Physiological_significance_of_HMP_Shunt2d_seminar_1[1].pptx

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1. 1 St MBBS 2nd phase seminar Date : 9th nov 22 (batch B ) Time : 11am --- 01 pm. 10 Nov 22 (batch A ) Venu :Biochemistry Demonstration hall MIMSR Medical college , Latur Department of biochemistry

2. Pyruvate dehydrogenase complex Roll no.95 and …..

3. Pyruvate Dehydrogenase 3 COMPONENTS : Pyruvate decarboxylase / dehydrogenase Dihydrolipoyl transacetylase Dihydro lipoyl dehydrogenase Co-factors / co-enzymes: Thiamine pyrophosphate lipoamide CoA FAD NAD

4. Pyruvate dehydrogenase

5. Regulation of PDH End product inhibition by - acetyl CoA - NADH Co-valent modification of PDH enzyme CLINICAL ASPECTS 1. Thiamine deficiency: - PDH activity decreased - pyruvate is converted to lactate -lactic acidosis 2. Inherited deficiency of glycolytic enzymes - Pyruvate kinase - aldolase

6. Physiological significance of HMP Shunt Roll no .33 and 78

7. The main purpose of the shunt pathway is to produce ribose phosphate and to generate NADPH. The oxidative phase of the pathway is seen in the following organs, where NADPH generation is required for lipid synthesis or steroid synthesis. Liver Adipose tissue Adrenal cortex Mammary glands Testes and ovaries Red blood cells Lens of eye. The nonoxidative phase is present in all tissues, and so synthesis of ribose is possible in all tissues of the body.

8. 1 .) To produce NADPH which is required for: i ) Reductive biosynthesis—fatty acids, cholesterol and steroid hormones ii )Free radical scavenging iii )Maintain RBC membrane integrity by keeping GSH in reduced state iv )Prevention of methemoglobin formation v ) Detoxification by hydroxylation vi )Maintain the transparency of lens vii )Bactericidal activity of macrophages 2 . )To produce ribose and deoxyribose for DNA and RNA synthesis Clinical importance of the shunt pathway Glucose-6-phosphate dehydrogenase deficiency Drug-induced hemolytic anemia Methemoglobinemia Thiamine deficiency leads to reduced transketolase activity

9. GPDH Deficiency The enzyme glucose-6-phosphate dehydrogenase (GPD) may be deficient in some persons. It is the most common enzyme deficiency seen in clinical practice. The defect is transmitted as an X-linked recessive trait. The deficiency will lead to drug-induced hemolytic anemia. The deficiency is manifested only when exposed to certain drugs or toxins, e.g. intake of antimalarial drugs like primaquine. Primaquin stimulates peroxide formation inside RBC. In GPD deficient cells, the level of NADPH is low; hence further production of peroxides will lead to cell lysis. Similarly, ingestion of toxic glycosides present in fava beans may have similar effect (Favism). Sulfa drugs and furadantin may also precipitate the hemolysis. This will lead to jaundice and severe anemia. Clinical significance ----- glucose -6- phosphate dehydrogenase deficiency .

10. METABOLIC FATE OF GLYCINE ROLL NO .102 AND …….

11. Synthesis of purine ring: Glycine contributes to the C 4 , C 5 and N 7 of the purine ring.

12. Synthesis of heme 3 . Formation of glutathione Glycine as conjugating agent Glycine as a neurotransmitter Detoxification Bile salt formation Constitute of collagen

13. Acts as a neurotransmitter In brain stem and spinal cord At moderate levels, disrupts neuronal traffic At very high levels causes overexitation Is a constituent of protein Seen at bends or turns In collagen, every 3 rd amino acid is glycine

14. Conjugates with bile acids glyco cholic acid glycochenodeoxy cholic acid Conjugates with benzoic acid

15. FORMATION FO CREATINE

16. Synthesis and degradation of creatine.

17. Primary hyperoxaluria :- Renal stone, colic & hematuria. Glycinemia and glycinuria (Defect cleavage of glycine) METABOLIC ERRORS OF GLYCINE

18. This process is known as beta-oxidation, because the oxidation and splitting of two-carbon units occur at the beta-carbon atom. The oxidation of the hydrocarbon chain occurs by a sequential cleavage of two-carbon atoms PREPARATIVE STEP The co-enzyme A is a complex molecule containing B complex vitamin pantothenic acid and a molecule of beta mercapto ethanolamine; this SH group forms thioester bond in acyl-CoA. To emphasize the function of the SH group, the CoA is sometimes written as CoA-SH. Beta oxidation of fatty acid

19. Preparative Step 1: Activation of Fatty Acids # fatty acids are activated to their coenzyme A (CoA) derivative. This activation is taking place in cytoplasm. #ATP is hydrolyzed to AMP and PPi and the energy from hydrolysis of PPi drives the reaction forward. Thus two high energy bonds are utilized in this reaction. # The enzyme is a thiokinase or fatty acyl-CoA synthetase (Step 0). # Three different enzymes, one each for short chain, medium chain and long chain fatty acids have been identified. # Small chain fatty acids may also be activated by thiophorase enzyme, using succinyl-CoA.

