Amino Acids – Nitrogen Disposal (01/05/2021) - Dr. Fahad Abdul Razak - PDF

(004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021...

(004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 of jaundice. OUTLINE Drug sample-may be blood or urine, but mostly urine specimen I. Case Study Blood alcohol level- should be less than.1% (depending on the country) II. Discussion CBG- Adults must be 70-80 (mostly 80) A. Steps in Amino Acid Disposal What was likely the precipitating factor of the patient’s 1. Transamination syndrome? 2. Glutamate Dehydrogenase Answer: Increased Nitrogen load precipitated by Upper GI Bleeding 3. Transport of Ammonia to the Liver (UGIB) III. Urea Cycle What is Asterexis? IV. Clinical Correlations Answer: Asterexis/ Flexing Tremor/ Liver Flap is characterized by A. Cirrhosis NON-rhythmic, asymmetric tremor with loss of voluntary control of extremities while in sustained position. B. Mechanism C. Laboratory (On PE, wrists can NOT be maintained in extension). This is D. Treatment checked by applying pressure while the wrist is extended. V. Other Metabolic Disorders Involving Urea Cycle Abnormal or uncontrolled jerking of the hand. In Bates’, it’s A. Hyperammonemia Type 1 referring to hepatic disease or liver cirrhosis. It is otherwise B. Hyperammonemia Type 2 known as flexing tremor or liver flap. C. Citrullinemia What is most likely the cause of the patient’s symptoms? D. Argininosucciniaciduria Answer: Hepatic Encephalopathy probably secondary to elevated E. Hyperargininemia Ammonia levels and history of liver cirrhosis. VI. Test Yourself VII. References II. DISCUSSION Amino Acids contain an amino group as part of their structure I. CASE STUDY The presence of the α-amino group keeps amino acids A 50-year old man with liver cirrhosis was brought to the safely locked away from oxidative breakdown. emergency room due to altered mental status, Recent history of the In order for the Carbons in Amino Acids to enter into the patient revealed that he had been disoriented and confused for the energy generating metabolic pathways, the Amino Group past few days with episodes of hematemesis and nausea. On must first be removed so they can be detoxified and physical examination, the patient was disoriented with icteric sclerae. excreted Abdomen was globular/distended with a note of fluid wave. Removing the α-amino group is essential for producing Hyperreflexia and Asterixis were apparent on neurological energy from any amino acid and is an obligatory step in the examination. Pertinent History: known history of Liver Cirrhosis. catabolism of all amino acids. Illicit drugs were NOT detected in urine and Blood Alcohol The Amino Acid is secreted mainly as Urea, but some are Levels were within normal range. No hypoglycemia was noted. also excreted as Free Ammonia in order to buffer urine However, Blood Ammonia levels noted to be elevated. 50 y/o male Chief complaint: altered mental status PE: General Status: Disoriented HEENT: Icteric Sclera Abdomen: Globular/Distended with noted fluid wave Neuro: (+) Hyperreflexia, (+) Asterixis Diagnostics results: NO illicit drugs noted in urine Blood Alcohol Levels: Normal CBG: Normal range Blood Ammonia: elevated Figure 1. Summary of Amino Acid catabolism Hematemesis- vomiting of blood Icteric sclerae – the white of the eyes are yellow. Usually a sign Page 1 of 8 CMED 1F  (004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 A. STEPS IN AMINO ACID DISPOSAL 1. Transamination 2. Glutamate Dehydrogenase 3. Transport of Ammonia to the Liver 1. Transamination Funneling of Amino Acid groups to Glutamate Catalyzed by Aminotransferase (AST/SGOT and ALT/SGPT) o Aminotransferase: (also called Transaminases) are found in the cytosol and mitochondria Co-factor: Pyridoxal Phosphate (derived from Vitamin B6) Figure 3. Reactions catalyzed during Amino acid catabolism (A) Alanine Reactions are reversible (depends on the concentration) Aminotransferase (ALT); (B) Aspartate Aminotransferase (AST) and can be used in the synthesis or the degradation of amino acids 2. Glutamate Dehydrogenase Net amino acid still the same Oxidative Deamination of Amino Acids (The amount of ammonia does not decrease in this step. Mainly done in Kidneys and Liver The aminotransferase means moving your amino group Reversible and depends on the concentration of reactants from one amino acid to another, and mostly for this first (glutamate, α-ketoglutarate, and ammonia) and the ratio of step is toward your glutamate.) oxidized to reduced coenzymes Most amino acids participate in transamination