Metabolism of Ammonia and Urea Cycle PDF

METABOLISM OF AMMONIA  NH3 TRANSPORT Overall pattern of N-removal from an L-amino acid  Ammonia is constantly being liberated in the metabolism of amino acids (mostly by transamination and deamination) and other nitrogenous compounds such as biogenic amines , amino group of purines and...

METABOLISM OF AMMONIA  NH3 TRANSPORT Overall pattern of N-removal from an L-amino acid  Ammonia is constantly being liberated in the metabolism of amino acids (mostly by transamination and deamination) and other nitrogenous compounds such as biogenic amines , amino group of purines and pyrimidines and by the action of intestinal bacteria (urease) on urea. Biogenic amines (five established biogenic amine neurotransmitters: the three catecholamines—dopamine, norepinephrine (noradrenaline), and epinephrine (adrenaline)—and histamine and serotonin).  NH3 formed in the tissues, a considerable quantity of NH3 is produced in the gut by intestinal bacterial flora, both 1. From dietary proteins, and 2. From urea present in fluids secreted into the GI tract. At the physiological pH, ammonia exists as ammonium (NH+4) ion. The portal vein or hepatic portal vein (HPV) is a blood vessel that carries blood from the gastrointestinal tract, gallbladder, pancreas and spleen to the liver. This NH3 is absorbed from the intestine into portal venous blood which contains relatively high concentration of NH3 as compared to systemic blood. Liver promptly removes the NH3 from the portal blood, so that blood leaving the liver is virtually NH3-free. This is essential since even small quantities of NH3 are toxic to CNS.  CLINICAL SIGNIFICANCE Severely impaired hepatic function or the development of collateral communications between portal and systemic veins as may occur in cirrhosis Liver, the portal blood may bypass the liver. Surgically produced shunting procedures socalled Eck-fistula or other forms of portocaval shunts are conducive to NH3 intoxication. CLINICAL ASPECT Hyperammonaeia Acquired Inherited hyperammonaeia hyperammonaeia  Hyperammonaeia Hyperammonaemia is associated with comatose states such as may occur in hepatic failure and mental retardation. Comatose states: A period of prolonged unconsciousness brought on by illness or injury or hepatic failure. Where a person is unresponsive and cannot be woken. Glasgow Coma Scale (GCS)  Acquired hyperammonaem ia It is usually the result of cirrhosis of the Liver with the development of a collateral circulation, which shunts the portal blood around the organ, thereby severely reducing the synthesis of urea.  Inherited hyperammonaeia Results from genetic defects in the urea cycle enzymes. Normal Blood Ammonia Level: In man, normal blood level of NH3 varies from 40 to 70 μg/100 ml. Free NH+4 (ammonium ion) concentration of fresh plasma is less than 20 μg per 100 ml.  The symptoms of NH3 intoxication include: Features of NH3 intoxication A peculiar flapping tremor (problem in muscle contraction Slurring of speech Blurring of vision In severe cases follows to coma and death. These resemble those of syndrome of hepatic coma, where blood and brain NH3 levels are elevated. flapping tremor  Why NH3 is Toxic? The cause of NH3 toxicity is not definitely known, Following associated biochemical changes are important. Increased NH3 concentration enhances amination of α-ketoglutarate, an intermediate in TCA cycle to form Glutamate in brain. This reduces mitochondrial pool of α-ketoglutarate ↓ consequently depressing the TCA cycle, affecting the cellular respiration.  Increased NH3 concentration enhances glutamine formation from Glutamate and thus reduces ‘braincell’ pool of glutamic acid. Hence there is decreased formation of inhibitory neurotransmitter GABA (γ-aminobutyric acid) ↓.  UREA FORMATION (KREBS-HENSELEIT CYCLE) Urea is the end product of protein metabolism (amino acid metabolism), in the tissues or from bacterial action in the gut is accomplished by the production of urea which is excreted in the urine. Urea is synthesized in liver and transported to kidneys for excretion in urine. Urea cycle is the first metabolic cycle that was elucidated by Hans Krebs and Kurt Henseleit (1932), hence it is known as Krebs- Henseleit cycle.  This cycle was described in more detail later on by Ratner and Cohen. The nitrogen of amino acids, converted to ammonia is toxic to the body. It is converted to urea and detoxified. Urea accounts for 80-90% of the nitrogen containing substances excreted in urine.  Urea has two amino ( NH2) groups, one derived from NH3 and the other from aspartate. Carbon atom is supplied by CO2. Present in the body  Intracellular Half of the body protein present in skeletal and connective tissues. Other half intracellular.  Each day human turnover 1-2% of their total body protein, principally the muscle protein  Proteins/Amino acids are nitrogen containing compound and major source of nitrogenous waste of our body and highly toxic for body.  Nitrogen present in form of amino group, nitrogenous waste taken to the liver and converted to less toxic water soluble compound UREA.  Transfer of an amino group Amino group of AA. Which contains nitrogen taken from AA and to transfer to some other molecules, can be Alanine or most commonly Alpha-Ketoglutarate and converted to Glutamate. Glutamate can freely circulate to blood carrying excess nitrogenous waste with it. This is the 1st step in urea cycle  Glutamate (only) reaches to Liver and under goes to Oxidative deamination (removal of amino group) to liberate the free amonia to urea synthesis.  