis, intracranial tumors and hemorrhage.Anaesthesia increases blood sugar depending on the degree and duration. B: Hypoglycemia:Causes of hypoglycemia:This is a state in which the blood glucose is below 40 mg % (“ true “ glucose) through glucose oxidase test.It can be caused by Overdosage of insulin hormone in a case of treatment of diabetes mellitus.Hypoactivity of thyroid hormones (myxoedema and creatinism),hypopituitarism (symmond’s diseases) and hypoadrenalism (addison’s diseases) can causes the fasting glucose to reduce.Severe liver diseasesGlycogen storage diseases (GSD) i.e. von gierk’s diseases which is due to liver phosphorylase enzyme deficiency hence impaired glucagon synthesis from glucose.In children spontaneous hypoglycemia and leucine sensitive hypoglycemia is experienced. THE HEXOSE MONOPHOSPHATE PATHWAY (HMP SHUNT)It is also known as the pentose phosphate pathway or 6 phosphogluconate pathway.Occurs in the cytosol of the cell.It includes two reactions, Irreversible oxidative reactions. Followed by a series of reversible sugar–phosphate interconversions.No ATP is directly consumed or produced in the cycle.Carbon 1 of glucose 6-phosphate is released as CO2, and two NADPH are produced for each glucose 6-phosphate molecule entering the oxidative part of the pathway. The rate and direction of the reversible reactions of the pentose phosphate pathway are determined by the supply of and demand for intermediates of the cycle. The pathway provides a major portion of the body’s NADPH, which functions as a biochemical reductant.It also produces ribose 5-phosphate, required for the biosynthesis of nucleotides and provides a mechanism for the metabolic use of five-carbon sugars obtained from the diet or the degradationof structural carbohydrates in the body.IRREVERSIBLE OXIDATIVE REACTIONSThe oxidative portion of the pentose phosphate pathway consists of three reactions that lead to the formation of ribulose 5-phosphate, CO2, and two molecules of NADPH for each molecule of glucose 6-phosphate oxidized. This portion of the pathway is particularly important in the liver, lactating mammary glands, and adipose, which are active in the NADPH-dependent biosynthesis of fatty acids (testes, ovaries, placenta and adrenal cortex, are active in the NADPH-dependent biosynthesis of steroid hormones and in erythrocytes, which require NADPH to keep glutathione reduced.Dehydrogenation of glucose 6-phosphateGlucose 6-phosphate dehydrogenase (G6PD) catalyzes an irre versible oxidation of glucose 6-phosphate to 6-phosphogluconolactone in a reaction that is specific for NADP+ as its coenzyme. The pentose phosphate pathway is regulated at the G6PD reaction. NADPH is a potent competitive inhibitor of the enzyme, and under most metabolic reactions the ratio of NADPH/NADP is sufficiently high to substantially inhibit enzyme activity. However, with increased demand for NADPH, the ratio of NADPH/NADP decreases and flux through the cycle increases in response to the enhanced activity of G6PD. Insulin up regulates expression of the gene for G6PD, and flux through the pathway increases in the well fed state. B. Formation of ribulose 5-phosphate6-Phosphogluconolactone is hydrolyzed by 6-phosphogluconolactone hydrolase. The reaction is irreversible and not rate-limiting. The oxidative decarboxylation of the product, 6-phosphogluconate is catalyzed by 6-phosphogluconate dehydrogenase. This irreversible reaction produces a pentose sugar phosphate (ribulose 5-phosphate), CO2 (from carbon 1 of glucose), and a second molecule of NADPH.REVERSIBLE NONOXIDATIVE REACTIONSThe nonoxidative reactions of the pentose phosphate pathway occur in all cell types synthesizing nucleotides and nucleic acids. These reactions catalyze the interconversion of sugars containing three to seven carbons. These reversible reactions permit ribulose 5-phosphate (produced by the oxidative portion of the pathway) to be converted either to ribose 5-phosphate (needed for nucleotide synthesis, or to intermediates of glycolysis—fructose 6-phosphate and glyceraldehydes 3-phosphate. For example, many cells that carry out reductive biosynthetic reactions have a greater need for NADPH than for ribose 5-phosphate. Transketolase (which transfers two-carbon units in a thiamine pyrophosphate (TPP)-requiring reaction) and transaldolase (which transfers three-carbon units) convert the ribulose 5-phosphate produced as an end product of the oxidative reactions to glyceraldehydes 3-phosphate and fructose 6-phosphate, which are intermediates of glycolysis. In contrast, under conditions in which the demand for ribose for incorporation into nucleotides and nucleic acids is greater than the need for NADPH, the nonoxidative reactions can provide the biosynthesis of ribose 5-phosphate from glyceraldehydes 3-phosphate and fructose 6-phosphate in the absence of the oxidative steps. USES OF NADPHThe coenzyme NADP+ differs from NAD+ only by the presence of a phosphate group on one of the ribose units. This small change in structure allows NADP+ to interact with NADP specific enzymes that have unique roles in the cell. For example, in the cytosol of hepatocytes the steady-state ratio of NADP /NADPH is approximately 0.1, which favors the use of NADPH in reductive biosynthetic reactions. This contrasts with the high ratio of NAD+ /NADH, which favors an oxidative role for NAD+. This section summarizes some important NADP+ or NADPH-specific functions.A. Reductive biosynthesisNADPH can be thought of as a high-energy molecule, much in the same way as NADH. However, the electrons of NADPH are destined for use in reductive biosynthesis, rather than for transfer to oxygen as is the case with NADH. Thus, in the metabolic transformationsof the pentose phosphate pathway, part of the energy of glucose 6-phosphate is conserved in NADPH—a molecule with a negative reduction potential that, can be used in reactions requiring an electron donor.Reduction of hydrogen peroxideHydrogen peroxide is one of a family of reactive oxygen species (ROS) that are formed from the partial reduction of molecular oxygen. These compounds are formed continuously as by-products of aerobic metabolism, through reactions with drugs and environmental toxins, or when the level of antioxidants is diminished, all creating the condition of oxidative stress. The highly reactive oxygen intermediates can cause serious chemical damage to DNA, proteins, and unsaturated lipids, and can lead to cell death. These ROS have been implicated in a number of pathologic processes, including reperfusion injury, cancer, inflammatory disease, and aging. The cell has several protective mechanisms that minimize the toxic potential of these compounds.Enzymes that catalyze antioxidant reactions: Reduced glutathione, a tripeptide-thiol (γ-glutamylcysteinylglycine) present in most cells, can chemically detoxify hydrogen peroxide. The is catalyzed by the selenium-requiring glutathione peroxidase, forms oxidized glutathione, which no longer has protective properties.The cell regenerates reduced glutathione in a reaction catalyzed by glutathione reductase, usingNADPH as a source of reducing equivalents. Thus, NADPH indirectly provides electrons for the reduction of hydrogen peroxide.Note:Erythrocytes are totally dependent on the pentose phosphate pathway for their supply of NADPH because, unlike other cell types, erythrocytes do not have an alternate source for this essential coenzyme.Additional enzymes, such as superoxide dismutase and catalase, catalyze the conversion of other toxic oxygen intermediates to harmless products. These enzymes serve as a defense system to guard against the toxic effects of reactive oxygen species. Antioxidant chemicals: A number of intracellular reducing agents, such as ascorbate , vitamin E and ?