reactions Glutamate dehydrogenase requires either NAD+ or NADP+ except Lysine and Threonine (lose α-amino groups by as coenzyme deamination) Formation of Free Ammonia Glutamate is the product from aminotransferase (first step). Dehydrogenase will remove the hydrogen group, so ammonium ion is released, and α-ketoglutarate is formed. Oxidative deamination is adding oxygen and removing an amino group. Glutamate transfers nitrogen by transamination reactions to α-keto acids to form their corresponding α-amino acids. Guanosine triphosphate (GTP) is an allosteric inhibitor of glutamate dehydrogenase, whereas ADP is an activator. Figure 2. Aminotransferase reaction using α-Ketoglutarate as the amino group acceptor. Figure 4. Oxidative Deamination by Glutamate Dehydrogenase 3. Transport of Ammonia to the Liver Ammonia is produced by almost all cells of the body Ammonia in the blood is usually toxic, particularly to the CNS. So, it is processed mostly in the liver. Page 2 of 8 CMED 1F  (004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 Ammonia travels to the liver, mainly in the form of alanine But for muscles, there is glucose-alanine pathway. This and glutamine. involves transamination of pyruvate to form alanine. Also produced by bacteria in the gut and travels to the liver The ALT will convert the alanine into glutamate, but if there is a need to transfer amino acids into the liver, the glutamate in the via the hepatic portal vein. muscle is converted. Once energy is used back into alanine, Only the liver has the capability to convert it to Urea that alanine will be transported into the blood back to the liver to be reused again. ALT is present both in the muscle and in the liver. In most tissues of the body, ammonia is transported in the form of glutamine. For the muscles, ammonia is transported in the form of glutamine and alanine. When muscles use alanine, it is transferred more frequently. In the liver, the alanine will be converted back to glutamate, the glutamate dehydrogenase will remove the ammonia group then the ammonia will be converted to urea through the urea cycle. III. UREA CYCLE Figure 5. Transport of Ammonia to liver from Peripheral Tissues Figure 6. Urea Cycle The first 2 reactions are done in the mitochondria. Others are Alanine and glutamine are the two ways to transport the done in the cytoplasm of the liver. ammonia across the body. Ammonium is added to bicarbonate. In other books, ammonia Glutamine synthetase (found in most tissues) converts plus carbon dioxide. glutamate to glutamine by adding extra ammonia to the glutamate. The first step is conversion of ornithine to citrulline by ornithine -The difference between glutamate and glutamine is that transcarbamoylase through the donation of a phosphate group glutamate (glutamic acid) has an oxygen group, or or inorganic phosphate from carbamoyl phosphate. The carboxyl group at the end, while glutamine usually has an carbamoyl portion of carbamoyl phosphate is transferred to extra ammonium group. ornithine transcarbamoylase (OTC) as the high-energy -Once in glutamine form, it will be transferred to the blood phosphate released as inorganic phosphate. Ornithine is towards the liver. Once in the liver, glutaminase will regenerated with each turn of the urea cycle (the same way convert it back to glutamate to be reused again and it will that oxaloacetate is regenerated by reactions of the citric acid separate ammonia to be converted to urea. cycle). NOTE that the FORMATION OF CARBAMOYL Page 3 of 8 CMED 1F  (004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 PHOSPHATE by carbamoyl phosphate synthetase I (CPS I) is driven by cleavage of two molecules of ATP (one of which serves as a phosphate donor and conversion of the second Summary of urea cycle: ATP to AMP and pyrophosphate, coupled to the hydrolysis of Net: ammonia + bicarbonate + one group of aspartate + pyrophosphate to orthophosphate which provides the driving 3 ATP will yield 1 urea, 1 fumarate , 2 ADP , 1 AMP and 4 inorganic phosphate force for synthesis of the amide bond and the mixed acid anhydride bond of carbamoyl phosphate). Ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial glutamate dehydrogenase. Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogen of urea. ALSO, Carbamoyl phosphate synthase I is the rate-limiting enzyme in urea cycle. ONLY ACTIVE in the presence of N- acetylglutamate (which is an allosteric activator that enhances the affinity of the synthase for ATP). Citrulline will exit from the mitochondria to the cytoplasm. Through various reactions, aspartate will be converted to argininosuccinate, then arginine, and finally to urea. Arginosuccinate synthetase combines citrulline with aspartate to form arginosuccinate. The α-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation arginosuccinate is driven by the cleavage of ATP to adenosine monophosphate (AMP) and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea. Arginosuccinate is cleaved by arginosuccinate lyase to yield arginine and fumarate. The arginine formed by this reaction Figure 7. Links between the urea cycle and citric acid cycle. serves as the immediate precursor of urea. Fumarate produced in the urea cycle is hydrated to malate, providing a link with several metabolic pathways. For example, the malate can be IV. CLINICAL CORRELATIONS transported into the mitochondria via the malate-aspartate shuttle, reenter the tricarboxylic acid cycle, and get oxidized to A. Cirrhosis oxaloacetate, which can be used for gluconeogenesis. -Chronic condition of the liver with diffuse parenchymal damage and regeneration leading to: Arginase hydrolyzes arginine to ornithine and urea and is Ο Distortion of liver architecture virtually exclusive to the liver. Therefore, ONLY THE LIVER Ο Increased resistance of blood flow through the liver CAN CLEAVE ARGININE, thereby synthesizing urea, whereas other tissues, such as kidney, can synthesize arginine by these reactions. Urea will be sent to kidneys where it will be excreted in the form of urine. Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered and excreted in the urine. A portion of the urea diffuses from the blood into the intestine and is cleaved to CO2 and NH3 by bacterial urease. This ammonia is partly lost in the feces and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from the blood into the gut. The intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in the number of intestinal bacteria responsible for the NH3 production. Figure 8. Comparison between Normal Liver and Liver Cirrhosis. Page 4 of 8 CMED 1F  (004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 When the liver is damaged, extra friable blood vessels are produced. These will increase resistance from the liver and shift it more towards systemic circulation. When that happens, instead of filtering the amino content in the body, ammonia starts to accumulate because ammonia is not yet converted to urea. Clinical manifestations include: -Malaise -Lethargy -Palmar erythema -Ascites -Jaundice (The first location that it is usually apparent is under the tongue, then eyes, and followed by the skin) -Hepatic encephalopathy in the late stages (Toxins accumulating in the bloodstream affecting the patient’s Figure 9. Systemic clinical manifestations of liver cirrhosis. mental status) When the liver is not able to detoxify toxins, it will lead to damaged liver. The common cause of liver damage and build up of ammonia, is usually toxins in the body. Acquired hyperammonemia: liver disease is a common Most common etiologies of cirrhosis are: cause of hyperammonemia in adults and may be due -Toxins such as alcohol, viral infections (eg. Hepatitis B to, for example, to viral hepatitis or to hepatotoxins such or C infection), metabolic diseases in children (e.g Wilson’s disease, hemochromatosis, or a1-antitrypsin deficiency) as alcohol. Cirrhosis of the liver may result in formation of collateral circulation around the liver. As a result, portal B. Mechanism blood is shunted directly into the systemic circulation and does not have access to the liver. Therefore, the -Normal ammonia: 5-50 umol/L conversion of ammonia to urea is severely impaired, -Hyperammonemia (caused by inability of the leading to elevated levels of ammonia. liver to detoxify ammonia in the body) -Exacerbated by increased ammonia load such as Congenital hyperammonemia: Genetic deficiencies of gastrointestinal bleeding each of the five enzymes of the urea cycle, with an -When there is gastrointestinal bleeding, the ammonia from overall incidence of estimated 1:25,000 live births. X- the bleeding gets reabsorbed into the body through linked ornithine transcarbamoylase deficiency is the normal pathways of absorption. It is like digesting the blood and reabsorbing the ammonia. most common of these disorders, predominantly affecting males, although female carriers may become symptomatic. All of the other urea cycle disorders follow an autosomal recessive inheritance pattern. In each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth. Page 5 of 8 CMED 1F  (004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 