[BIOCHEM]LAB_207_NUCLEOTIDE-METABOLISM.pdf

(007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 OUTLINE I. CASE NO.1: ADENOSINE DEAMINASE DEFICIENCY...

(007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 OUTLINE I. CASE NO.1: ADENOSINE DEAMINASE DEFICIENCY A. Objectives B. Discussion 1. Nucleotide Structure a. Nitrogenous base b. Pentose monosaccharide c. Phosphate groups 2. Functions C. Catabolism of the Purine Mononucleotides 1. Guanosine What is the likely diagnosis? a. Guanosine and deoxyguanosine → o Probable severe combined immunodeficiency guanine o Adenosine deaminase (ADA) deficiency b. Guanine → xanthine What is the biochemical basis for this disorder? c. Xanthine oxidase → uric acid 2. Adenosine o Adenosine deaminase deficiency which is a. Adenosine and deoxyadenosine → involved in purine catabolism inosine/deoxyinosine ▪ ADA is an enzyme involved in the b. Inosine/deoxyinosine → breakdown of purines. Specifically, it is hypoxanthine responsible for the deamination of c. Hypoxanthine → xanthine → uric adenosine and deoxyadenosine to acid inosine and deoxyinosine. Individuals 3. Uric Acid with mutations in the ADA gene, typically D. Clinical Correlation inherited in an autosomal recessive E. Adenosine Deaminase Deficiency manner, exhibit decreased ADA F. Clinical Manifestation enzymatic activity together with G. Diagnostics elevations in adenosine and H. Treatment deoxyadenosine. Deoxyadenosine is II. CASE NO. 2: GOUTY ARTHRITIS highly cytotoxic, particularly to A. Objectives lymphocytes. Thus, ADA-deficient B. Discussion individuals exhibit a severe combined 1. Uric Acid and Urate immunodeficiency and suffer from 2. Causes of Hyperuricemia secondary complications associated a. Metabolic abnormalities → with a compromised immune system. overproduction [de novo synthesis] Potential treatment options: b. Defects in the purine salvage - Haploidentical bone marrow transplantation, pathway - ADA enzyme replacement therapy c. Inflammation - ADA gene therapy. C. Clinical Correlation D. Clinical Manifestations A. OBJECTIVES E. Diagnostics 1. Describe the catabolic pathway of purine synthesis F. Treatment 2. Explain metabolic basis of immunodeficiency III. APPENDIX B. DISCUSSION 1. Nucleotide structure I. CASE NO. 1: ADENOSINE DEAMINASE a. Nitrogenous base DEFICIENCY − purines [A/G] − pyrimidines [C/T/U] A 6-month-old baby is brought to the hospital by with a chief complaint of decreased sensorium. On history patient was noted to have decreased activity level, lack of appetite, and worsening diaper rash that has not resolved with usual antifungal treatment. Delivery: The baby was born at term and no noted complications. Past Medical History: Frequent URTI symptoms, watery stools. Differential diagnosis: Immuno-compromised Physical Examination: the patient is afebrile but is noted to be lethargic with no lymphadenopathy. Mild hepatosplenomegaly and hypotonia are noted on physical examination. Chest radiography demonstrates an absent thymic shadow but normal heart silhouette. Laboratory values revealed elevated levels of adenosine and deoxyadenosine in the urine and blood. Page 1 of 8 CMED 1F  (007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 Signalling molecules b. Pentose monosaccharide (ribose or deoxyribose) Hepls with movement of genetic information of metabolic (DNA/RNA) process - 1 + 2 → nucleoside More phosphate groups- more energy ATP carries more energy than ADP, AMP. De novo synthesis- inside the body c. Phosphate groups (mono, di or triphosphates) - synthesis purine nucleotides mainly in the liver but can be all throughout body - Roles - RNA and DNA synthesis - Structural components coenzymes - Signaling molecules C. CATABOLISM OF THE PURINE - Helps with movement of genetic information of metabolic processes MONONUCLEOTIDES To produce GMP, you need guanine; To produce IMP, Hydrolysis of the phosphate by a nucleotidase → nucleoside inosine is required and to produce AMP, adenine group is needed. 