MBBS UREA CYCLE, STEPS, CLINICAL ASPECTS

Urea cycle for MBBS/BDS students

1. UREA CYCLE, HYPERAMMONEMIA Dr. Apeksha N i raula Assistant Professor Department of Biochemistry Institute of Medicine TUTH

2. OBJECTIVES Transamination Deamination Reactions (Ammonia Formation) Ammonia Transport Disposal of Ammonia – Urea cycle H y perammonemia

3. Proteins are linear hetero polymers of Lα – Amino acids, which are linked by peptide bonds Nitrogen (N) is characteristic of proteins Amino acids are not stored by the body Hence, they must be obtained from the diet, synthesized de novo, or produced from normal protein degradation Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded

4. Amino Acids are classified in 3 categories: Essential (Can’t be synthesized in body) Non-Essential (Can be synthesized in body) Semi-Essential Total amino acids are 20 Essential amino acids are 10

5. Body contains 100g of protein present freely inside the body Amino Acid Pool Amino acids of pool are in dynamic state These participate in different metabolic reactions occurring in the body AMINO ACID POOL

6. 50% of the pool is formed of : Glutamate Glutamine 10% of pool is formed of Essential amino acids: SOURCES: Protein turnover Ingestion

7. PROTEIN TURNOVER: The amount of protein degraded and synthesized constantly in the body is called as protein turnover (300-400g) Factors: Ubiquitin tags a protein and increase its degradation Amino acid sequence of Proline, Glutamine, Serine and Threonine increases the degradation Above factors are responsible for affecting Protein turnover

8.  GENERAL ASPECTS OF AMINO ACID METABOLISM Amino acids undergo 2 steps along metabolism These are: Transamination Deamination

9. The transfer of an amino group (-NH2 ) from an amino acid to a keto acid is known as Transamination F eatures: The reaction is catalyzed by transaminases All reactions require PLP derivative of B6 No free NH3 is liberated, just transfer occurs Nitrogen is finally concentrated in Glutamate Only Glutamate undergoes oxidative deamination to liberate ammonia. Serum transaminases can be used for diagnostic and prognostic purposes TRANSAMINATION

10. The removal of amino group as NH3 is called as Deamination Mostly it occurs in: Liver Kidneys DEAMINATION

11.  Deamination is of 2 types: Oxidative deamination (coupled with oxidation) Non-oxidative deamination Effect of Protein Rich Meal: After ingestion of protein rich meal glutamate level is increased, which leads to increased levels of NH3 in body

12.  Ammonia originates in the catabolism of amino acids that are primarily produced by the degradation of proteins – dietary as well as existing within the cell Other sources of Ammonia: Bacterial degradation of urea in the intestinal lumen Action of renal glutaminase on glutamine in renal tubular cells Action of intestinal glutaminase on glutamine in intestinal mucosal cells Release of amino groups of purines and pyrimidines as ammonia during catabolism of these nitrogen bases Ammonia Production

13. Elimination of Ammonia Ammonia is toxic, especially for the CNS, because it reacts with α- ketoglutarate, thus making it limiting for the Citric acid cycle decrease in the ATP level Liver damage or metabolic disorders associated with elevated ammonia can lead to tremor, slurred speech, blurred vision, coma, and death Normal concentration of ammonia in blood: 30-60 µM

14. 1. Decreased glucose utilization and ATP generation: In the brain cell mitochondria, excess ammonia may cause the reductive amination of -ketoglutarate by glutamate dehydrogenase, forming glutamate depletes 𝝰-ketoglutarate, a key intermediate of the TCA cycle, and leads to its impairment As a result, there is severe inhibition of glucose utilization and a fall of ATP generation [Concept not universally accepted] Glutamate depletion: Ammonia exerts an inhibitory effect on the activity of glutaminase, resulting in the depletion of glutamate, an excitatory neurotransmitter, in the neuronal cells Glutamine, synthesized and stored in glial cells, is the most likely precursor of glutamate (it is transported into the neurons and hydrolyzed by glutaminase) Biochemical Basis of Ammonia Toxicity

15. Ammonia inhibits glutaminase and depletes the glutamate concentration Moreover, intracellular accumulation of glutamine causes osmotic shifts of water into the cell, resulting in edema and swelling of astrocytes This may aggravate the encephalopathy of hyperammonaemia 3. Neuronal dysfunction: Hyperammon em ia increases the permeability of the neuronal membrane to K + and Cl – ions to cause neuronal dysfunction 4. Accumulation of excitotoxins: Increased transport of tryptophan across the blood-brain barrier and accumulation of its metabolites, which are excitotoxins, are also implicated in ammonia toxicity Two of such tryptophan-derived metabolites are serotonin and quinolinic acid.

16. Urea Cycle The urea cycle (also known as the Ornithine cycle ) Liver: Only organ where urea synthesis occurs Discovered by Hans Krebs and Kurt Henseleit, 1932 Urea cycle was the first cyclic pathway to be identified Cycle of biochemical reactions occurring in many animals that produces urea (NH2) 2 CO from ammonia (NH 3 )

17. Major excretory product in humans, accounting for an average of 86% of nitrogen eliminated The rest of the nitrogen is eliminated as follows: 4.5% by creatinine , 2.8% as ammonium ions , 1.7% as uric acid , and 5.0% as other compounds About 30 g urea is excreted per day ; the amount excreted is dependent on protein intake Higher the protein intake, more is the urea synthesis and excretion