-carotene, are able to reduce and detoxify oxygen intermediates in the laboratory.Consumption of foods rich in these antioxidant compounds has been correlated with a reduced risk for certain types of cancers, as well as decreased frequency of certain other chronic health problems. Cytochrome P450 monooxygenase systemMonooxygenases (mixed function oxidases) incorporate one atom from molecular oxygen into a substrate (creating a hydroxyl group), with the other atom being reduced to water. In thecytochrome P450 monooxygenase system, NADPH provides the reducing equivalents required by this series of reactions. This system performs different functions in two separate locations in cells. The overall reaction catalyzed by a cytochrome P450 enzyme is:R-H + O2 + NADPH + H+→ R-OH + H2O + NADPWhere R may be a steroid, drug, or other chemical. [Note:Cytochrome P450s (CYPs) are actually a superfamily of related, heme-containing monooxygenase enzymes that participate in a broad variety of reactions. The name P450 reflects the absorbance 450 nm by the protein.Phagocytosis by white blood cellsPhagocytosis is the ingestion by receptor-mediated endocytosis of microorganisms, foreign particles, and cellular debris by cells such as neutrophils and macrophages (monocytes).It is an important body defense mechanism, particularly in bacterial infections. Neutrophils and monocytes are armed with both oxygen-independent and oxygen-dependent mechanisms for killing bacteria.Oxygen-independent mechanism: Uses pH changes in phagolysosomes and lysosomal enzymes to destroy pathogens.Oxygen-dependent system: This includes the enzymes, NADPH oxidase and myeloperoxidase (MPO) that work together in killing bacteria. This system is the most potent of the bactericidal mechanisms. An invading bacterium is recognized by the immune system and attacked by antibodies that bind it to a receptor on a phagocytic cell. After internalization of the microorganism has occurred, NADPH oxidase, located in the leukocyte cell membrane, is activated and reduces molecular oxygen from the surrounding tissue into superoxide (O2–?), a free radical. The rapid consump tion of molecular oxygen that accompanies formation of superoxide is referred to as the respiratory burst.GLUCOSE 6-P DEHYDROGENASE DEFICIENCYThis is an inherited disease characterized by hemolytic anemia caused by the inability to detoxify oxidizing agents. G6PD deficiency is the most common disease-producing enzyme abnormality in humans, affecting more than 400 million individuals worldwide.This deficiency has the highest prevalence in the Middle East, tropical Africa and Asia, and partsof the Mediterranean.G6PD deficiency is X-linked, and is, in fact, a family of deficiencies caused by more than 400 different mutations in the gene coding for G6PD. Only some of these mutations cause clinical symptoms.Note:(In addition to hemolytic anemia, a clinical manifestation of G6PD deficiency is neonatal jaundice appearing 1–4 days after birth.The jaundice, which may be severe, typically results from increased production of unconjugated bilirubin).The life span of individuals with a severe form of G6PD deficiency may be somewhat shortened as a result of complications arising from chronic hemolysis.This negative effect of G6PD deficiency has been balanced in evolution by an advantage in survival—an increased resistance to falciparum malaria shown by female carriers of the mutation. [Note: Sickle cell trait and ?-thalassemia minor also confer resistance.] Role of G6PD in red blood cellsDiminished G6PD activity impairs the ability of the cell to form the NADPH that is essential for the maintenance of the reduced glutathione pool. These results in a decrease in the cellular detoxification of free radicals and peroxides formed within the cell.Glutathione also helps maintain the reduced states of sulfhydryl groups in proteins, including hemoglobin. Oxidation ofThose sulfhydryl groups leads to the formation of denatured proteins that form insoluble masses (called Heinz bodies) that attach to the red cell membranes.Additional oxidation of membrane proteins causes the red cells to be rigid (less deformable), and they are removed from the circulation by macrophages in the spleen and liver.Although G6PD deficiency occurs in all cells of the affected individual, it is most severe in erythrocytes, where the pentose phosphate pathway provides the only means of generatingNADPH. Other tissues have alternative sources for NADPH production (such as NADP+ -dependent malate dehydrogenases), that can keep glutathione reduced. The erythrocyte has no nucleus or ribosomes and cannot renew its supply of the enzyme. Thus, red blood cells are particularlyvulnerable to enzyme variants with diminished stability.B. Precipitating factors in G6PD deficiencyMost individuals who have inherited one of the many G6PD mutations do not show clinical manifestations, that is, they are asymptomatic. However, some patients with G6PD deficiency develop hemolytic anemia if they are treated with an oxidant drug, ingest fava beans, or contracta severe infection.Oxidant drugs: Commonly used drugs that produce hemolytic anemia in patients with G6PD deficiency are best remembered from the mnemonic AAA—Antibiotics (for example, sulfa methoxazole and chloramphenicol), Antimalarials(eg , primaquine but not quinine), and Antipyretics (eg, acetanilide but not aceta minophen).Favism:Some forms of G6PD deficiency, for example the Mediterranean variant, are particularly susceptible to the hemolytic effect of the fava (broad) bean, a dietary staple in the Mediterranean region. Favism, the hemolytic effect of ingesting fava beans, is not observed in all individuals with G6PD deficiency, but all patients with favism have G6PD deficiency. Infection:Infection is the most common precipitating factor of hemolysis in G6PD deficiency. The inflammatory response to infection results in the generation of free radicals in macro phages,which can diffuse into the red blood cells and cause oxidative damage.C Properties of the variant enzymesAlmost all