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NUCLEIC ACID METABOLISM AND DISORDERS CBME 6.1-6.4 DJT 22.pptx
- 1. Edited August 2022 Nucleic acid metabolism Dr. Dhiraj J. Trivedi
- 2. Nitrogen Base, Nucleoside, Nucleotide and Nucleic acid Metabolism
- 3. Competencies for Nucleic acid metabolism • BI 6.2 – Describe and discuss the metabolic process in which nucleotides are involved • BI 6.3 – Describe common disorders associated with nucleotide metabolism • BI 6.4 – Discuss the lab results of analyte associated with gout and Lesch-Nyhan syndrome
- 4. SLO - Nucleic acid metabolism • Know names of purine and pyrimidine bases • Know the names of Purine and Pyrimidine in NTs • Know the names of sugars present in NTs • Know role of phosphate in NTs • Know how Nucleosides and Nucleotides are formed • Know nomenclature of Nucleoside and Nucleotides • Know what is free nucleotides & their biological function • What are cyclic nucleotides and their functions
- 5. SLO - Nucleic acid metabolism • Out line of digestion and absorption of Nucleotides. • Synthesis of nucleotides [De Novo and Salvage ] • Source of Atoms in Purine ring • Over view synthesis of purine nucleotide – IMP • Conversion of IMP to AMP and GMP • Reactions of Salvage pathway • Importance of Salvage pathway and associated disorders. • What is synthetic analogues of Nucleotide and their functions
- 6. SLO - Nucleic acid metabolism Cont… • Degradation of purine nucleotides, Reaction over view. • Uric acid – associated disorders, • Gout – Types, Signs & Symptoms, Diagnosis and management • How alcoholism invites GOUT? • Hypouricaemia, Xanthinuria, ADA deficiency, SCID, LNS • Source of atoms in pyrimidine nucleotide • Synthesis of pyrimidine nucleotides. [De novo and Salvage] • Catabolism of Pyrimidine nucleotides. • Orotic aciduria- condition, Signs & Symptoms, Diagnosis and management • Logic for Anticancer role
- 7. Nitrogenous base in Nucleic acid • Purine Bases • Nine membered ring • Adenine - A • Guanine - G • Pyrimidine Bases • Six membered ring • Cytosine – C • Uracil – U • RNA • Thymine – T • DNA
- 8. Sugar in Nucleic acid • Ribose • Pentose sugar • RNA • Deoxy Ribose • Pentose sugar • DNA Also contain phosphate group
- 9. Base + Sugar =Nucleosides Ribonucleosides Adenine + Ribose Adenosine Guanine + Ribose Guanosine Uracil + Ribose Uridine Cytosine + Ribose Cytidine Xanthine + Ribose Xanthosine Hypoxanthine + Ribose Inosine Deoxyribonucleosides Adenine + Deoxyribose Deoxyadenosine Guanine + Deoxyribose Deoxyguanosine Cytosine + Deoxyribose Deoxycytidine Thymine + Deoxyribose Deoxythymidine
- 10. Base + Sugar + Phosphate =Nucleotides Ribonucleotides Adenosine + Phosphate [Pi] Adenylic acid [AMP] Guanosine + Phosphate [Pi] Guanylic acid [GMP] Uridine + Phosphate [Pi] Uridylic acid [UMP] Cytidine + Phosphate [Pi] Cytidylic acid [CMP] Inosine + Phosphate [Pi] Inosinic acid [IMP] Deoxy ribonucleotides Deoxyadenosine + Phosphate [Pi] Deoxy adenylic acid [d-AMP] Deoxyguanosine + Phosphate [Pi] Deoxy Guanylic acid [d-GMP] Deoxycytidine + Phosphate [Pi] Deoxy Cytidylic acid [d-CMP] Deoxythymidine + Phosphate [Pi] Deoxy thymidylic acid [d-TMP]
- 11. Functions of Nucleotides - ATP 1. Constituent of DNA & RNA 2. Energy currency of body 3. AMP allosteric regulator 4. cAMP – second messenger 5. PAPS – Phospho Adenosine Phospho Sulphate – S donor 6. SAM – S-Adenosine methionine – CH3 group donor 7. NAD , NADP & FAD – Coenzymes – Active vitamins Nucleotides which are not polymerised to form nucleic acid, DNA & RNA. They are free hence free nucleotides.