18. The first two reactions take place in the Mitochondria , and the rest occur in Cytosol Formation of Carbamoyl Phosphate [ CPS-I ] Synthesis of Citrulline [ ORNITHINE TRANSCARBAMOYLASE ] Synthesis of Argininosuccinate [ ARGINOSUCCINATE SYNTHETASE ] Cleavage of Argininosuccinate [ ARGINOSUCCINASE ] Cleavage of Arginine [ ARGINASE ]

19. Reaction 1: Formation of Carbomyl Phosphate Carbamoyl phosphate is formed from ammonia and carbon dioxide in an energy-requiring reaction Two ATP molecules are required to drive this reaction forward The enzyme catalyzing this step, Carbamoyl phosphate synthetase-I (CPS-I) is rate-limiting for the pathway

20. It is present in very high concentration in liver mitochondria and its Km for ammonia (250 ℳM) is not much higher than the physiological ammonia concentration These properties enable the enzyme to effectively remove ammonia quantitatively from its environment N-Acetyl glutamate (NAG) is an obligatory positive effector of CPS-I

21. Difference between CPS- I and CPS-II

22. Reaction 2: Synthesis of Citrulline Carbamoyl phosphate, a high energy mixed anhydride, condenses with ornithine to form citrulline The reaction is catalyzed by a mitochondrial enzyme ornithine transcarbamoylase Citrulline diffuses out of the inner mitochondrial membrane so that the subsequent reactions of the urea cycle take place in the cytosol

23.  Reaction 3: Condensation of Citrulline with Aspartate This is a complex condensation reaction between citrulline and aspartate to form argininosuccinate Reaction is catalyzed by the enzyme argininosuccinate synthetase Free energy is required in this reaction which is provided by pyrophosphate cleavage of ATP Irreversible Reaction

24. The carbon skeleton of aspartate is released as fumarate while its nitrogen remains in the cycle, forming one of the nitrogen side chains of arginine Reaction is catalyzed by the enzyme argininosuccinate lyase (also known as argininosuccinase) Reaction 4: Cleavage of Argininosuccinate

25. In the last step, arginine is hydrolyzed by the enzyme arginase to form urea The other product of this reaction, ornithine, enters the mitochondrial matrix to participate in the urea cycle again Reaction 5: Formation of Urea

26. Overall Equation of Urea Cycle

27. Synthesis of fumarate (reaction 4) is important because it links urea cycle to the citric acid cycle Fumarate is converted to malate which is in turn oxidized to oxaloacetate Oxaloacetate can condense with acetyl CoA to form citrate, the first intermediate of TCA cycle Urea cycle is linked to TCA cycle: Krebs Bicycle

28. The stoichiometry of urea synthesis is as below: Formation of one molecule of urea is powered by three ATPs and requires one molecule each of ammonia, carbon dioxide and aspartate In all, hydrolysis of four high energy phosphate groups is required in each cycle: two are needed to drive the formation of carbamoyl phosphate and two for the formation of argininosuccinate  Energetics of Ureagenesis

29. However, the net energy expenditure may fall to only one ATP if fumarate (formed in the fourth step) is converted to malate When this malate is oxidized to oxaloacetate, one NADH molecule is generated that can give rise to three ATP molecules through the electron transport chain Thus, the energy expenditure becomes one (4–3)= 1 ATP molecule for each molecule of urea formed

30. Coarse Regulation Occurs by induction-repression mechanism The urea cycle enzymes are induced or repressed depending on the metabolic needs of the body In starvation , urea cycle enzymes are induced; activities are elevated by 10–20-fold It permits increased formation of urea in response to increased catabolism of the proteins that occurs in starvation Moreover, cellular energy falls low in starvation, which activates the glutamate dehydrogenase This results in increased production of ammonia whic h is channeled into urea cycle A protein rich diet also accelerates urea cycle through the activation of rate limiting enzyme, carbamoyl-phosphate synthetase Control of Urea Cycle

31. Occurs by Allosteric-modulation Major regulatory enzyme of the urea cycle is Carbamoyl phosphate synthetase-I , which is subject to allosteric activation by N-acetyl glutamate (NAG) Transfer of the acetyl group from acetyl CoA to glutamate by the enzyme NAG synthase (NAGS) forms this compound NAG synthase is under positive allosteric modulation by arginine and product inhibition by NAG Fine Regulation

32. A high glutamate level also leads to increased NAG synthesis; this situation occur when more amino acids are degraded High glutamate level leads to increased NAG, hence enhanced activity of CPS-I, and thereby increased rate of ureagenesis

33.  Synthesis of urea provides the major route for the removal of toxic ammonia from body Blockage of any of the steps of urea synthesis, therefore, results in accumulation of ammonia in the blood; the condition is known as hyperammonaemias Can be due to genetic deficiency of an enzyme of the urea cycle (i.e. familial hyperammonaemia) or due to some acquired defect (i.e. acquired hyperammonaemia) Defects of Urea Cycle

34. Genetic deficiency of each of the five enzymes of urea cycle have been described with an overall prevalence of 1: 30,000 live births Hyperammonemia type I (Carbamoyl Phosphate Synthetase) Hyperammonemia type II (Ornithine Transcarbamoylase) – X-linked Citrullinemia (Argininosuccinate Synthetase) Argininosuccinic aciduria (Argininosuccinase) Hyperargininaemia (Arginase) Familial Hyperammonaemia

35. Seen in several disease processes, such as alcoholism, hepatitis or biliary cirrhosis, and is characterized by progressive loss of hepatocytes Acquired Hyperammonaemia

36. Detection of increased ammonia levels in blood and/or urine Glutamine level is also elevated because excess ammonia is diverted into glutamine synthesis The immediate substrate of the deficient enzyme also is elevated in blood and urine in familial hyperammonaemia Diagnosis of Hyperammonemia

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