G6PD variants are caused by point mutations in the gene for G6PD. Some mutations do not disrupt the structure of the enzyme’s active site and, hence, do not affect enzymic activity.However, many mutant enzymes show altered kinetic properties, eg , variant enzymes may show decreased catalytic activity, decreased stability, or an alteration of binding affinity for NADP, NADPH, or glucose 6-phosphate. The severity of the disease usually correlates with the amount of residual enzyme activity in thepatient’s red blood cells. G6PDA– is the prototype of the moderate (Class III) form of the disease. The red cells contain an unstable but kinetically normal G6PD, with most of the enzyme activity present in the reticulocytes and younger erythrocytes. The oldest cells, therefore, have the lowest level of enzyme activity, and are preferentially removed in a hemolytic episode. G6PD Mediterranean is the prototype of a more severe (Class II) deficiency in which the enzyme has decreased stability resulting in decreased enzymic activity. Class I mutations (rare) are the most severe and are associated with chronic nonspherocytic anemia, which occurs even in the absence of oxidative IC: PROTEIN METABOLISMGLUCOGENIC AND KETOGENIC AMINO ACIDS:Glucogenic amino acids:These are the amino acid that are the precursor for the synthesis of carbohydrates i.e. arginine, phenylalanine e.t.cKetogenic amino acid:These are the amino acids that are precursor for the synthesis of ketone compounds like acetyl CoA, Acetoacetate e.t.c. which are the precursor fo the synthesis of biomolecules like lipids. METABOLISM OF AMINO ACIDS,SPECIALIZED PRODUCTS AND THEIR METABOLIC DISORDER.GLYCINE:A nonessential, glycogenic amino acid.Actively involved in the synthesis of many specialized product i.e. Heme, purines, creatineIncorperated in the body for the synthesis of proteins,synthesis of serine and glucose, particitipate in one carbon metabolism.Synthesis of glycine:Synthesized from serine in the presence of hydroxymethyl transferase which is tetrahydrofolate dependentCan be obtained from threonine catalysed by threonine aldolaseGlycine can be synthesized from one carbon compoun (N5N10-methylene THF),CO2 and NH3 in the precence of glycine synthase.SYNTHESIS OF SPECIALIZED PRODUCT:Formation of purine ring: utilized in the formation of position 4 and 5 opf carbon and position 7 nitrogen in the purine ring.Synthesis of heme (porphyrine ): glycine condences with succinyl CoA to form δ-amino levulinate which is the precursor for Heme synthesis.Succinyl CoA +glycine δ-amino levulinate (ALA)Biosynthesis of creatinine:glycine mmethinine and arginine are required for the synthesis of creatinineTransfer of guanidino group of arginine to glycine in the presence of arginine transamidase to produce guanidoacetate (glycocyamine).S-Adenosylmethionine (active methionine) transfers methyl group from glycocyamine to produce creatine which occurs in the liver.Creatine is phosphorylated to phosphocreatine (cratine phosphate) in the presence of creatine kinase and stored in the muscles as a high energy compound.Clinical importance:Serum: creatine 0.2-0.6 mg/dl creatinine 0.6-1mg/dlUrine:creatine 0-50mg/day creatinine 1-2g.dayMetabolic disorders of glycine metabolism:GLYCINURIA:A state in whichh there is high excreation of glycine in urine above 0.5-1 g/day due to defective rena reabsorbtion.Characterized by increased tendency for the formation of oxalate real stones.Primary hyperoxaluria:Defect due to glycine transaminase impairment in glyoxalate oxidation to formateCharactcetrized by increased urinary oxalate leading oxalate stoness.(oxalosis) depositing of oxalate is observed in various tissues.PHENYLALANINE AND TYOSINE :They are aromatic amino acid,phenylalanine is essential amino acids while tyrosine is non-essential amino acids.Metabolism of phenylalanine occurs through tyrosineTyrosine can be incorperated into various proteins like epinephrine, norepinephrine, dopamine(catecholamine), thyroid hormones and pigments like melanine.Conversion of phenylalanine to tyrosine:Under normal circunstances degradation of phenyalanine occurs through tyrosine.Phrnylanalanine is hydroxylated by phenylalanine hydroxylase (present in the liver)to produce tyros;ine. This is irreversible reaction and requires bio[pterine as the coenzyme(tetrahydrobiopterine) which is oxidised to dihydrobiopterine which is then regenerated by NADPH dependent dihydrobiopterine reductase. The failure or blockage of phenyalanine hydroxylase may result in phenylketonuria.Degradation of tyrosine or phenylalanine:Phenylalanine is converted to tyrosineTyrosine undergoes transamination to give p-hydroxyphenypyruvate in the precence of tyrosin transaminase (PLP sdependent).p-hydroxyphenypyruvate undergoes oxidative decarboxylase in presence of p-hydroxyphenypyruvate hydroxylase or dioxygenase (copper containing enzyme) to produce homogentisateHomogentisate is converted to 4-maleylacetoacetate a reaction catalysed by homogentisate oxygenase which requiresmolecular oxygen to break the aromatic ring.Isomerization of 4-maleylacetoacetate to 4 fumarylacetoacetate reaction catalysed by maleylacetoacetate isomerase.4 fumarylacetoacetate undergoes hydrolysis to yeild fumarate and acetoacetate which are the precursor in lipid synthesis ,TCA cycle and glucose synthesis.SYNTHESIS OF MELANIN:Synthesis of melanin occors in the melanosomes present in the melanocytes.Tyrosin is the precusor for melaniin and tyosinase(copper containing) is the primary exzyme that is involved in the synthsis if the pigment.Tyrosine gets concerted to3,4 –dihydroxyphenylalanine (DOPA) in the rteaction catalysed by tyrosinase.DOPA gets converted to dopaquinone and in subsequent reactions itforms leucodopachrome followed by 5,6 dihydroxyindole. Oxidation process takes place,Tyrosinse converts the 5,6 dihydroxyindole .melanochrome is synthesized from indole quinone which on polymerization we get melanin.Alternatively, dopaquinone iscondenced with cysteine and red melanin is generated.BIOSYNTHESIS OF THYROIOD HORMONES:Thyroid hormones are synthesized from tyrosine. Tetraiodothyronine(thyroxine) and triiodothyronine homones.Iodinization of tyrosine ring occurs to produce mono and diiodotyrosine from which triiodothyronine (t3)and thyroxine (T4) are synthesizedProtein thyroglobulin undergoes proteolytic breakdown to release the free T3and T4 hormones.Disorders of tyrosine and phenylalanine metabolism:PHENYLKETONURIA:Due to hepatic enzyme phenylalanine hydroxylase.this leads to accumulation of phenylalaninein tissues and blood. And in urine there is elevated amount of phenylpyruvate and other keto acids.Biochemical manifestation:Effects on central nervous system-mental retardation,failure to walk,failure to grow tremor.failureto transport other aromatic amino acid like tryptophan and tyrosine,and leads to defectin myelin formation.Effect on pigmentation-inhibition of tyrosinase