- 12. Cyclic Nucleotides Mono-phosphate nucleotide Cyclic bond arrangement between the sugar and phosphate groups regulate cell function by controlling the activity of protein kinases Cyclic nucleotides are important second messenger cAMP (Adenosine-3’ 5’ -cyclic monophosphate) cGMP (Guanosine-3’ 5’ -cyclic monophosphate)
- 13. Digestion and Absorption of Nucleotides Non Essential Not utilized Catabolized and excreted Dietary Nucleoprotein Nucleic acids Nucleotides Nucleoside Free purine / Free Pyrimidine Ribose / Deoxyribose Gastric and Pancreatic Proteolytic enzymes Pancreatic and intestinal RNase and DNase Intestinal Nucleotidase Nucleosidase Amino acids Phosphate
- 14. Synthesis of Nucleotides Salvage pathway Direct formation De Novo synthesis Step by step synthesis Liver RBC, Brain cell, Leukocytes Energy consuming process Energetically less expensive Many enzymes are required Takes time Can not use preformed base few enzymes are required Quick process Can recycle preformed base
- 15. Purine
- 16. De novo synthesis of purine nucleotides Synthesis of inosine monophosphate [IMP] Purine ring is constructed on preformed Ribose 5 P Site : LIVER [cytoplasm] End product : Ribonucleotide De novo does not occur in-Brain, RBC, PMNL, Bone marrow
- 17. Requirements for purine synthesis • PRPP : 5’-Phosphorybosyl-1-pyrophosphate • Energy: Six ATP molecules • Amino acids: Glycine, Glutamine, Aspartic acid • CO2 from HCO3 • Vitamin: Formyl Tetra hydro- folate ( H4F) • Enzymes: 11 different enzymes
- 18. SOURCES OF INDIVIDUAL ATOMS IN PURINE RING C6 C2 C8 C5 C4 N3 N1 N7 N9 Aspartate Glycine CO2 Glutamine N-formyl H4F N-formyl H4F
- 19. 5 Phosphoribosyl amine Glycinamide ribosyl 5 phosphate Formyl glycinamide Ribonucleotide FGAR Formyl glycinamidine Ribonucleotide FGAM Purine synthesis Ribose 5 P 5 Phospho Ribosyl 1-pyrophosphate Amino imidazole Ribonucleotide AIR 5-amino-4-carboxyamino Imidazole ribonucleotide ACAIR N-succinyl-5-amino Imidazole carboxamine ribonucleotide SAICAR Amino imidazole Carboxamide ribonucleotide AICAR Formyl Amino imidazole Carboxamide ribonucleotide FAICAR IMP Inosine mono phosphate Glutamine Glycine NF-THF Glutamine CO2 Aspartate NF-THF 1 2 3 4 5 6 7 8 9 10 11 PRPP synthase PRPP glutamyl amidotransferasesynthase +
- 20. Conversion of IMP to AMP Inosine 5’ mono phosphate IMP Adenylosuccinate AMP GTP GDP + Pi Aspartic acid Adenylo Succinate synthetase Fumarate Adenylosuccinase A to A
- 21. Conversion of IMP to GMP Inosine 5’ monophosphate IMP H2O NAD+ NADH + H+ IMP dehydrogenase Xanthosine monophosphate Glutamine Glutamate ATP AMP + PPi GMP GMP synthase G to G
- 22. Effect of purine synthesis inhibitors in human • Extremely toxic to tissue • Decreases replication of cell. • Affect Bone marrow, GI tract, Immune system and hair follicles. • Anticancer drugs methotrexate causes adverse effects like… – Anemia, Scaly skin, GI tract disturbance immunodeficiency and baldness
- 23. Synthetic analogues of Nucleotides • They are antimetabolites, • Synthesized by substituting or altering heterocyclic ring • 5-Fluorouracil, 5-Iodouracil, 6-mercaptopurine • Allopurinol – Gout • Arabinosylcytosine [cytarabine] – Cancer • Azathioprine – Immunosuppressive –organ transplant • Zidovudine [AZT] and Didanosine – Anti HIV • Remdesivir – Corona virus
- 24. Salvage pathway • Tissue :- Erythrocytes, Brain, Polymorpho nuclear leucocytes (De Novo synthesis not operating) Definition:- Recycling / Reutilization of free purine or pyrimidine bases derived from degradation of nucleotides to synthesize corresponding nucleotide
- 25. Salvage pathway Advantage: Economies energy expenditure -6 ATP Quick process Can recycle preformed base Enzymes: - APRTase: Adenosine Phospho Ribosyl Transferase HGPRTase: Hypoxanthine Guanosine Phospho Ribosyl Transferase Substrate: - PRPP --- Starting material