D. Treatment -treatment varies depending on etiology -Common therapies include: Ο Avoidance of liver toxins Ο Salt restriction Ο Procedures to reduce portal pressure Ο Give medication to increase ammonia excretion such as lactulose (given in hepatic encephalopathy) V. OTHER METABOLIC DISORDERS INVOLVING Figure 10. Summary of blood ammonia catabolism. Blood ammonia levels rise, UREA CYCLE NH3 builds up inside the cells and drives the GDH reaction to form Glutamate, thus depleting a-Ketoglutarate -> decreased energy production in Kreb’s cycle. A. Hyperammonemia Type 1 – a consequence of carbamoyl phosphate synthase I deficiency, this -When there is a lot of ammonia, this will shift the relatively infrequent condition (estimated frequency reaction backwards. 1:62,000) probably is familial. -Instead of producing alpha-ketoglutarate using glutamate dehydrogenase, glutamate is produced instead. B. Hyperammonemia Type 2 – a deficiency of ornithine -Without the production of alpha-ketoglutarate, there will be transcarbamoylase produces this X chromosome-linked less alpha-ketoglutarate that will enter the Kreb’s cycle. deficiency. The mothers also exhibited -This would result in less energy and would facilitate other hyperammonemia and an aversion to high-protein foods, symptoms like malaise and decreased mental status. cerebrospinal fluid, and urine, probably due to enhanced -It will also decrease the amount of energy in the cells, glutamine synthesis in response to elevated levels of which may lead to cell death. tissue ammonia. C. Laboratory C. Citrullinemia – rare disorder, plasma and cerebrospinal fluid citrulline levels are elevated and 1-2g of citrulline -ALT is more specific for acute hepatic injury are excreted daily. One patient lacked detectable -AST is more sensitive for acute hepatic injury probably arginosuccinate synthase activity. In another, the Km for due to increased amount in liver citrulline was 25 times higher than normal. Citrulline and - ALT (alanine aminotransferase) is abundant arginosuccinate, which contains nitrogen destined for across the body. ALT is also present in the muscles. urea synthesis, serve as alternative carriers of excess Even if you have acute MI (myocardial infarction) nitrogen. Feeding arginine enhanced excretion of or heart attack, there might be an increase in ALT. citrulline in these patients. Similarly, feeding benzoate -Meanwhile, AST is more sensitive to liver damage diverts ammonia nitrogen to Hippurate via glycine. because it is more abundant in the liver. D. Argininosucciniaciduria – rare characterized by Normal values for ALT and AST:7-41 units per liter. elevated levels of argininosuccinate in blood, -Slight elevation is usually irrelevant cerebrospinal fluid, and urine is associated with friable, -It becomes clinically significant when the values tufted hair (trichorrhexis nodosa). Both early-onset types are 4 times elevated. Anything around 150-160 are known. The metabolic defect is absence of units per liter would be clinically significant. argininosuccinase. Diagnosis by measurement activity can be performed on umbilical cord or amniotic fluid These enzymes are measured thru serum once the liver cells. As for citrullinemia, feeding arginine and benzoate is damaged. Usually, when the liver is damaged, these promotes nitrogen excretion. enzymes are leaked into the blood. E. Hyperargininemia – characterized by elevated blood When liver cirrhosis gets worse, the liver will run out of these enzymes. For chronic liver disease, do not expect and cerebrospinal fluid arginine levels, low erythrocyte the ALT and AST to be very high. It may either be normal levels of arginase, and a urinary amino acid pattern or even decreased eventually. resembling that of lysine-cystinuria. This pattern may reflect competition by arginine with lysine and cystine for reabsorption in the renal tubule. A low-protein diet lowers plasma ammonia levels and abolishes lysine- cystinuria. Page 6 of 8 CMED 1F  (004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 VI. TEST YOURSELF a) Carbamoyl phosphate synthetase I b) Ornithine transcarbamoylase 1. Urea production occurs almost exclusively in ___________ c) Argininosuccinate synthetase a) Kidneys d) Argininosuccinate lyase b) Liver e) Arginase c) Blood d) Urine 2. Which is the first amino group entering into urea cycle? a) carbamoyl phosphate b) Ornithine c) Citrulline d) Argininosuccinate 3. The first enzyme in the pathway carbamoyl phosphate synthase I, is allosterically activated by ___________ a) N-acetyl glutamate b) Acetyl coA c) Glutamate d) Carbamoyl phosphate 4. A second amino group is transferred to citrulline from ___________ a) Aspartate b) Glutamate c) Alanine d) Guanine 5. In liver disease, the enzymes AST and ALT leak into the blood from damaged liver cells. Both of these enzymes have which one of the following in common? a) They both transfer ammonia to α-keto acids to form amino acids. b) They both form intermediates of glycolysis from amino acids. c) They both require thiamine pyrophosphate as a cofactor. d) They both catalyze irreversible reactions. e) They both convert α-ketoglutarate to glutamate. 