Nitrogen Metabolism 2022-1(1) PDF

PHARM 282 INTRO. BIOCHEMISTRY/MOLECULAR BIOLOGY II Protein and amino acid metabolism II - Nitrogen metabolism and the urea cycle INTRODUCTION Humans are totally dependent on other organisms for converting atmospheric nitrogen into forms available to the body Before atmos...

PHARM 282 INTRO. BIOCHEMISTRY/MOLECULAR BIOLOGY II Protein and amino acid metabolism II - Nitrogen metabolism and the urea cycle INTRODUCTION Humans are totally dependent on other organisms for converting atmospheric nitrogen into forms available to the body Before atmospheric nitrogen can be utilized by animals it must be ''fixed," that is, reduced from N2 to NH3 by microorganisms, plants, and electrical discharge from lightning – Nitrogen fixation is carried out by bacterial nitrogenases forming reduced nitrogen, NH4+ which can then be used by all organisms to form amino acids Reduced nitrogen enters the human body as – Dietary free amino acids, protein and the ammonia produced by GI bacteria Two principal enzymes glutamate dehydrogenase and glutamine synthetase are found in all organisms – effect the conversion of ammonia into the amino acids, glutamate and glutamine, respectively 2 Overview of the flow of nitrogen in the biosphere 3 Incorporation of Nitrogen into Amino Acids A healthy adult eating a varied and plentiful diet is generally in "nitrogen balance," a state where the amount of nitrogen ingested each day is balanced by the amount excreted – In the well fed condition, excreted nitrogen comes mostly from digestion of excess protein or from normal turnover Protein turnover is defined as the synthesis and degradation of protein Under some conditions the body is either in negative or positive nitrogen balance Nitrogen Balance = Nitrogen intake - Nitrogen loss In negative nitrogen balance more nitrogen is excreted than ingested – This occurs in starvation and certain diseases During starvation carbon chains of amino acids from proteins are needed for gluconeogenesis; ammonia released from amino acids is excreted mostly as urea and is not reincorporated into protein A negative nitrogen balance can be used as part of a clinical evaluation of4 malnutrition  A diet deficient in an essential amino acid also leads to a negative nitrogen balance, since body proteins are degraded to provide the deficient essential amino acid. Positive nitrogen balance – occurs in growing children, who are increasing their body weight and incorporating more amino acids into proteins than they break down – Positive nitrogen balance also occurs in pregnancy and during re- feeding after starvation 5 The Glutamate Deydrogenase Reaction The glutamate dehydrogenase utilizes both nicotinamide nucleotide cofactors; NAD+ in the direction of nitrogen liberation and NADP+ for nitrogen incorporation In the forward reaction glutamate dehydrogenase is important in converting free ammonia and α-KG to glutamate, forming one of the 20 amino acids required for protein synthesis However, it should be recognized that the reverse reaction is a key anapleurotic process linking amino acid metabolism with TCA cycle activity – In the reverse reaction, glutamate dehydrogenase provides an oxidizable carbon source used for the production of energy as well as a reduced electron carrier, NADH 6 Direction of reactions: The direction of the reaction depends on the relative concentrations of – Glutamate – α-ketoglutarate – Ammonia – the ratio of oxidized to reduced coenzymes For example, after ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation and the formation of ammonia – The reaction can also be used to synthesize amino acids from the corresponding α- ketoacids 7 AIIosteric regulators: ATP and GTP are allosteric inhibitors of glutamate dehydrogenase, whereas ADP and GDP are activators of the enzyme – Thus, when energy levels are low in the cell, amino acid degradation by glutamate dehydrogenase is high, facilitating energy production from the carbon skeletons derived from amino acids 8 D-Amino acid oxidase: D-Amino acids are found in plants and in the cell walls of microorganisms, but are not used in the synthesis of mammalian proteins – D-Amino acids are, however, present in the diet, and are efficiently metabolized by the liver – D-Amino acid oxidase is an FAD-dependent enzyme that catalyzes the oxidative deamination of these amino acid isomers – The resulting α-ketoacids can enter the general pathways of amino acid metabolism, and be reaminated to L-isomers, or catabolized for energy 9 The Glutamine Synthetase Reaction The reaction catalyzed by glutamine synthetase is: 10 Glutamine synthetase reaction is also important in several respects It produces glutamine, one of the 20 major amino acids In animals, glutamine is the major amino acid found in the circulatory system(~50 %) – Free ammonia is toxic and is preferentially transported in the blood in the form of amino or amide groups – Its role there is to carry ammonia to and from various tissues but principally from peripheral tissues to the kidney, where the amide nitrogen is hydrolyzed by the enzyme glutaminase; this process regenerates glutamate and free ammonium ion, which is excreted in the urine – Ammonia arising in peripheral