- 26. Adenine Guanine Hypoxanthine PRPP PRPP PRPP AMP + PPi GMP + PPi IMP + PPi APRTase HGPRTase HGPRTase + + + Reactions of Salvage pathway PRPP PRPP Lesch – Nyhan syndrome
- 27. Lesch – Nyhan syndrome Deficiency of enzyme Hypoxanthine Guanine Phospho Rybosyl Transferase [HGPRTase] • Sex linked metabolic disorder (males only affected) • Biochemistry– • HGPRTase deficiency results in PRPP accumulation and decreased GMP, IMP level • Increase synthesis of purine • Symptoms: 1st year • Aggressive behavior , Self mutilation • Mental retardation, Learning disability • Impaired renal function, Gout Disorder:-
- 28. Lesch – Nyhan syndrome • Diagnosis: • Increase Serum Uric acid – Hyperuricaemia • Increase urine uric acid level – Uricosuria , Orange red urine, UA crystals in urine • Increased Urate to Creatinine ratio in urine • Deficiency of HGPRTase enzyme in RBC and other tissue, Megaloblastic anemia • Genetic study • Treatment: Symptomatic • Allopurinol • Alkalization of urine • Benzodiazepines for neurological problem • Protecting devices against self mutilation L N S = Lack of Nucleotide Salvage
- 29. Case study- Lesch-Nyhan syndrome Mother brought her six months old child to paediatric clinic for his unusual urge to bite lips, finger and failure to develop mentally. Serum uric acid was elevated. His peripheral blood smear showed megaloblastic anaemia. Questions: 1. What is the disorder child is suffering from? 2. What is the biochemical defect? 3. How uric acid is produce? What is normal serum uric acid level? Ans: 1. Child is having Lesch-Nyhan syndrome. [compulsive self-mutilation cardinal sign] 2. Complete deficiency of salvage pathway enzyme HGPRTase. [Regeneration of purine nucleotides is blocked] 3. Uric acid is the end product of purine metabolism. In this disease purine accumulates and degraded to uric acid causing increased uric acid level in blood. Normal uric acid level in children is 2.0 to 5.5mg/dl.
- 30. H –Hyperuricaemia G – Gout P – Pissed /Drunk behavior R – Recessive disorder T – dysTonia ase – Allapurinol
- 31. DEGRADATION OF PURINES AMP Adenosine Pi Phosphatase Inosine H2O NH4 Adenosine deaminase Hypoxanthine Pi Ribose 1-P Purine Nucleoside phosphorylase Xanthine O2 H2O2 Xanthine oxidase Uric acid Xanthine oxidase O2 H2O2 Allopurinol − − Site: Liver, Spleen, Kidney Intestinal mucosa Deficiency SCID
- 32. DEGRADATION OF PURINES GMP Guanosine Pi Phosphatase Guanine Xanthine Pi Ribose 1-P Purine Nucleoside phosphorylase O2 H2O2 Xanthine oxidase Uric acid Guanine deaminase NH4 Allopurinol −
- 34. Disorders of Purine Metabolism • Hyperuricemia > 7.0 mg/dl • GOUT • LESCH NYHAN SYNDROME -LNS • SCID – Severe Combined Immunodeficiency Syndrome
- 35. URIC ACID End product of purine metabolism Normal serum level: 3 to 7 mg/dl [male] 6.0 [female] Daily excretion : 500 – 700 mg/day Elevation in the serum uric acid concentration – HYPERURICEMIA Result in GOUT , Renal Stone Lactic acidosis and Ketoacids decrease excretion
- 36. Case study - Gout A 36 year old male patient presented with complaints of pain while passing urine, swollen metatarsophalangeal joint of great toe. Laboratory report revealed serum uric acid level of 14 mg/dl and urine show 10 to 15 crystals /low power field brown crystals of uric acid. 1. Name the disease. And state on what basis? 2. Enumerate the biochemical causes for above condition 3. Suggest the drug of choice and mechanism of drug action Ans: 1. Presented case is of Gout [signs are hyperuricaemia, uric acid nephrolithiasis and acute inflammatory arthritis] 2. Biochemical cause is increased uric acid synthesis. It can be due to defect in enzymes like PRPP synthase, HGPRTase or glucose 6 phosphatase. 3. Treatment: 1. Anti-inflammatory drug Colchicine [relieves pain] 2. Uricosuric agent [which increase excretion] like probenecid or sulfinpyrazone 3. Allopurinol [inhibit key enzyme uric acid synthesis] Competitive inhibitor of OX enzyme.