leads albinismDiagnosis:Done byand Guthrie test and ferric chloride test and green colour is obtained. Normal values in phenylalanine in plasma PKU20-65mg/dl Treatment:Dietary intake of phenylalanine should be measured in plasma levels and adjusted.TYROSENEMIA TYPE II: (RICHNER-HANART SYNDROME)Due to blockage of tyrosine transaminase hence accumulation and excreation of tyrosine and its metabolitesCharacterised in skkin(dermatitis) and eye lessionNeonatal tyrosenemia:Due to p-hydroxyphenylpyruvatedioxygengase. Can be controlled byascorbic acidALKAPTONURIA:Defect on the enzyme homogentisate oxidase hence accumulation of homogentisate in blood and tissues. Alkapton is the pigment produced incase of accumulation and deposit occurs in the connective tissues,bones and various organs resulting in ochronosisDiagnosis:Benedict’s test,carry out ferric chloride and silver nitrate test in urineTreatment:Consumption of proteins with low phenylalanine contents Tyosinisis or tyrosinemia type I:Due to deficiency of Fumarylacetoacetate hydroxylase and maleylacetoacetate isomerase which may lead to liver failure, rickets, renal tibular dysfunction and polyneuropathyTreatment:Recommended diets with low tyrosine, phenylalanine and methionineALBINISM:Causes:Deficiency or lack of enzyme tyrosinaseSecrease in melanosomes of melanocytesImpairment in melanin polymerizationLack of protein matrix in melanosomesLimitation of substrate (tyrosine) availabilityPresence of inhibitors of tyrosinaseThe common cause of albinism is defect on tyrosinase enzyme responsible for the synthesis of melanin.Clinical manifestation:Melanin protect the body from sun rays hence albinos havesensitive to sunlight and susceptible to skin cancer(carcinoma). Photophobia is associated with lack of the pigment in the eyes.UREA CYCLE:Site of metabolism is liver and urea is the end product of protein metabolism.Metabolism discovered or elucidated by hans kreb and kurt henseleit in 1932Urea has two amino groups one from ammonia and other from aspartate and carbon is supplied from carbon dioxide.Catalitic enzymes:Two enzymes are found in mitochondria and others in cytosol.Steps in urea cycle:Synthesis of carbomoyl phosphate:Synthesized by condesation carbon dioxide(CO2) with ammonium ions (NH4+) to form carbomyl phosphate in the presence of carbomy phosphate synthetase a rate limiting enzyme requiring N-Acetylglutamate.Formation of citrulline:Citrulline is synthesized when ornithine is condence with carbomyl phosphate in the presence of orhinthine transcarbomylase. The citrulline synthesized at this point is transported into cytosol. Oornithine and citrulline are basic aminoSynthesis of arginosuccinate:Citrulline is condensed aspartate in the presence arginosuccinate synthetase to yeild arginosuccinate. This is a step for second amino group incorperation and ATP is requirded and is cleaved to yeild AMP and PPiCleavage of arginosuccinate:Arginosuccinate is cleaved by arginosuccinase to give fumarate and arginine. Arginine is immediate precursor for urea synthesis. Fumarate enters into TCA cycle, gluconegenesis.Formation of urea:Arginase leaves arginine to yield urea and ornithine. Ornithine enters into mitochondriafor re-use.Arginase is activated by Mn2+CLINICAL SIGNIFICANCE OF UREAA moderately active man consuming about 300 gm carbohydrates, 100 gm of fats and 100 gm of proteins daily must excrete about 16.5 gm of N daily. 95 per cent is eliminated by the kidneys and the remaining 5 per cent,for the most part as N, in the faeces.1. Normal LevelThe concentration of urea in normal blood plasma from a healthy fasting adult ranges from 20 to 40mg%.2.Increase of LevelsIncreases in blood urea may occur in a number of diseases in addition to those in which the kidneys are primarily involved. The causes can be classified as:PrerenalMost important are conditions in which plasma vol/ body-fluids are reduced:Salt and water depletion,Severe and protracted vomiting as in pyloric and intestinal obstruction,Severe and prolonged diarrhoea,Pyloric stenosis with severe vomiting,Haematemesis,Haemorrhage and shock; shock due to severe burns,Ulcerative colitis with severe chloride loss,In crisis of Addison’s disease (hypoadrenalism).RenalThe blood urea can be increased in all forms of kidney diseases:In acute glomerulonephritis.In early stages of type II nephritis (nephrosis) the blood urea may not be increased, but in later stages with renal failure, blood urea rises.Other conditions are malignant nephrosclerosis,chronic pyelonephritis and mercurial poisoning.In diseases such as hydronephrosis, renal tuberculosis; small increases are seen but depends on extent of kidney damage.Postrenal DiseasesThese lead to increase in blood urea, when there is obstruction to urine flow. This causes retention of urine and so reduces the effective filtration pressure at the glomeruli; when prolonged, produces irreversible kidney damage.Causes:Enlargement of prostate,Stones in urinary tract,Stricture of the urethra,Tumours of the bladder affecting urinary flow.Decreased levels: Decreases in blood urea levels are rare. It may be seen:In some cases of severe liver damage,Physiological condition: Blood urea has been seen to be lower in pregnancy than in normal nonpregnant women.REGULATION OF UREA CYCLE:The rate limiting enzyme is carbamoyl phosphate synthase. Requires NAG (N-acetyl glutamate) synthesized from acetyl CoA and Glutamate. Increase of NAG increases synthesis of urea in the liver.Carbamoylphosphate synthase I and Glutamate dehydrogenase located in the nitochondria coordinate the synthesis of NH3 for the synthesis of cabamoyl phosphate.The remaining four enzymes particitipate in the synthesis of urea depending on the concentration of the substrates.INTERGRATION OF UREA CYCLE WITH TCA CYCLE:The formation of fumarate in urea cycle is the intergrating point to TCA cycle.Oxaloacetate undergoes transamination to produce aspartate which enters urea cycle as the second source of amino group to synthesis urea.ATP (12) generated in the TCA cycle while 4 ATP are utilized in urea cycle.CO2 and H2O are the end product that are formed on complete oxidation of various metabolites whareby CO2 generated is utilized in the urea synthesis.Metabolic disorders of urea cycle:Hyperammonemia- a disorder of increased amount of ammonia in blood.DisorderEnzyme involvedHyperammonemia type 1Carbamoyl phosphate synthase IHyperammonemia type IIOrnithinetranscarbamoylaseCitrullinemiaArginosuccinate synthaseArginosuccinicArginosuccinaseHyperargininemiaArginase Blood urea clinical significance:Pre-renal-associated with increased protein breakdown hence negative nitrogen balance observed during diabetic coma, thyroxicosis, leukemiableeding disordersRenal-increased in cases of patients suffering from acute glomerulonephritis, chronic nephritis, nephrosclerosis,polycystic kidney.Post renal-elevated in cases of urinary tract obstraction