tissue is carried in a non-ionizable form which has none of the neurotoxic or alkalosis-generating properties of free ammonia 11  Amino and amide groups from these 2 substances (glutamate and glutamine), are freely transferred to other carbon skeletons by transamination and transamidation reactions Therefore, glutamate plays the central role in nitrogen metabolism, serving as both a nitrogen donor and nitrogen acceptor 12 13 THE UREA CYCLE THE UREA CYCLE 14 OVERVIEW Urea is the major disposal form of amino groups derived from amino acids – accounts for about 80 – 90 % of the nitrogen-containing components of urine One nitrogen of the urea molecule is supplied by free NH3, and the other nitrogen by aspartate – Glutamate is the immediate precursor of both ammonia (through oxidative deamination by glutamate dehydrogenase) and aspartate nitrogen (through transamination of oxaloacetate by aspartate aminotransferase) The carbon and oxygen of urea are derived from CO2 The cycle starts and finishes with ornithine Urea is produced by the liver, and then is transported in the blood to the kidneys for excretion in the urine 15 Overview Ammonia (first nitrogen for urea) enters the cycle after condensation with bicarbonate to form carbamoyl phosphate which reacts with ornithine to form citrulline Aspartate (the donor of the second urea nitrogen) and citrulline react to form argininosuccinate, which is then cleaved to arginine and fumarate Arginine is hydrolyzed to urea and ornithine is regenerated – Urea is then transported to the kidney and excreted in urine The cycle requires 3 ATPs to excrete each two nitrogen atoms – It is therefore more energy efficient to incorporate ammonia into amino acids than to excrete it The major regulatory step is the initial synthesis of carbamoyl phosphate, and the cycle is also regulated by induction of the enzymes involved 16 17 The reactions of the urea cycle which occur in the mitochondrion are contained in the red rectangle. All enzymes are in red, CPS-I is carbamoyl phosphate synthetase-I, OTC is 18 ornithine transcarbamoylase Reactions of the cycle The series of reactions that form urea is known as the Urea Cycle or the Krebs-Henseleit Cycle First two reactions leading to the synthesis of urea occur in the mitochondria, whereas the remaining cycle enzymes are located in the cytosol 1. Formation of carbamoyl phosphate: – Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I (CPSI) is driven by cleavage of two molecules of ATP – 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 nitrogens of urea – CPS I requires N-acetylglutamate as a positive allosteric activator CPS II participates in the biosynthesis of pyrimidines and does not require N-acetyl-glutamate, and occurs in the cytosol 2. Formation of citrulline: – Carbamoyl phosphate reacts with ornithine to form citrulline, catalyzed by ornithine transcarbamoylase (OTC) – Ornithine and citrulline are basic amino acids that participate in the urea cycle They are not incorporated into cellular proteins, because there are no codons for these amino acids – Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the citric acid cycle – The release of the high-energy phosphate of carbamoyl phosphate as inorganic phosphate drives the reaction in the forward direction – Citrulline, is transported across the mitochondrial membranes in exchange for cytoplasmic ornithine and enters the cytosol 20 3. Synthesis of argininosuccinate: – Citrulline condenses with aspartate to form argininosuccinate – The α-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea – The formation of argininosuccinate is driven by the cleavage of ATP to AMP and pyrophosphate (PPi) This is the equivalent of hydrolysis of two molecules of ATP since PPi is irreversibly cleaved to 2 Pi This is the third and final molecule of ATP consumed in the formation of urea 4. Cleavage of argininosuccinate: – Argininosuccinate is cleaved to yield arginine and fumarate The arginine formed by this reaction 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 transported into the mitochondria via the malate shuttle and reenter the TCA cycle 21 22 5. Cleavage of arginine to ornithine and urea: – Arginase cleaves arginine to ornithine and urea, and occurs almost exclusively in the liver Thus, whereas other tissues, such as the kidney, can synthesize arginine by these reactions, only the liver can cleave arginine and, thereby, synthesize urea Ornithine reenters the mitochondrion for another turn of the cycle The inner mitochondrial membrane contains a citrulline/ornithine exchange transporter 6. Fate of urea: – 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 23  In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from 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 these patients Oral administration of neomycin reduces the number of intestinal bacteria responsible for this NH3 production The bacterial source of ammonia in the intestines can also be decreased by a compound that acidifies the colon, such as lactulose, a poorly absorbed synthetic disaccharide that is metabolized by colonic bacteria to acidic products This promotes excretion of ammonia in feces as protonated ammonium ions 24 25 Overall stoichiometry of the