- 37. What is GOUT? Metabolic disease due to over production (25%) Decreased renal excretion (75%) of URIC ACID Severe hyperuricemia ( above 7mg/dl ) Na-urate crystals get deposited in soft tissues Result in inflammation in joints – GOUTY ARTHRITIS Excruciating pain in great toe, Erythema – Red & hot joint Mostly affecting males and post menopausal women G- Great toe, O- Osteoarthritis, U- Uric acid, T- Tender joint/Tophi
- 38. Inborn error of metabolism Incidence: 1 in 500 live birth Over production of uric acid Due to enzyme defect Types of GOUT PRIMARY GOUT SECONDARY GOUT Due to various disease causing Increased synthesis or Decreased excretion
- 39. Over production of uric acid due to enzyme defects Increase PRPP Synthetase – loss of feedback inhibition Increased PRPP Glutamyl amidotransferase – feedback inhibition Increased Glutathione reductase – Stimulate HMP Shunt Decrease HGPRTase – increased PRPP synthesis Decrease Glucose 6 phosphatase- Von Gierke’s disease (GSD-I) divert G6P to HMP shunt – increase Ribose Types of GOUT PRIMARY GOUT
- 40. Increased synthesis or decrease excretion Increased synthesis Leukemia, Lymphoma, polycythemia, cancers, trauma, starvation, Psoriasis Decreased excretion- Renal impairment, Alcoholism, Lactic acidosis, Ketoacidosis Types of GOUT SECONDARY GOUT
- 41. Signs and Symptoms of GOUT Inflammation In Joints Of Big Toe, Small Toe And Ankle Gout-Early Stage: No Joint Damage Gout-Late Stage: Arthritic Joint Sudden onset of severe pain Swelling, Warmth, Reddish- erythema
- 42. How Alcoholism causes Gout? Case: Alcohol induced Gout CASE-NA: 1: A 45 year old man attended medical outdoor clinic. He complained of aching joints since two days post alcohol party at friend’s farm-house. His physical examination revealed swelling, tenderness, asymmetric tophus on his big toe of right foot with red ness around. Blood and urine analysis indicates striking elevated levels of uric acid.
- 43. Ethanol CH3CH2OH Acetaldehyde Acetate Acetyl CoA NAD+ NADH H+ NAD+ NADH H+ ADH ALDH TCA CO2 + H2O Degradation of ethanol
- 44. Ethanol CH3CH2OH Acetaldehyde Acetate Acetyl CoA Glucose Pyruvate Lactate Oxaloacetate NAD+ NADH H+ NAD+ NADH H+ Low NAD/ NADH ratio ─ ─ ─ Gluconeogenesis Hypoglycaemia X X X Low ATP/AMP Lactic acidosis ↓ UA excretion Hyper Uricaemia GOUT ADH ALDH Impact of ethanol on carbohydrate metab TCA CO2 + H2O X
- 45. Ethanol CH3CH2OH Acetaldehyde Acetate Acetyl CoA Glucose Pyruvate Lactate Oxaloacetate NAD+ NADH H+ NAD+ NADH H+ Low NAD/ NADH ratio ─ ─ ─ Gluconeogenesis Hypoglycaemia X X X Ketone bodies Ketoacidosis Low ATP/AMP Lactic acidosis ↓ UA excretion Hyper Uricaemia GOUT Lipolysis Fatty liver TG + + + ─ Starvation in alcoholics + ADH ALDH ↓ UA excretion Hyper Uricaemia Liver cirrhosis Impact of ethanol on Lipid metab
- 46. Ethanol CH3CH2OH Acetaldehyde Acetate Acetyl CoA Glucose Pyruvate Lactate Oxaloacetate NAD+ NADH H+ NAD+ NADH H+ Low NAD/ NADH ratio ─ ─ ─ Gluconeogenesis Hypoglycaemia X X X Ketone bodies Ketoacidosis Low ATP/AMP Lactic acidosis ↓ UA excretion Hyper Uricaemia GOUT Lipolysis Fatty liver TG + + + ─ Starvation in alcoholics + ADH ALDH Cell Toxic Cell lysis Neurodegenerative changes ↑ UA Hyper Uricaemia GOUT Down excitatory NT R Up Inhibitory NT R Memory loss Sleepiness Depression More craving alcohol ↓ UA excretion Hyper Uricaemia Liver cirrhosis Liver cirrhosis Cell Toxicity
- 47. Ethanol CH3CH2OH Acetaldehyde Acetate Acetyl CoA Glucose Pyruvate Lactate Oxaloacetate NAD+ NADH H+ NAD+ NADH H+ Low NAD/ NADH ratio ─ ─ ─ Gluconeogenesis Hypoglycaemia X X X Ketone bodies Ketoacidosis Low ATP/AMP Lactic acidosis ↓ UA excretion Hyper Uricaemia GOUT Lipolysis Fatty liver TG + + + ─ Starvation in alcoholics + ADH ALDH Cell Toxic Cell lysis Neurodegenerative changes ↑ UA Hyper Uricaemia GOUT Down excitatory NT R Up Inhibitory NT R Memory loss Sleepiness Depression More craving alcohol ↓ UA excretion Hyper Uricaemia Liver cirrhosis Liver cirrhosis Low NAD/ NADH ratio Oxidative stress ROS