1. Guanosine- a. Guanosine and deoxyguanosine → guanine - Phosphorolytic cleavage pentose sugar by purine nucleoside phosphorylase b. Guanine → xanthine - Guanine deaminase: hydrolysis of amino group c. Xanthine oxidase → uric acid - Catalyzed by xanthine oxidase 2. Adenosine: - a. Adenosine and deoxyadenosine → inosine/deoxyinosine (nucleoside) - Catalyzed by adenosine deaminase and deoxyinosine b. Inosine/deoxyinosine → hypoxanthine - Catalyzed by purine nucleoside phosphorylase - Removal of the pentose sugars from the nucleosides c. Hypoxanthine → xanthine → uric acid - Catalyzed by xanthine oxidase. Uric acid 3. Uric Acid - Not very soluble in blood - Excreted in urine End product of purine catabolism is always uric acid which is toxic to our body. “SALVAGE PATHWAY” recycles this component. All other mammals possess the enzyme uricase that converts uric acid to allantoin that is easily eliminated through urine. Overproduction of uric acid, generated from the metabolism of purines, has been proven to play 2. Functions emerging roles in human disease. RNA and DNA synthesis Structural components coenzyme Page 2 of 8 CMED 1F  (007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 multiple cells D. CLINICAL CORRELATION o Affects development of non-immune phenotypes o Non-immune phenotypes include: - neurologic problems and hearing loss - lung disease hepatocellular damage - kidney disease - bone disorders These nonimmune phenotypes are thought to be associated with accumulation of the ADA substrate adenosine, which is a potent cell-signaling molecule. It is clear that both the immune and nonimmune phenotypes in ADA deficiency are resultant from the accumulation of the ADA substrates deoxyadenosine (immunotoxi6c) and adenosine (abnormal cell signaling). First immunodeficiency disease that molecular defect was discovered First inherited disease to be treated with gene therapy Decreased levels and function of T and B lymphocytes and can include Natural Killer (NK) T cells The monogenetic nature of this disorder together with the relatively well defined biochemical basis of the disorder E. ADENOSINE DEAMINASE DEFICIENCY have led to effective therapies that target the restoration ADA deficiency is a rare autosomal recessive genetic of ADA enzymatic activity and the lowering of ADA disorder that results in severe combined immunodeficiency disease substrates. (SCID) characterized by decreased levels and function of T and B F. CLINICAL MANIFESTATION lymphocytes as well as decreased levels of Natural Killer (NK) T cells. Sibling death in infancy (e.g., multiple deaths during infancy The most severe form of the disease, typically associated with due to infection or unexplained deaths) or previous near complete lack of ADA enzymatic activity, is seen in infants miscarriages in the mother where secondary infections resultant from a compromised immune Family history of SCID or other primary immunodeficiency system appear in the first 6 months of life. Family history of consanguinity Autosomal recessive mutation 1st genetic → inherited immunodeficiency Most patients present before 3 - 6 months of age o Metabolic cause found Poor feeding Failure to thrive Chronic diarrhea Recurrent infections, especially pneumonia Very small/absent thymus death by 18 to 24 months G. DIAGNOSTICS Low CD3 T-cell count, usually < 300/μL o normal lymphocyte count in a newborn is ~2500/μL Flow cytometry for lymphocyte subsets to quantify CD3/4/8 T-cells, CD19 B-cells, and CD16/56 NK-cells Abnormal lymphocyte proliferation studies to mitogens Low quantitative immunoglobulins (initially normal due to maternal transfer of IgG) No antibody response to Elevations in adenosine and deoxyadenosine→ cytotoxicity vaccination to cells Newborn screening for T-cell receptor excision circles o Phosphorylation of deoxyadenosine to dATP (TREC) → activation of apoptotic pathways (cell death) H. TREATMENT Isolation accounts for the loss of lymphocytes Enzyme replacement in ADA deficiency Adenosine →activation of adenosine receptors on Page 3 of 8 CMED 1F  (007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 ○ an effective treatment for ADA deficiency. It involves the injection of purified ADA protein that has been modified with polyethylene glycol (PEG- ADA). PEG-ADA injections effectively lower the levels of ADA substrates and alleviate the symptoms of ADA deficiency Gene therapy ○. ADA deficiency was the first case of gene therapy in humans ○ involves removal of the patient’s bone marrow followed by the stable transfer of a “good” ADA gene into the bone marrow cells that are then transferred back into patients, where they expand and populate the immune system. The metabolic disturbances are corrected, as are many of the other phenotypes seen. Bone marrow transplantation II. CASE 2: GOUTY ARTHRITIS Purine catabolism is important as uric acid is toxic, the blood is already saturated with the amount of uric acid, since it is not very A 43-year-old man came into the emergency department with severe soluble it easily crystalizes when above normal levels. The salavge right big toe pain. Apparently well until early in the morning when he pathway exist to decrease the amount of purines that are being woke up with severe pain in his right big toe. No history of trauma to degraded to uric acid, it also recycles hypoxanthine back into inositol the toe and no previous history of such pain in other joints Patient phosphate and guanine to GMP to be use in other systems or had a drinking spree that night before. reactions without this pathway uric acid build up is inevitable. Uric acid is ussual present in the blood at ionizedbfirm and at physiogic ph it is not soluble. A. OBJECTIVES 1. Reiterate purine metabolism from previous case 2. Explain mechanism of action of allopurinol and colchicine B. DISCUSSION Purine bases are used in many important biologic processes including the formation of nucleic acids (RNA and DNA), energy currency (adenosine triphosphate [ATP]), cofactors (nicotinamide adenine dinucleotide, flavin adenine dinucleotide), and cellular signaling (guanosine triphosphate [GTP], ATP, adenosine). Purines On Physical examination, he was found mild distress are both synthesized de novo and taken in through the diet. Their secondary to the pain in his right toe. The right big toe was swollen, degradation is a ubiquitous process; however, increased levels of the warm, red, and exquisitely tender. Remaining PE was normal. enzymes that carry out the metabolism of purine bases suggest that Synovial fluid was obtained and revealed rod- or needle-shaped purine catabolism is higher in the liver and the gastrointestinal tract. crystals that were negatively birefringent under polarizing microscopy, Abnormalities in purine biosynthesis and degradation are consistent with gout. associated with numerous disorders suggesting that the regulation of What is the likely diagnosis? purine levels is essential. o Gouty arthritis How would you make a definite diagno bysis? 1. Uric Acid and Urate o Arthrocentesis to obtain synovial fluid analysis to Present in blood as ionized urate form at physiologic pH check for monosodium urate crystals within the synovial Urate is not very soluble in an aqueous environment leukocytes or in material derived from tophi under polarizing Blood already very near saturation microscopy. o Increased amounts → uric acid crystal formation What is the pathophysiology of this disorder? o Elevated levels of purine end product uric acid probably due to decreased excretion by kidney/ increased production of uric acid result in precipitation of urate crystals in the joints. Page 4 of 8 CMED 1F  (007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 c. Inflammation 2. Causes of hyperuricemia a. Metabolic abnormalities → overproduction [de novo synthesis] o Overproduction of purine nucleotides →degradation→hyperuricemia Just like with concentration of there is a lot of GMP, IMP, AMP this pushes the reaction toward degradation or what we have least of -Urate aggregate→ form crystals(can occur other places , so would push towards