urea cycle Aspartate + NH3 + HCO3 - + 3ATP + H20= Urea + fumarate + 2ADP + AMP + 2 Pi +PPi + 2H+ – Therefore, the synthesis of urea is irreversible, with a large, negative ∆G – One nitrogen of the urea molecule is supplied by free NH3, and the other nitrogen by aspartate – Glutamate is the immediate precursor of both ammonia (through oxidative deamination by glutamate dehydrogenase) and aspartate nitrogen (through transamination of oxaloacetate by aspartate aminotransferase) In effect, both nitrogen atoms of urea arise from glutamate, which, in turn, gathers nitrogen from other amino acids 26 Regulation of the urea cycle In general, the urea cycle is regulated by substrate availability; the higher the rate of ammonia production, the higher is the rate of urea formation - feed-forward regulation N-acetylglutamate is an essential activator for carbamoyl phosphate synthetase I - the rate-limiting step in the urea cycle N-acetylglutamate is synthesized from acetyl CoA and glutamate in a reaction for which arginine is an activator Therefore, the intrahepatic concentration of N-acetylglutamate increases after ingestion of a protein-rich meal, which provides both the substrate (glutamate) and the regulator of N-acetylglutamate synthesis – The ability of a high-protein diet to increase urea cycle enzyme levels is another type of feed-forward regulation This leads to an increased rate of urea synthesis 27  The steady-state concentration of N-acetylglutamate is set by the concentration of its components: – acetyl-CoA – glutamate – arginine, which is a positive allosteric effector of N-acetylglutamate synthetase 28 METABOLISM OF AMMONIA METABOLISM OF AMMONIA 29 OVERVIEW Ammonia is produced by all tissues during the metabolism of a variety of compounds, and it is disposed off primarily by formation of urea in the liver However, the level of ammonia in the blood must be kept very low, because even slightly elevated concentrations (hyperammonemia) are toxic to the CNS There must, therefore, be a metabolic mechanism by which nitrogen is moved from peripheral tissues to the liver for ultimate disposal as urea, while at the same time low levels of circulating ammonia must be maintained 30 Sources of ammonia 1. From amino acids: – Many tissues, but particularly the liver, form ammonia from amino acids by the aminotransferase and glutamate dehydrogenase reactions previously described 2. From glutamine: – The kidneys form ammonia from glutamine by the action of renal glutaminase – Most of this ammonia is excreted into the urine as NH4+, which provides an important mechanism for maintaining the body's acid-base balance – Ammonia is also obtained from the hydrolysis of glutamine by intestinal glutaminase The intestinal mucosal cells obtain glutamine either from the blood or from digestion of dietary protein 31 3. From bacterial action in the intestine: – Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine – This ammonia is absorbed from the intestine by way of the portal vein and is almost quantitatively removed by the liver via conversion to urea 4. From amines: – Amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters, give rise to ammonia by the action of amine oxidase (eg. the degradation of catecholamines) 5. From purines and pyrimidines: – In the catabolism of purines and pyrimidines, amino groups attached to the rings are released as ammonia 32 Transport of ammonia in the circulation Although ammonia is constantly produced in the tissues, it is present at very low levels in blood – This is due both to the rapid removal of blood ammonia by the liver, and the fact that many tissues, particularly muscle, release amino acid nitrogen in the form of glutamine or alanine, rather than as free ammonia Formation of urea in the liver is quantitatively the most important disposal route for ammonia – Urea travels in the blood from the liver to the kidneys, where it passes into the glomerular filtrate Glutamine provides a nontoxic storage and transport form of ammonia – The ATP-requiring formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in the muscle and liver, but is also important in the nervous system, where it is the major mechanism for the removal of ammonia in the brain – Glutamine is found in plasma at concentrations higher than other amino acid – Circulating glutamine is removed by the kidneys and deaminated by glutaminase 33 34 Hyperammonemia The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation – levels of serum ammonia are normally low (5 to 50 µmol/L) However, when the liver function is compromised, due either to genetic defects of the urea cycle, or liver disease, blood levels can rise above 1000 µmol/L Such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS – For example, elevated concentrations of ammonia in the blood cause the symptoms of ammonia intoxication, which include tremors, slurring of speech, somnolence, vomiting, cerebral edema, and blurring of vision – At high concentrations, ammonia can cause coma and death 35 Neurotoxicity Associated with Ammonia Ammonia is neurotoxic – Failure to make urea via the urea cycle or to eliminate urea through the kidneys leads to marked brain damage – Result of either of these events is a buildup of