i.e. tumor,stones TOPIC: METABOLISM OF NUCLEIC ACIDSNucleotides consist of a nitrogenous base, a pentose and a phosphate. The pentose sugar is D-ribose in RNA and 2-deoxy D-ribose in DNA. They participate in almost all the biochemical processes, either directly or indirectly. They are structural components of nucleic acids, coenzymes and are involved in the regulation of several metabolic reactions. Steps of BiosynthesisFormation of 5-phosphoribosyl-1-pyrophosphate (PRPP): The process begins with D-Ribose-5'-P obtained from HMP-shunt pathway. PRPP is synthesized by the enzyme PRPP synthase from D- ribose-5'-P and ATP. Formation of 5'-phosphoribosyl-1 amine (PRA): The amide group of glutamine is transferred to C of PRPP by the enzyme Glutamine PRPP amido transferase. Phosphate group is replaced by –NH group. This gives the N-9 of purine ring.Formation of glycinamide ribotide, GAR ( 5'-Phosphoribosylglycinamide):Glycine condenses with PRA using ATP as energy source to form glycinamide ribotide (GAR). The reaction is catalysed by the enzyme Glycinamide kinosynthase. This provides C-4, C-5 and N-7 of the Purine ring.Formation of formylglycinamide ribotide, FGAR ( 5'-phosphoribosyl-N-formyl-glycinamide):The amino nitrogen of glycinamide is formylated by N10-formyl tetrahydrofolate catalysed by the enzyme formyl transferase. The formyl carbon becomes C-8 of the purine ring.Formation of a-N-formylglycinamidine ribotide, FGAM: ( 5'-phosphoribosyl-N-formylglycinamidine): Another amide group of glutamine is transferred to FGAR to form FGAM. Phosphoribosylglycinamidine synthase is the enzyme that catalyses the reaction and ATP provides the energy. The reaction contributes N-3 of Purine ring.Formation of 5-Aminoimidazole riboside, AIR ( 5'-phosphoribosyl-5-aminoimidazole): This reaction is catalysed by the enzyme aminoimidazole ribosyl phosphate synthetase (AIR-Psynthetase), which brings about the dehydratative closure of the ring, by removal of a molecule of H2O. ATP is required for the reaction. Formation of 5-aminoimidazole-4-carboxylic acid ribotide, C-AIR (also called 5'-phosphoribosyl-5aminoimidazole-4 carboxylate): This reaction uses CO2 to carboxylate AIR. It contributes to C-6 of the purine nucleus. Neither Biotin nor ATP is required for carboxylation. Formation of 5-aminoimidazole-N-succinyl carboxamide ribotide, 5-AISCR. (5-'phosphoribosyl-5-aminoimidazole-4-N succino carboxamide):This reaction is catalysed by the enzyme succinyl carboxamide synthetase. ATP is used to condense Aspartic acid with aminoimidazole carboxylate-5-Phosphate. This contributes to N-1 of the purine nucleus.Formation of 5-aminoimidazole-4-carboxamide ribotide, 5-AICAR. ( 5'-Phosphoribosyl 5- aminoimidazole-4-carboxamide): 5-AISCR under-goes cleavage by the cleaving enzyme adenylosuccinate lyase to form 5-AICAR and fumarate. Formation of 5-formamidoimidazole-4-carboxyamide ribotide, 5-FICR. (5'-phosphoribosyl-5-formamidoimidazole-4 carboxamide): Carbon-2 (C-2) the final carbon of the Purine ring is donated by N10-formyl tetrahydrofolate in a reaction catalysed by the enzyme formyl transferase and forms 5-FICR.Formation of inosinic acid (IMP): 5-FICR undergoes a dehydrative ring closure, by elimination of one molecule of water. The reaction is catalysed by the enzyme IMP cyclohydrolase to form Inosinic acid (IMP). Formation of Other Purine Nucleotides1. Formation of AMP from IMPThis is brought about in 2 steps:The enzyme adenylosuccinate synthetase catalyses the condensation of Aspartic acid with IMP to form adenylosuccinate. GTP provides the required energy.Adenylosuccinate is then cleaved by the cleaving enzyme adenylosuccinate lyase to form AMP and fumarate.Formation of GMP from IMP: This is brought about in 2 steps:The enzyme IMP dehydrogenase oxidises IMP to xanthosine monophosphate or xanthylic acid (XMP). ? In the second stage, the amide group of glutamine is transferred to C-2 of Xanthine Monophosphate catalysed by the enzyme. GMP synthetase to form GMP.A. Purine Salvage PathwaysTwo pathways are available.1. One-step Synthesis? Formation of GMP and IMP: Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase)catalyses the one-step formation of the nucleotides from either guanine or hypoxanthine, using PRPP as the donor of the ribosyl moiety.Regulation: The enzyme HGPRTase is regulated by the competitive inhibition of GMP and IMP respectively.? Formation of AMP: The enzyme Adenine phosphoribosyl transferase (APRTase) in similar way catalyses the formation of AMP from adenine, ribosyl moiety is donated by PRPP.Regulation: The enzyme APRTase is regulated by the competitive inhibition of AMP. 2. Two-Step Synthesis(Nucleoside phosphorylase-nucleoside kinase pathway)Under some conditions, it is possible for purine bases to be salvaged by a two-step process as under:Nucleoside Phosphorylase is an enzyme that brings about nucleoside breakdown. But the reaction is readily reversible and can form back ‘nucleoside’ which is rather a favourable pathway. Once the nucleoside is formed, a kinase enzyme may phosphorylate it to the 5'-nucleotide.Formation of AMPAdenine is the only purine that may be salvaged by the two-step pathway. Guanosine and inosine kinases have not been detected in animal cells.PURINE SALVAGE CYCLEThis is a cycle in which GMP and IMP as well as their deoxyribonucleotides are converted into their respective nucleosides by a purine 5'nucleotidase enzyme. The nucleosides formed can be hydrolytically cleaved producing the corresponding sugar phosphates and setting free the N- bases. The guanine and hypoxanthine then can be phosphoribosylated again to complete the cycle.Regulation of Purine Synthesis? PRPP synthetase enzyme regulates purine synthesis. It is allosterically inhibited by the feedback effects of PRPP and a number of purine nucleotides such as AMP, GMP, ADP, GDP, NAD and FAD.? Glutamine PRPP amidotransferase enzyme is for the rate-limiting step of purine synthesis. It is regulated by feedback inhibitory effects of AMP and GMP.?A proper balance between the Adenine and guanine concentration is maintained by adenylosuccinate synthetase and IMP dehydrogenase respectively.CATABOLISM OF PURINESPurines are Catabolised to Uric AcidAn average of 600 to 800 mg of uric acid is excreted by human beings most of it is found in urine. Guanine and Adenine nucleotides have their separate enzymes until the formation of a common product xanthine. The final reaction is the conversion of xanthine to uric acid by xanthine oxidase. A. Adenine Nucleotide Catabolism1. In the Liver and Heart MuscleAn enzyme purine-5’-nucleotidase hydrolyses adenylate (AMP). As a result a nucleoside,Adenosine is obtained.Adenosine deaminase removes ammonia from adenosine and gives inosine.Purine nucleoside phosphorylase phosphorolyses inosine to ribose-1-P and hypoxanthine.Xanthine oxidase then converts hypoxanthine to xanthine and xanthine to uric acid. Molecularoxygen is reduced