guanine, xanthine and eventually uric acid. than knee, like carliage or ear, wrist, elbows etc) → engulfed I.e to much purine= to much uric acid via degradation process. by macrophages→ rupture →release of lysozymes → o Examples: inflammation(decreases ph in the are to promote lactic acid - ↑ activity of 5-phosphoribosyl-1-pyrophosphate which would lead to more inflammation and then erosion) (PRPP) synthetase → production of PRPP which would and erosion produce more of our IMP. - Precursor of both purine and pyrimidine de novo C. CLINICAL CORRELATION biosynthesis Gout is a disorder that occurs when uric acid crystallizes in the joints of the body, usually the great toe or large joints. - Increase the rate of degradation lead to increased Hyperuricemia is a clinical condition characterized by elevated levels purine nucleotide production and hence increased uric acid of uric acid. This leads to the formation of sodium urate crystals, which production are found primarily in the joints of the extremities and in the renal b. Defects in the purine salvage pathway interstitium. The presence of urate crystals is associated with extreme Lesch-Nyhan Syndrome swelling and tenderness in the joints of the extremities. This condition This syndrome is associated with mental is often referred to as gout or gouty arthritis. In this condition, elevated retardation and self-destructive behavior, which levels of uric acid are detectable in the blood and urine, and definitive diagnosis can be made by observing the presence of urate crystals in may be associated with inadequate production of synovial fluid removed from affected joints. The preference of urate purine nucleotides through the salvage pathway in crystal formation in the joints of the extremities, such as the big toe, is certain neuronal cells.. In addition, patients with thought to be associated with the decreased temperature of the Lesch-Nyhan syndrome have gout resulting from extremities that aids in urate crystal formation when levels exceed the inability to salvage purine bases, which leads solubility. to increased levels of uric acid. Hyperuricemia and D. CLINICAL MANIFESTATIONS gout can also arise from numerous undefined mechanisms that include dietary issues. Peripheral (initially) mono-arthritis then progress to o X-linked recessive (easier for males to polyarthritis (when not controlled or managed) be affected that females) - Most common is Metatarsophalangeal [MTP] o HGPRT deficiency (aka. Podagra), ankle, knee or foot. o Swelling usually o responsible for reforming IMP and GMP at night time from hypoxanthine and guanine Occasional Fever, malaise o decrease in salvage back into purine Chronic Kidney Disease nucleotide pool - Formation of urate crystals in kidneys o increased activity of PRPP synthetase - Uric acid stone formation → ↑ de novo purine synthesis →↑uric Tophi formation acid - Deposition of urate stones in soft tissues Page 5 of 8 CMED 1F  (007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 Colchicine - Inhibit microtubule formation - Inhibit phagocytic activity of macrophage - side effect nitropenia - not used at normal times - This prevents the urate crystals from rupturing the phagocytes and causing inflammation in the joints. Additional drugs used for the treatment of gout include alloxanthine, another inhibitor of xanthine oxidase. Uricosuric agents, such as probenecid or sulfinpyrazone, that increase renal excretion of uric acid, are used in patients who are“underexcretors” of uric acid. Purine catabolic pathway. The enzyme steps inhibited by allopurinol E. DIAGNOSTICS are indicated. Arthrocentesis showing negative birefringent crystals - (shows yellow under polarized light) Increased serum uric acid (not really dependent on this as patients come during pain so serum levels are normal) - Normal 5-6mg/dL Increased urinary excretion of uric acid [>800mg/24hours] Rat bite erosions over cortical bones F. TREATMENT Avoid high protein diets Avoid alcohol Treat underlying cause if secondary (kidney or liver dysfunction, gene disorder) Allopurinol - Decrease uric acid production by inhibiting xanthine oxidase - The administration of allopurinol is an effective treatment of gout because it decreases the amount of uric acid produced, which, in turn, alleviates the amount of sodium urate crystals that are formed. TEST YOURSELF 1. What catalyzes the reaction from adenosine to Inosine monophosphate and the reaction from Inosine monophosphate to hypoxanthine? 