circulating levels of ammonium ion Aside from its effect on blood pH, ammonia readily traverses the brain blood barrier and in the brain is converted to glutamate via glutamate dehydrogenase, depleting the brain of α-KG As the α-KG is depleted, oxaloacetate falls correspondingly, and ultimately TCA cycle activity comes to a halt In the absence of aerobic oxidative phosphorylation and TCA cycle activity – Irreparable cell damage and neural cell death ensue – In addition, the increased glutamate leads to glutamine formation 36 Neurotoxicity Associated with Ammonia Ammonia toxicity will lead to brain swelling due, in part, to an osmotic imbalance due to high levels of both ammonia and glutamine in the astrocytes – The ammonia levels inhibit glutaminase, leading to glutamine elevation Additionally, high levels of glutamine alter the permeability of the mitochondrial membrane, leading to an opening of a pore in the mitochondrial membrane (the mitochondrial permeability transition pore), which leads to cell death Another toxic effect of ammonia is a lowering of glutamate levels (due to the high activity of the glutamine synthetase reaction) This depletes glutamate stores which are needed in neural tissue since glutamate is both a neurotransmitter and a precursor for the synthesis of γ- aminobutyrate: GABA, another neurotransmitter Therefore, reductions in brain glutamate affect energy production as well as neurotransmission 37 Hyperammonemia The two major types of hyperammonemia: 1.Acquired hyperammonemia: Liver disease is a common cause of hyperammonemia in adults It may be a result of an acute process, for example, viral hepatitis, ischemia, or hepatotoxins Cirrhosis of the liver caused by alcoholism, hepatitis, or biliary obstruction may result in formation of collateral circulation around the liver As a result, portal blood is shunted directly into the systemic circulation and does not have access to the liver The detoxification of ammonia (that is, its conversion to urea) is, therefore, severely impaired, leading to elevated levels of circulating38 ammonia 2. Hereditary hyperammonemia: – Also called Urea Cycle Disorders (UCDs) – Genetic deficiencies of each of the five enzymes of the urea cycle have been described, with an overall prevalence estimated to be 1 in 30,000 live births – Ornithine transcarbamoylase deficiency, which is X-linked, is the most common of these disorders – All of the other urea cycle disorders follow an autosomal recessive inheritence pattern – In each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth – Blood chemistry will also show elevations in glutamine – All inherited deficiencies of the urea cycle enzymes result in mental retardation – Most urea cycle disorders are associated with hyperammonemia, however argininemia and some forms of argininosuccinic aciduria do not present with elevated ammonia 39 Urea Cycle Disorders (UCDs) 1. N-Acetylglutamate synthase deficiency 2. Carbamoyl phosphate synthetase deficiency 3. Ornithine transcarbamoylase deficiency 4. Citrullinemia (Deficiency of argininosuccinic acid synthase) 5. Argininosuccinic aciduria (Deficiency of argininosuccinic acid lyase) 6. Argininemia (Deficiency of arginase) 7. Hyperornithinemia, hyperammonemia, homocitrullinuria syndrome (Deficiency of the mitochondrial ornithine transporter) 40 Carbamoyl Phosphate Synthetase and N-acetylglutamate Synthetase Deficiencies – Hyperammonemia has been observed in infants with 0–50 % of the normal level of carbamoyl synthetase activity in their livers – In addition to other treatments, these infants have been treated with arginine, on the hypothesis that activation of N-acetylglutamate synthetase by arginine would stimulate the residual carbamoyl phosphate synthetase – This enzyme deficiency generally leads to mental retardation – A case of N-acetylglutamate synthetase deficiency has been described and treated successfully by administering carbamoyl glutamate, an analog of N-acetylglutamate, that is also able to activate carbamoyl phosphate synthetase – This treatment mitigates the intensity of the disorder but brain damage is irreversible 41 Ornithine Transcarbamoylase (OTC) Deficiency – The most common deficiency involving urea cycle enzymes is lack of ornithine transcarbamoylase – Mental retardation and death often result, but the occasional finding of normal development in treated patients suggests that the mental retardation usually associated is caused by the excess ammonia before adequate therapy – OTC deficiency is inherited in an X-linked recessive manner, meaning males are more commonly affected than females – OTC deficiency is diagnosed using a combination of clinical findings and biochemical testing, while confirmation is often done using molecular genetics techniques – In addition to ammonia and amino acids appearing in the blood in increased amounts, orotic acid also increases, presumably because carbamoyl phosphate that cannot be used to form citrulline diffuses into the cytosol, where it condenses with aspartate, ultimately forming orotate 42 OTC Deficiency Treatment goal for individuals affected with OTC deficiency is the avoidance of hyperammonemia This can be accomplished through a strictly controlled