- 48. Diagnosis of GOUT Serum Uric acid - > 7.0mg/dl CBC – increase count, ESR- Increase, Renal function tests - Synovial fluid show Uric acid crystal X – Ray joints C – Reactive Protein [CRP] - Inflammation CT Scan
- 49. Treatment of gout • Decrease uric acid production • Allopurinol – • competitive inhibitor of xanthine oxidase Increase uric acid excretion • Colchicine – anti inflammatory drug • Uricosuric drug -- probenecid • Phenylbutazone • Indomethacin • Oxyphenbutazone • corticosteroids
- 50. Case study - Gout A 36 year old male patient presented with complaints of pain while passing urine, swollen metatarsophalangeal joint of great toe. Laboratory report revealed serum uric acid level of 14 mg/dl and urine show 10 to 15 crystals /low power field brown crystals of uric acid. 1. Name the disease. And state on what basis? 2. Enumerate the biochemical causes for above condition 3. Suggest the drug of choice and mechanism of drug action Ans: 1. Presented case is of Gout [signs are hyperuricaemia, uric acid nephrolithiasis and acute inflammatory arthritis] 2. Biochemical cause is increased uric acid synthesis. It can be due to defect in enzymes like PRPP synthase, HGPRTase or glucose 6 phosphatase. 3. Treatment: 1. Anti-inflammatory drug Colchicine [relieves pain] 2. Uricosuric agent [which increase excretion] like probenecid or sulfinpyrazone 3. Allopurinol [inhibit key enzyme uric acid synthesis] Competitive inhibitor of OX enzyme.
- 51. Severe Combined Immuno Deficiency - SCID • Deficiency of Adenosine Deaminase [ADA] • Adenosine accumulate • Adenosine converted to ATP and dATP • dATP inhibit Ribonucleotide reductase • Reduce d-nucleotide synthesis • Affect DNA synthesis, • T and B lymphocytes • Causes Immunodeficiency What is the cause of Immunodeficiency? Bubble Boy disease
- 52. PYRIMIDINE
- 53. PYRIMIDINE SYNTHESIS Requirements: Carbamoyl phosphate [CPS-II] PRPP: ATP: Amino acid: Aspartic acid, Glutamine Cofactor: FAD, NAD, Mg++ Pyrimidine ring is synthesized as free and then incorporated in to nucleotide
- 54. SOURCES OF INDIVIDUAL ATOMS IN PYRIMIDINE RING C4 C2 C5 C6 N1 N3 Aspartate CO2 Glutamine
- 55. De Novo pyrimidine synthesis 2 ATP + CO2 + Glutamine CarbamoyI Phosphate Carbamoyl aspartate Dihydroorotate Orotate Orotidine 5’ monophosphate OMP Uridine 5’ monophosphate UMP Aspartate Carbamoyl phosphate Synthetase- II 2 ADP + Pi + Glutamate Pi Aspartate transcarbamoylase Dihydroorotase H2O Dihydroorotate dehydrogenase NAD+ NADH + H+ Mitochondrial Orotate phosphoribosyl transferase PRPP PPi OROTIC ACIDURIA OMP decarboxylase CO2
- 56. Orotic aciduria • Rare autosomal recessive condition • Error in de novo synthesis of pyrimidine • Deficiency of Orotate phosphoribosyl transferase and • OMP decarboxylase [ together called UMP synthase] • Manifest in first year of life • Growth Retardation, Neurological abnormality, • Urinary excretion of Orotic acid • Treated by UTP feeding
- 57. How UTP prevents Orotic aciduria? 2 ATP + CO2 + Glutamine Carbamoyl Phosphate Orotate OMP UMP Carbamoyl phosphate Synthetase- II 2 ADP + Pi + Glutamate Orotate phosphoribosyl transferase OROTIC ACIDURIA OMP decarboxylase UTP Feed back Inhibition CPS-II ─ +
- 58. DEGRADATION OF PYRIMIDINES Cytosine Uracil NH4 Cytidine deaminase Dihydrouracil NADP Dihydropyrimidine dehydrogenase Ureidopropionate H2O Dihydro pyrimidine amidohydrolase CO2 + NH3 Acetate Ureiodopropinase H2O NADPH + H
- 59. Thank you