6. Which one of the following occurs in the urea cycle? a) Carbamoyl phosphate is derived directly from glutamine and CO2. b) Ornithine reacts with aspartate to generate argininosuccinate. c) The α-amino group of arginine forms one of the nitrogens of urea. d) Ornithine directly reacts with carbamoyl phosphate to form citrulline. e) N-acetylglutamate is a positive allosteric effector of ornithine transcarbamoylase. 7. An infant who appeared normal at birth began to develop lethargy, hypothermia, and apnea within 24 hours. An analysis of blood components indicated high levels of ammonia and citrulline, and low levels of urea. The most likely defective enzyme in this child is which one of the following? Page 7 of 8 CMED 1F  (004) AMINO ACIDS – NITROGEN DISPOSAL DR. FAHAD ABDUL RAZAK | 01/05/2021 Answers: VII. REFERENCES 1. B (Explanation: Urea production occurs almost exclusively 1. Biochemistry by Lehninger, 6th Edition in the liver and is the fate of most of the ammonia channeled there. 2. Harper’s Illustrated Biochemistry, 26th Edition The urea passes into the bloodstream and thus to the kidneys and is 3. Lippincott’s Illustrated Reviews Biochemistry, 6th Edition excreted into the urine). 2. A (Explanation: The carbamoyl phosphate, which functions as an activated carbamoyl group donor enters the urea cycle). 3. A (Explanation: The first enzyme in the pathway carbamoyl phosphate synthase I, is allosterically activated by N-acetyl glutamate). 4. A (Explanation: The second amino group enters from aspartate generated in mitochondria by transamination and transported into the cytosol). 5. E. (Explanation: These transaminases convert amino acids to their corresponding α-keto acids in reactions that are readily reversible. α-Ketoglutarate and glutamate serve as the other α-keto acid/amino acid pair. Pyruvate (the end product of glycolysis) is the α-keto acid corresponding to alanine, and oxaloacetate (an intermediate of the TCA cycle) is the partner of aspartate. PLP is the cofactor. Thus, AST will convert aspartate and α-ketoglutarate to oxaloacetate and glutamate, and ALT will convert alanine and α- ketoglutarate to pyruvate and glutamate). 6. D. (Explanation: Carbamoyl phosphate within the mitochondria is formed from NH4+, CO2, and ATP. Carbamoyl phosphate synthetase II catalyzes carbamoyl phosphate synthesis from glutamine for pyrimidine synthesis in the cytoplasm. Carbamoyl phosphate reacts with ornithine to form citrulline, which reacts with aspartate to form argininosuccinate. Fumarate is released from argininosuccinate, and arginine is formed. Urea is produced from the guanidinium group on the side chain of arginine, not from the amino group on the α-carbon. Ornithine is regenerated. N-Acetylglutamate is an allosteric activator of carbamoyl phosphate synthetase I. Ornithine transcarbamoylase is not a regulated enzyme in mammals, and in bacteria N-acetylglutamate is not an allosteric effector of ornithine transcarbamoylase. 7. C. (Explanation: The patient has a defect in argininosuccinate synthetase. Citrulline, the substrate for the reaction, accumulates and can be measured in the blood. A carbamoyl phosphate synthetase I deficiency would block carbamoyl phosphate formation, and citrulline would neither be synthesized nor accumulated. An ornithine transcarbamoylase deficiency would lead to orotic acid accumulation (carbamoyl phosphate made in the mitochondria would diffuse into the cytoplasm, thereby activating pyrimidine synthesis and overproducing ortoic acid). An argininosuccinate lyase deficiency would lead to elevated argininosuccinate, which is not observed, and an arginase deficiency would lead to elevated arginine, which was also not observed. Defects in argininosuccinate lyase and arginase also do not have as elevated ammonia levels as do defects in previous enzymes of the cycle, since two nitrogens have been disposed of in the synthesis of argininosuccinate. Page 8 of 8 CMED 1F