low-protein diet, as well as preventative treatment with nitrogen scavenging agents such as sodium benzoate and sodium phenylacetate The goal is to minimize the nitrogen intake while allowing waste nitrogen to be excreted by alternate pathways Arginine is typically supplemented as well, in an effort to improve the overall function of the urea cycle Liver transplant is considered curative for this disease Experimental trials of gene therapy resulted in the death of one participant and have been discontinued 43 44 Argininosuccinate Synthetase The inability to condense citrulline with aspartate results in accumulation of citrulline in blood and its excretion in urine (citrullinemia) Inheritance is autosomal recessive and about 50 % of cases are severe, due to hyperammonemia There are heterogeneous mutations that cause this deficiency, and three distinct types In Type I, the enzyme usually has an altered Michaelis constant, and the enzyme is affected in both liver and kidney In Type II the kidney is not affected, and the residual enzyme in liver is kinetically normal Type III argininosuccinate synchetase deficiency is caused by lack of transcription of the gene 45 Argininosuccinate Lyase Deficiency Argininosuccinic aciduria, also called argininosuccinic acidemia, is an inherited disorder that causes the accumulation of argininosuccinic acid (also known as "ASA") in the blood and urine – Impaired ability to split argininosuccinate to form arginine resembles argininosuccinate synthetase deficiency in that the substrate, in this case argininosuccinate, is excreted in large amounts – The severity of symptoms in this disease varies greatly so that it is hard to evaluate the effect of therapy, which includes dietary supplementation with arginine – In patients with early-onset disease therapy with a low-protein diet and arginine supplementation has resulted in good outcomes 46 Arginase Deficiency (Argininemia) – Arginase deficiency is rare but causes many abnormalities in development and function of the CNS – Arginine accumulates and is excreted as well as precursors of arginine and products of arginine metabolism may also be excreted – Unexpectedly, some urea is also excreted; this has been attributed to a second type of arginase found in the kidney – A diet including essential amino acids but excluding arginine has been used effectively – Type I affects liver, but not kidney, brain or intestinal trace – The enzyme involved in Type I argininemia is the enzyme found in cytosol and contributes to the production of urea – The enzyme identified as responsible for Type II argininemia is found in kidney mitochondrial matrix – The formation of nitric oxide from arginine and polyamines from ornithine are affected by a deficiency in this enzyme 47 Ornithine translocase deficiency also called hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome Caused by mutations in the gene that provides instructions for making the mitochondrial ornithine transporter This transports ornithine across the inner membrane of mitochondria to the mitochondrial matrix, where it participates in the urea cycle Mutations in this gene result in a mitochondrial ornithine transporter that is unstable or the wrong shape, and which cannot bring ornithine to the mitochondrial matrix This failure of ornithine transport causes an interruption of the urea cycle and the accumulation of ammonia, resulting in the signs and symptoms of ornithine translocase deficiency 48 GENERAL TREATMENT STRATEGIES FOR UREA CYCLE DISORDERS Very key to treatment is early diagnosis and treating aggressively with compounds that can aid in nitrogen removal Low-protein diets are essential to reduce the potential for excessive amino acid degradation Arginine supplementation has also proved beneficial Once argininosuccinate has been synthesized, the two nitrogen molecules destined for excretion have been incorporated into the substrate; the problem is that ornithine cannot be regenerated 49  For disorders before this step, drugs are used that form conjugates with amino acids and conjugated amino acids are excreted and the body then has to use its nitrogen to resynthesize the excreted amino acid The two compounds most frequently used are benzoic acid and phenylbutyrate (active component is phenylacetate) Benzoic acid, after activation, reacts with glycine to form hippuric acid, which is excreted As glycine is synthesized from serine, the body now uses nitrogens to synthesize serine, so more glycine can be produced Phenylacetate forms a conjugate with glutamine, which is excreted This conjugate removes two nitrogens per molecule and requires the body to resynthesize glutamine from glucose, thereby using another two nitrogen molecules. 50 Urea cycle defects are excellent candidates for treatment by gene therapy since the defect only has to be repaired in one cell type (in this case, the hepatocyte) 51 52 Question: Using the following information about five newborn infants (identified as I to V) who appeared normal at birth but developed hyperammonemia after 24 hours, determine which urea cycle enzyme might be defective in each case. All infants had low levels of blood urea nitrogen (BUN). (Normal citrulline levels are 10 to 20 µM.) 53