2. What recycles the purines back to Inosine monophosphate (IMP) or Guanosine monophosphate (GMP)? 3. What enzyme catalyzes the reaction from Hypoxanthine to Xanthine and the uric acid? 4. Why is it the big toe mostly the first site to be affected and the first symptom to appear in gouty arthritis? Page 6 of 8 CMED 1F  (007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 5. What is the first immunodeficiency disease that molecular defect 14. In humans, the principal catabolic product of pyrimidines is: was discovered and the first inherited diseases to be treated with a. Uric acid d. beta-Alanine gene therapy? b. Allantoin e. Urea 6. A 1-year-old female patient is lethargic, weak, and anemic. Her c. Hypoxanthine height and weight are low for her age. Her urine contains an 15. Complete hydrolysis of nucleic acids will not yield: elevated level of orotic acid. Activity of uridine monophosphate a. Guanosine d. Phosphoric acid synthase is low. Administration of which of the following is most b. Ribose e. All of the above likely to alleviate her symptoms c. Deoxyribose a. Adenine b. Guanine c. Phosphate d. Thymidine monophosphate synthase deficiency; B; B; C; B; E; D; A ornithine transcarbamylase deficiency but not in uridine 7. A 42-year-old male patient undergoing radiation therapy for E; Blood ammonia level would be expected to be elevated in prostate cancer develops severe pain in the metatarsal phalangeal less solubility of uric acid; Adenosine deaminase deficiency; D; of the lower temperature in lower extremities which leads into joint of his right big toe. Monosodium urate crystals are detected phosphorylase; Salvage pathway; Xanthine oxidase; Because by polarized light microscopy in fluid obtained from this joint by Adenosine deaminase (ADA) and Purine nucleoside arthrocentesis. This patient’s pain is directly caused by the overproduction of the end product of which of the following REFERENCES metabolic pathways? 1. Chatterjea, M.N. & Shinde, R. (2012). Textbook of Medical a. de Novo pyrimidine biosynthesis Biochemistry (8th ed.). India: Jaypee Brothers Medical b. Pyrimidine degradation Publishers. c. De novo purine biosynthesis 2. Ferrier, D. R. (2017). Lippincott illustrated reviews: d. Purine salvage Biochemistry. Philadelphia: Wolters Kluwer. e. Purine degradation 3. Toy, E. C. (2015). Case files: Biochemistry. New York: Mc 8. What laboratory test would help in distinguishing an orotic aciduria Graw-Hill. caused by ornithine transcarbamylase deficiency from that caused by uridine monophosphate synthase deficiency? 9. A drug which prevents uric acid synthesis by inhibiting the enzyme xanthine oxidase is: a. Aspirin d. Probenecid b. Allopurinol e. Phenyl butazone c. Colchicine 10. Inosinic acid is the biological precursor of: a. Cytosine and uric acid b. Adenylic acid and guanylic acid c. Orotic acid and uridylic acid d. Adenosine and thymidine e. Uracil and thymidine 11. The probable metabolic defect in gout is: a. A defect in excretion of uric acid and kidney b. An overproduction of pyrimidines c. An overproduction of uric acid d. An underproduction of purine e. Rise in calcium leading to deposition of calcium urate 12. Synthesis of GMP from IMP requires the following: a. Ammonia, NAD+, ATP b. Glutamine, NAD+, ATP c. Ammonia, GTP, NADP+ d. Glutamine, GTP, NADP+ e. Glutamine, UTP, NADP+ 13. In humans, the principal breakdown product of purines is: a. Ammonia d. Urea b. Allantoin e. Uric acid c. Alanine Page 7 of 8 CMED 1F  (007) NUCLEOTIDE METABOLISM DR. FAHAD ABDUL RAZAK | 01/26/21 APPENDIX Page 8 of 8 CMED 1F