20. Preparative Step 2: Role of Carnitine Fatty acids are activated in the cytoplasm; but beta oxidation is in mitochondria . So transport of fatty acids through the mitochondrial membrane is essential. The long chain fatty acyl-CoA cannot pass through the inner mitochondrial membrane. Therefore a transporter, carnitine is involved in transfer of fatty acids. Carnitine is β- hydroxy- γ- trimethyl ammonium butyrate: (CH3) 3 –N+–CH2–CHOH–CH2–COOH.

21. Carnitine is synthesized in the liver and kidneys from lysine and methionine. During growth or pregnancy, the requirement of carnitine might exceed its natural production. Human genetic disorders, affecting different steps of carnitine metabolism will cause deficiency of fatty acid oxidation. During the aging process, carnitine concentration in cells diminishes. Bones are particularly affected adversely, leading to osteoporosis in elderly subjects. Administration of carnitine is capable of improving the clinical condition. The classical presentation of primary carnitine deficiency is hepatomegaly, elevated transaminases, and hyperammonemia . The causes for secondary carnitine deficiency are organic acidurias, and drug-induced (valproic acid, zidovudine).

22. Preparative Step 3: Carnitine Acyl Transferase The enzyme carnitine acyl transferase-I ( CAT-I ) will transfer the fatty acyl group to the hydroxyl group of carnitine to form acyl carnitine. The reaction occurs on the cytosolic side of inner mitochondrial membrane. Preparative Step 4: Translocase A protein translocase will carry the acyl carnitine across the membrane to the matrix of mitochondria. On the matrix side of the membrane another enzyme, carnitine acyl transferase-II ( CAT-II ) will transfer the acyl group back to co-enzyme A molecule. Carnitine is returned to the cytosolic side by the translocase

23. Clinical Applications Medium chain and short chain fatty acids do not require carnitine for transport across the inner mitochondrial membrane. So, medium chain and short chain fatty acids are easily oxidized. Carnitine deficiency is reported in preterm infants, in whom impaired fatty acid oxidation is noticed. So more glucose is utilized, resulting in episodes of hypoglycemia

24. Deficiency of translocase: It leads to defective metabolism of long-chain fatty acids. In this condition, muscle cramps are precipitated by fasting, exercise and high fat diet. Inherited CAT-I deficiency affects only the liver resulting in reduced fatty acid oxidation and ketogenesis with hypoglycemia . CAT-II deficiency affects primarily skeletal muscle and, when severe, the liver. The sulfonylurea drugs ( glibenclamide and tolbutamide ), used in the treatment of type 2 diabetes mellitus, reduce fatty acid oxidation and, therefore, hyperglycemia by inhibiting CPT-I.

25. The fatty acyl-CoA is dehydrogenated to a transenoyl CoA with FAD accepting the hydrogen atoms (Step 1). FADH2 when oxidised in electron transport chain will produce 1.5 ATP molecules. Reactions of beta oxidation of fatty acid

26. This is catalyzed by an enoyl-CoA hydratase (step 2). This step forms a beta-hydroxy fatty acyl-CoA. The L isomer alone is formed during the hydration of the trans double bond

27. The beta-hydroxy fatty acyl-CoA is again oxidized to form beta-keto fatty acyl-CoA. (Step 3). This dehydrogenase acts only on L isomer. The NADH when oxidized in electron transport chain will generate 2.5 ATPs.

28. The beta-keto fatty acyl-CoA now undergoes thiolytic cleavage, splitting off a molecule of acetyl-CoA and leaving behind a fatty acid with 2 carbon atoms less (Step 4). The newly formed fatty acyl-CoA will sequentially undergo further cycles of steps 1, 2, 3 and 4 of beta-oxidation until the fatty acid is completely converted to acetyl-CoA.

29. Summary of β-oxidation of palmitic acid (16 C). It undergoes 7 cycles, which give rise to 8 molecules of acetyl-CoA. β-oxidation of fatty acids. See that the first step is FAD dependent and the third step is NAD+ dependent

30. Energetics of Beta-Oxidation (ATP Yield) Palmitic acid (16 C) needs 7 cycles of beta-oxidation. So, it gives rise to 8 molecules of acetyl-CoA. Every molecule of acetyl-CoA when oxidized in the TCA cycle gives 10 molecules of ATP. Each molecule of FADH2 produces 1.5 molecules of ATP and each NADH generates 2.5 molecules of ATP, when oxidized in the electron transport chain. Hence, the energy yield from one molecule of palmitate may be calculated as: 8 acetyl CoA × 10 = 80 ATP 7 FADH2 × 1.5 = 10.5 ATP 7 NADH × 2.5 = 17.5 ATP Gross total = 108 ATP Net yield = 108 minus 2 =106 ATP (In the initial activation reaction, the equivalents of 2 high energy bonds are utilized). The efficiency of beta oxidation is about 33%.