Reactions of Urea Cycle Urea synthesis is a five-step cyclic process, with five distinct enzymes. The first two enzymes are present in mitochondria while the rest are localized in cytosol.  Location of enzymes It is partly mitochondrial and partly cytosolic. One mol. of NH3 and one mol. of CO2 are converted to one mol. of urea for each turn of the cycle and orinithine is regenerated at the end The overall process in each turn of cycle requires 3 mols of ATP.  Stages of Urea cycle The reactions of urea cycle can be studied in five sequential enzymatic reactions. Reaction 1: Synthesis of carbamoyl-phosphate. Reaction 2: Synthesis of citrulline. Reaction 3: Synthesis of argininosuccinate. Reaction 4: Cleavage of argininosuccinate. Reaction 5: Cleavage of arginine to form ornithine and urea. eaction 1: Synthesis of Carbamoyl-P (Mitochondri (N-Acetylglutamate) Mitochondrial carbamoyl phosphate synthetase I catalyses the ATP-dependant conversion to carbamoyl phosphate. Role of N-acetyl Glutamate (AGA): Exact role of N-acetyl glutamate is not known. Its presence brings about some conformational changes in the enzyme molecule and affects the affinity of the enzyme for ATP.  Reaction 2: Synthesis of Citrulline: (Mitochondrial) Ornithine has 2 nitrogen containing group as amino acid but not involved in protein synthesis. Ornithine serve as carrier and picks up ammonia from carbomyl phosphate and form bigger molecule known as citruline. One molecule of Inorganic phosphate is released.  The citrulline then leaves the mitochondria to enter the cytoplasm for rest of the Urea cycle. Citrulline goes in few more reactions to produce the urea and liberates back the ornithine, and then moves to mitochondria for further cycle. Reaction 3: Synthesis of Argininosuccinate: (cytosolic) Citrulline get condensed with aspartate and produces the argininosuccinate in an ATP-dependant reaction catalysed by argininosuccinate synthetase and produces AMP and release IP. Aspartate is another amino acid which serve another amino group that get attached to citrulline.  Uptill now we are just adding the molecules one after another to produce the complex molecule argininosuccinate.  Reaction 4: Cleavage of Argininosuccinate (Cytosolic) Fate of fumarate: The fumarate is converted to oxaloacetate (OAA) via the fumarase and malate dehydrogenase reactions and then transaminated to regenerate aspartate to participate in the cycle. Reaction 5: Cleavage of Arginine to Ornithine and Urea mitochondria Arginine The last reaction of the urea cycle completes the cycle. It is catalysed by the enzyme arginase, which is found only in the liver cells. Arginase catalyses hydrolysis of the guanidine group of arginine, releasing urea and regenerating ornithine. Overview Interrelation between urea and tricarboxylic acid (TCA) cycle The production of fumarate in urea cycle is the most important integrating point with TCA cycle. Fumarate is converted to malate and then to oxaloacetate in TCA cycle. Oxaloacetate undergoes transamination to produce aspartate which enters urea The urea cycle is linked to the TCA cycle through the production of fumarate. Amino acid catabolism, is therefore directly coupled to energy production.  Characteristic Features It is a cyclic process, five reactions which involves ornithine, citrulline, aspartic acid and arginine. Liver in mammals and all of the enzymes involved have been isolated from Liver tissue. Kidneys: Urea cycle operates in a limited extent. Kidney can form up to ariginine but cannot form urea, as enzyme arginase is absent in kidney tissues. Brain: Brain can synthesize urea from citrulline, but lacks the enzyme for forming citrulline from ornithine. Thus, neither the kidneys nor the brain can form urea in significant amounts.  Overall reaction and energetics The urea cycle is irreversible and consumes 4ATP. Two ATP are utilized for the synthesis of carbamoyl phosphate. One ATP is converted to AMP and PPi to produce arginosuccinate which equals to 2 ATP.  Metabolic disorders of urea cycle Metabolic defects associated with each of the five enzymes of urea cycle have been reported. All the disorders invariably lead to a build-up in blood ammonia (hyperammonemia), leading to toxicity. The clinical symptoms associated with defect in urea cycle enzyme include vomiting, lethargy, irritability, ataxia and mental retardation.  Blood urea—clinical importance In healthy people, the normal blood urea concentration is 10-40 mg/dl. Higher protein intake marginally increases blood urea level. About 15-30 g of urea (7-15 g nitrogen) is excreted in urine per day. Blood urea estimation is widely used as a screening test for the evaluation of kidney (renal) function. Elevation in blood urea may be broadly classified into three categories.  Elevation in blood urea 1. Pre-renal 2. Renal 3. Post-renal  1. Pre-renal This is associated with increased protein breakdown, as observed after major surgery, prolonged fever, diabetic coma, thyrotoxicosis etc. In leukemia also, blood urea is elevated.  2. Renal In renal disorders like acute glomerulonephritis, chronic nephritis, nephrosclerosis, polycystic kidney, blood urea is increased.  3. Post-renal Whenever there is an obstruction in the urinary tract (e.g. tumors, stones, enlargement of prostate gland etc.), blood urea is elevated. This is due to increased reabsorption of urea from the renal tubules. The term ‘uremia’ is used to indicate increased blood urea levels due to renal failure. Azotemia represents an elevation in blood urea/or other nitrogen metabolites which may or may not be associated with renal diseases.