at each stage to the superoxide (O2–) which is converted to H2O2 by superoxide dismutase.2. In the Skeletal MuscleAdenylate deaminase converts AMP into inosine monophosphate (IMP).Inosine monophosphate is hydrolysed by purine-5-nucleotidase to inosine.Inosine is changed to uric acid as mentioned above. GMP inhibits adenylate deaminase to reduce the catabolism of AMP.B. Guanine Nucleotide Catabolism1. In the Liver, Spleen, Kidneys, PancreasGMP is hydrolysed by purine-5’-nucleotidase into guanosine.Purine nucleoside phosphorylase phosphorolyses guanosine into Ribose -1-Phosphate and guanine.Guanine deaminase deaminates guanine to xanthine and produces NH3.Oxidation of xanthine to uric acid is brought about by xanthine oxidase.2. In the Liver MainlyGuanosine deaminase deaminates guanosine into xanthosine.Xanthosine is then phosphorolysed to Ribose -1-Phosphate and xanthine by purine nucleoside phosphorylase.Xanthine is then oxidised to uric acid by xanthine oxidase.Further Catabolism of Uric Acid (Nonprimates)In many nonprimate animals uric acid may be oxidized and decarboxylated by uricase, a hepatic copper containing enzyme to allantoin.Some fishes carry uricase as well as allantoinase. This converts allantoin into allantoic acid.Amphibians and other such animals contain allantoinase which converts allantoic acid into ureidoglycolate. Ureidoglycolate is further cleaved by ureidoglycolase into urea and glyoxylate.Urea is further converted to NH3 and CO2 in crustaceans by an enzyme urease found in their liver.URIC ACID METABOLISM AND CLINICAL DISORDERS OF PURINE The main site of uric acid formation is the liver from where it is carried to the kidneys.Miscible PoolIt is the quantity of uric acid present in body water.In normal subjects an average of 1130 mg of uric acid is present. Plasma contains higher concentration of uric acid compared to other body compartments containing water.TurnoverThis is the rate at which uric acid is synthesized and lost from the body. Normally, 500–600 mg of uric acid is synthesised. Not all is excreted in urine; some uric acid is excreted in bile. Some is converted to urea and ammonia by the intestinal bacteria.Distribution: It is very irregularly distributed in the body. Serum contains 3 to 7 mg/dl. Average valuesare slightly higher in males. Red cells contain half as much uric acid as serum. Muscles also contain less amounts compared to blood.Dietary effects: Uric acid excretion continues at a rather steady rate during starvation and during a purine-free diet owing to the so-called endogenous purine metabolism. The ingestion of foods high in nucleoproteins such as glandular organs produces a marked increase in urinary uric acid. Diets like milk, eggs and cheese, with low purine contents causes practically no increase in urinary uric acid.Effect of hormones:Administration of the glucocorticoid hormones and ACTH increases the excretion of uric acid in urine.Excretion of uric acid: Uric acid in the plasma is filtered by the glomeruli but is later partially reabsorbed by the renal tubules. Glycine is believed to compete with uric acid for tubular reabsorption. Certain uricosuric drugs such as salicylates, block reabsorption of uric acid. Lactic acid competes with uric acid in its excretion.Hence in lactic acidosis uric acid is retained, and can produce gout. There is now conclusive evidence for tubular secretion of uric acid by kidney. Thus uric acid is cleared both by(a) glomerular filtration; (b) by tubular secretion.Clinical disordersUric acid is the end product of purine metabolism in humans. Normal concentration in the serum of adults is 3-7 mg/dl.Hyperuricemia refers to the elevation in the serum uric acid concentration. L DISORDERS: Gout:It is a metabolic disease associated with overproduction of uric acid. It is a chronic disorder characterised by:Excess of uric acid in blood (Hyperuricemia).Deposition of sodium monourate in alveolar and non alveolar structures producing so called tophi. Recurring attacks of acute arthritis. These are due to deposition of monosodium urate in and around the structures of the affected joints.Types: There are two main types of gout:Primary gout,Secondary gout.1. Primary GoutHere the hyperuricaemia is not due to increased destruction of nucleic acid. The essential abnormality is increased formation of uric acid from simple carbon and nitrogen compounds without intermediary incorporation into nucleic acids.Primary metabolic gout: It is due to inherited metabolic defect in purine metabolism leading to excessive rate of conversion of glycine to uric acid.X-linked recessive defects enhancing the de novo synthesis of purines and their catabolism can also lead to hyperuricaemia. For example, defects of PRPP synthetase may make it feedback resistant. X-linked recessive defects of hypoxanthine guanine phosphoribosyl transferase may reduce utilisation of PRPP in the salvage pathway. Increased intracellular PRPP enhances de novo purine synthesis.Primary renal gout: It is due to failure in uric acid excretion.2. Secondary Gout a. Secondary metabolic gout:It is due to secondary increase in purine catabolism in conditions like leukaemia, prolonged fasting and polycythemia.Secondary renal gout: Occurs due to defective glomerular filtration of urate due to generalised renal failure. Treatment of Gout: Consist of:(a) Palliative Treatment and(b) Specific TreatmentPalliative treatment:This involves; Bed rest in acute stage,Diet—Purine free diet,Restricting alcohol consumption.? Anti-inflammatory DrugsColchicine: One of the nonspecific anti-inflammatory drugs. It has no effect on urate metabolism or excretion. Colchicine therapy is instituted during acute attack.Mechanism:Suppresses the synthesis and secretion of the chemotactic factor that is produced in urate crystal-induced inflammation.Dosage:Available as 0.5 mg tablet.In acute gout:One tablet hourly till symptoms are relieved or diarrhoea occurs.Long-term management: One tablet 3 to 4 times a week.NSAIDS: Drugs like: Indomethacin, Diclofenac, Naproxen, Piroxicam, Fenoprofen, Flurbiprofen,Ibuprofen, Rofecoxabin, etc. These drugs inhibit the synthesis of prostaglandins which are important mediators of the inflammatory response. These drugs have been found effective in treating patients having recurrent attacks of acute gout and also for terminating acute attack of gout.(b) Specific TreatmentAim: To lower the uric acid level of blood.Methods: The above can be achieved in three ways:By increasing the renal excretion of uric acid (uricosuric drugs).By decreasing the synthesis of uric acid using enzyme inhibitor.By increasing oxidation of uric acid.Uricosuric Drugs A uricosuric agent is one that enhances the renal excretion of uric acid probably by specific inhibition of its tubular reabsorption or secretion.Drugs used are:SalicylatesEffects vary with dosage. In low dosage of 1 to 2 gm/day, salicylates cause uric acid retention but in higher dosage 5 to 6 gm/day it has uricosuric effect. Longterm therapy with high dosage is not desirable due to the side effects.Probenecid (Benemide)– It is an efficient and harmless uricosuric drug.