31. The availability of free fatty acid (FFA) regulates the net utilization through beta-oxidation. The level of FFA, in turn, is controlled by glucagon:insulin ratio. Glucagon increases FFA level and insulin has the opposite effect. CAT-I is the regulator of entry of fatty acid into mitochondria. Malonyl-CoA inhibits CAT-I activity. Thus during de novo synthesis of fatty acid, beta-oxidation is inhibited. Regulation of Beta-Oxidation

32. Glucose Tolerance Test (GTT ) ROLL NO .147 and

33. Oral Glucose Tolerance Test (OGTT) It is artificial , because in day to day life, such a large quantity of glucose does not enter into blood. However, the GTT is well- standardised ; highly useful to diagnose diabetes mellitus in doubtful cases. Preparation of The Patient   The patient should have good carbohydrate diet (more than 150 g carbohydrate) for 3 days prior to the test. This is important Otherwise carbohydrates may not be tolerated even in a normal person. Patient should not take food after 8 PM the previous night. Should not take any breakfast. This is to ensure 12 hours fasting. The patient must be made comfortable and should be seated during the test.

34. Patient is asked to come early in morning, without any breakfast Blood and urine sample collected at 0 hr (Fasting ,F) Glucose Load Give glucose solution to drink. Dose: 75 g anhydrous glucose (82.5 g of glucose monohydrate) in 250-300 ml of water. In children, glucose dose 1.75 g /kg body weight. Sample Collection ½ hour intervals for next 2½ hours. (Total six samples, including 0-hr sample). Blood samples , Glucose estimation,( Quantitative.) Mini Glucose Tolerance Test hr and 2 hr samples

35.   Normal Persons Criteria for diagnosing diabetes Criteria for diagnosing IGT Fasting < 110 mg/dl > 126 mg/dl 110 to 126 mg/dl 1 hr (peak) after glucose < 160 mg/dl Not Prescribed Not Prescribed 2 hr after Glucose < 140 mg/dl > 200 mg/dl 140 to 199 mg/dl Plasma Glucose Levels in OGTT in Normal Persons and in Diabetic Patients

36. 1. If the fasting plasma sugar is more than 126 mg /dl, on more than one occasion 2. Or, if 2-hr post-glucose load value of GTT is more than 200 mg /dl (even at one occasion). 3. If the random plasma sugar level is more than 200 mg/dl , on more than one occasion . Diagnosis should not be based on a single random test alone; it should be repeated. Impaired Glucose Tolerance (IGT) Impaired Glucose Regulation (IGR) Blood sugar values are above normal level, but below the diabetic levels. Fasting plasma glucose level is between 110 and 126 mg/dl Such persons need careful follow up because IGT progresses to frank diabetes Diagnostic Criteria for Diabetes Mellitus

37. Gestational Diabetes Mellitus (GDM) carbohydrate intolerance is noticed, for the first time, during a pregnancy. A known diabetic patient, who becomes pregnant, is not included in this category. increased risk for subsequent development of frank diabetes. After the child birth, the women should be re-assessed and accordingly classified as having either diabetes mellitus or normal glucose tolerance. Fasting and 2-hr values are normal. Exaggerated rise in blood glucose following the ingestion of glucose is seen One or two urine samples may give a positive Benedict's test. Increased rate of absorption of glucose from the intestine. Also seen in hyperthyroidism . Alimentary Glucosuria

38. Lowering of renal threshold. Glucose tolerance is normal. Blood sugar levels are within normal limits. Low renal threshold Physiological in pregnancy ; harmless Renal Glucosuria Reducing Substances in Urine detected by Benedict's test . Blue colour = absence of sugar sugar Green = 0.5% (+ ) Yellow = 1%; (1%=1 g per 100 ml). (++ ) Orange = 1.5% (+++ ) Red = 2% or more of sugar( ++++ )

39. Following are the results of glucose tolerance test (GTT). Interpret the results. Time (in hours) Blood glucose (mg%) Urine sugar(Benedict’s test) 0 (fasting) 80 Blue 0.5 120 Blue 1.0 140 Blue 1.5 100 Blue 2.0 65 Blue 2.5 75 Blue

40. Following are the results of oral GTT performed with a 50-year-old. Interpret the result.

41. Time (in hours) Blood glucose (mg%) Urine sugar(Benedict’s test) 0 (fasting) 80 Blue 0.5 125 Yellow 1.0 135 Orange 1.5 105 Greenish yellow 2.0 85 Green 2.5 80 Blue

42. Following are the results of oral GTT performed with a 55-year old person. Interpret the results. Time (in hours) Blood glucose (mg%) Urine sugar(Benedict’s test) 0 (fasting) 200 Greenish yellow 0.5 275 Orange 1.0 320 Brick red 1.5 340 Brick red 2.0 300 Brick red 2.5 260 Orange