– Lowers the uric acid level. Fall is immediate and sustained.Dose: Available as 500 mg tablet. ? tablet twice daily for the first week and then one tablet twice daily. Not recommended for children. Therapy is continued for 10 to 12 weeks and patients can return to normal activities.Halofenate: The drug has good uricosuric effect. It also has a hypolipaemic effect. It liberates urates from urate binding sites of proteins of plasma and removes uric acid by normal excretion. The drug can be safely used for short-term and long-term therapy.2. Enzyme Inhibitor? Allopurinol (zyloprin) drug of choice: It has similar structure like hypoxanthine. Acts by competitive inhibition on “xanthine oxidase” and thus uric acid synthesis is impaired. The drug causes a rapid fall in serum uric acid level and an increase in concentration of hypoxanthine and xanthine in blood. Both xanthine and hypoxanthine are more soluble and so are excreted easily in urine. Allopurinol is acted upon by Xanthine oxidase and converted to alloxanthine.Dosage: Available as 100 mg tablet.Maintenance: 200 to 600 mg daily. Not recommended in children.? The drug can be used in secondary hyperuricaemia.? Allopurinol also has an inhibitory action on the enzyme tryptophan pyrrolase.Drugs Increasing Uric Acid Oxidation? Urate oxidase: The drug can be used in lowering uric acid level by oxidising uric acid.Dosage: 10,000 IU daily for 10 days. It shows a significant decrease in uric acid level. Can be used in severe gout with renal involvement and secondary hyperuricaemia. Lesch-Nyhan SyndromeAffects only males due to the defect of hypoxanthine-guanine phosphoribosyltransferase. The enzyme is almost absent and leads to increased purine salvage pathway from PRPP. This can result in severe gout, renal failure, poor growth, spasticity and tendency for self-mutilation.Xanthinuria: An autosomal recessive deficiency of xanthine oxidase, blocks the oxidation of hypoxanthineand xanthine to uric acid. It can cause xanthine lithiasis and hypouricaemia.Adenosine deaminase deficiency: An autosomal recessive deficiency of adenosine deaminase. It is associated with severe immunodeficiency and both T cells and B cells (lymphocytes) are deficient. There occurs an accumulation of deoxyribonucleotides which inhibit further production of precursors of DNA synthesis especially dCTP. Hypouricaemia occurs which is due to defective breakdown of purine nucleotides. Recently Gene replacement therapy has been used successfully in a few cases.Nucleoside phosphorylase deficiency: An autosomal recessive deficiency of purine nucleoside phosphorylase, causes the urinary excretion of guanine and hypoxanthine nucleosides. There is reduced production of uric acid. There is severe deficiency of cell mediated and humoral immunity.In von-Gierke’s disease: Deficiency of G-6-phosphatase leads to elevated rate of pentose formation in HMP. Thisacts as a good substrate for PRPP synthetase and enhances the synthesis of purines followed by their catabolism to uric acid.Increase lactic acid competes with uric acid excretion resulting to retention of uric acid.YNTHESIS OF PYRIMIDINESSYNTHESIS OF PYRIMIDINESThe synthesis of pyrimidines is much simpler compared to purines. Aspartate and glutamine (amide group) and CO2 contibute to atoms in the formation of pyrimidine ring. Materials required for pyrimidine synthesis;yrimidinesCarbamoyl phosphate: Synthesised from CO2 and glutamine.PRPP: 5-phosphoribosyl-1-pyrophosphateVarious enzymes: Carbamoyl phosphate synthetase II, Transcarbamoylase, Dihydro-orotase, Dehydrogenase, Transferase and Decarboxylase. ATP: For energy, amino acid: Aspartic acid and Cofactors: FAD+, NAD+, and Mg++.Steps of SynthesisSynthesis of carbamoyl phosphate: The synthesis of pyrimidine ring begins with the formation of carbamoyl phosphate from glutamine, CO2 and ATP, catalysed by the enzyme carbamoyl phosphate synthetase II. The enzyme is present in cytosol and does not require N-acetyl glutamate (NAG). The reaction is controlled by the feed-back inhibitory effect of pyrimidine nucleotide UTP.SECTION THREEFormation of carbamoyl aspartic acid (CAA): The enzyme apartate transcarbamoylase then transfers carbamoyl group from carbamoyl phosphate to aspartic acid to form carbamoyl aspartic acid (CAA).Committed and rate limiting step.? Enzyme is oligomeric.? Enzyme is inhibited (“feedback” allosteric inhibition) by CTP and UTP.? Inhibition can be reversed by ATP.Formation of dihydro-orotic acid: The reaction is catalysed by the enzyme dihydro-orotase, which removes a molecule of water (dehydration) from carbamoyl aspartic acid and brings about the closure of the ring to produce dihydro-orotic acid.Formation of orotic acid: The next step is oxidation of dihydro-orotate which is brought about by the enzyme dihydro-orotate dehydrogenase. The enzyme carries Fe, S, FMN and FAD in its prosthetic group. NAD+ is required as a coenzyme.Formation of orotidylic acid: Under the influence of the enzyme Orotate phosphoribosyl trans-ferase, 5phosphoribosyl group from 5-phospho-ribosyl-1pyrophosphate (PRPP) is transferred to orotic acid to produce orotidylic acid. Mg++ ions are required in the reaction.Formation of uridylic acid (UMP): Orotidylic acid undergoes decarboxylation, catalysed by the enzyme Orotidylate decarboxylase, and forms uridylic acid (UMP).? UMP is the first pyrimidine nucleotide to be formed.? Other pyrimidine nucleotides viz. UDP, UTP, CTP and d-UDP are synthesised from UMP.? UMP is the “feedback” inhibitor of the decarboxylase enzyme. Formation of Other Pyrimidine NucleotidesFormation of UDP and UTP? UMP is phosphorylated by ATP to form UDP, catalysed by the enzyme nucleoside monophosphokinase? UDP can be further phosphorylated by ATP to form UTP.Formation of CTPCTP can be synthesised from UTP, catalysed by the enzyme CTP synthetase. The reaction requires ATP for energy and glutamine.Formation of dUDP (Deoxyuridine Diphosphate).dUDP is formed from UDP by the action of the enzyme Ribonucleoside reductase. The reaction requires Thioredoxin (Iron-sulphur protein) and NADPH.Synthesis of Thymine DeoxyribonucleotidesdTMP is synthesised from dUMP. dUMP may arise from the hydrolysis of dUDP by phosphatase. Alternatively, dCMP may be produced by phosphorylation of circulating deoxycytidine with the help of ATP. The enzyme is deoxycytidine kinase.Thymidylate synthetase methylates dUMP into dTMP by transferring methyl group from N5, N10methylene H4 folate, the C5 of the uracil moiety.Thymidylate synthetase is covalently inhibited by folate antagonists like aminopterin and amethopterin which competitively inhibits DH2-folate reductase.dTMP can be changed to dTDP by transphosphory-lation with the help of ATP, Mg++ using thymidylate kinase. This is the synthesis of thymine and pseudouridine ribonucleotides. This occurs in TψU loop of each t-RNA molecule. UMP residue is precursor.Synthesis of Pyrimidine DeoxyribonucleotidesReduction of ribose to deoxyribose is achieved by ribonucleotide reductase. This enzyme transfers electrons from iron-sulphur containing protein called thioredoxin to the 2’-C of ribose moiety of purine or pyrimidine nucleotide diphosphate. NADPH reduces the oxidized thioredoxin to its reduced state by thioredoxin reductase.The enzyme contains FAD as its prosthetic group. Rate limiting step in dTTP synthesis. dTDP is then phosphorylated to dTTP by thymidine diphosphokinase using ATP and Mg++.CATABOLISM OF PYRIMIDINES1. Cytosine and UracilThe first step of the catabolism of pyrimidines is dephosphorylation to the nucleosides by 5’- nucleotidases. Pyrimidine nucleosides are then phosphorolysed into free pyrimidines and pentose 1 phosphate with the help of Pi and nucleoside phosphorylases.Uracil, is then reduced to 5,6-dihydrouracil by dihydrouracil dehydrogenase using NADPH.Cytosine will form uracil by deaminase.The hydrolysis of 5,6-dihydrouracil is the next step.This is done hydrolytically by hydropyrimidine hydrase to produce ?-ureidopropionic acid. The next step is further hydrolysis by ureidopropionase into CO2, NH3 and β-alanine. CLINICAL DISORDERS OF PYRIMIDINE METABOLISMOrotic Aciduria: It is of 2 types:Type I orotic aciduria: It is an autosomal recessive genetic disorder of a protein acting as both orotate phosphoribosyltransferase and OMP decarboxylase. Orotate fails to be converted to uridylate. This results in accumulation of orotate in blood elevating its level, Growth retardation and megaloblastic anaemia.Type II orotic aciduria: It is autosomal recessive, affecting OMP decarboxylase and is characterized by megaloblastic anaemia and the urinary excretion of asididine in higher concentrations than orotate.Other Causes of Orotic AciduriaOrotic aciduria that accompanies Reye Syndrome:Probably it is a consequence of the inability of severely damaged mitochondria to utilise carbamoyl phosphate, which then becomes available for cytosolic overproduction of orotic acid.Associated with deficiency of urea cycle enzyme:Increased excretion of orotic acid, uracil and uridine, sometimes accompanies a deficiency of liver mitochondrial ornithine transcarbamoylase. Excess carbamoyl-P passes to cytosol where itstimulates pyrimidine nucleotide biosynthesis resulting to mild orotic aciduria, which may get aggravated by high N2 foods.Drug Related Orotic AciduriaAllopurinol: Allopurinol an alternative substrate for orotate phosphoribosyl transferase competes with orotic acid. The resulting nucleotide product also inhibits orotidylate decarboxylase resulting in orotic aciduria and orotidinuria.6-Azauridine: 6-Azauridine, following its conversion to 6-azauridylate, also competively inhibits orotidylate decarboxylase resulting to increased excretion of orotic acid (orotic aciduria) and orotidine (orotidinuria).TOPIC LIPID METABOLISM:OXIDATION OF FATTY ACIDS:The oxidation of fatty acids occurs in the mitochondria and liver. ACC: acetyl-CoA carboxylase (ACC)PKA: cAMP-dependent protein kinase (PKA),Fatty Acids Are Activated Before Being Catabolized. Fatty acids must first be converted to an active intermediate before they can be catabolized. This is the only step in the complete degradation of a fatty acid that requires energy from ATP. In the presence of ATP and coenzyme A, the enzyme acyl-CoA synthetase (thiokinase) catalyzes the conversion of a fatty acid (or free fatty acid) to an “active fatty acid” or acyl-CoA, which uses one high-energy phosphate with the formation of AMP and PPI. The PPi is hydrolyzed by inorganic pyrophosphatase with the loss of a further high-energy phosphate, ensuring that the overall reaction goes to completion. Acyl-CoA synthetases are found in the endoplasmic reticulum, peroxisomes, and inside and on the outer membrane of mitochondria.Carnitine plays a major role in the transport of long-chain fatty acids through the inner mitochondrial membrane. Long-chain acyl-CoA cannot pass through the inner mitochondrial membrane, but its metabolic product, acylcarnitine, can.General reactions of fatty acid oxidationBeta oxidation of palmitic acid:The fatty acid is activated from free palmitic acid to palmitoyl CoAIt undergoes oxidation to yield 8 successful acetyl C0A that enters the tricarboxylic acid cycle. Utilization and excretion of the ketone bodies:Under metabolic conditions associated with a high rate of fatty acid oxidation, the liver produces considerable quantities of acetoacetate and D(_)-3-hydroxybutyrate (?-hydroxybutyrate). Acetoacetate continually undergoes spontaneous decarboxylation to yield acetone. These three substances are collectively known as the ketone bodies (also called acetone bodies or [incorrectly*] “ketones”). Acetoacetate and 3-hydroxybutyrate are interconverted by the mitochondrial enzyme D()-3-hydroxybutyrate dehydrogenase; the equilibrium is controlled by the mitochondrial [NAD+]/[NADH] ratio, ie, the redox state.Transport of ketone bodies from the liver and pathways of utilization and oxidation occurs in extra hepatic tissues.Hypolipidemic drugs and possible mechanism of actionDISORDERS OF FATTY ACID OXIDATION:Refsum’s diseases:Is a genetically inherited disorder (autosomal recessive) due to phytanate α- oxidase deficiency which converts phytanic acid to pristanic acid hence accumulation in tissues and blood (indicated by an increase in 20% of the total fatty acids). It affects both the ages from childhood to adults. It s clinical manifestations are:Neurological symptoms like neuropathy with distal muscular atrophy and progressive paresis of the distal parts of extremeties.Sensory disturbances which may include parenthesiae and severe knee pains.Eye manifestation like night blindness, concentric narrowing of visual fields and typical pigmentary retinitis.Increased CSF proteins whilst cell count is usually normal.Treatment: Reduction or omitting the intake of phytols diet which is a precursor of phytanic acids.REFERENCES: Lehniger’s principles of Biochemistry (2nd edn 2000) by D.L.Nelson and M.M Cox, Macmillan, Worth Pub Inc, NY.Biochemistry (4th edn 1992) by Lubert Stryer WH Freeman & co., NYHarper’s Biochemistry (25th edn) by R.K.Murray and others, Appleton and Lange, Stanford.Fundamentals of Biochemistry (1999) by Donalt Voet, Judith G Voet and Charlotte W Pratt, John Willey & Sons, New York.Medical Biochemistry by M.Chattergen & Rana Shinde. T.Palwer 2002 K.Wilson and J.Walker 2002 5th Edition.Biochemistry A problems Approach by Wood.Wilson. Benbow.Hood. 2nd Edition.Textbook of Biochemistry by D M Vasudevan and SrikumariEssentials of Medical Biochemistry by R.C. GuptaLippincot’s illustrated reviews ................
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