Protein Metabolism and Disorders of Ammonia Processing

Question 1

Essential Amino Acids

Answer 1

Question 2

Answer 2

Question 3

Protein Balance

Answer 3

Protein balance describes the relationship between the synthesis and degradation (proteolysis) of proteins.

Question 4

Answer 4

Question 5

Answer 5

Question 6

Answer 6

Question 7

Answer 7

• Regulation of protein turnover and nitrogen economy is complex. Insulin and glucocorticoids (cortisol in humans) participate in this regulation.
• Insulin increases synthesis and decreases degradation of endogenous proteins to favor the maintenance of body protein pools.
• Insulin-like growth factor is important for promoting protein synthesis during growth.
• Cortisol, released during stress or starvation, is counterregulatory (opposes the effects) to insulin and result in peripheral tissue protein catabolism.
• As many amino acids, especially alanine, are precursors for the synthesis of glucose, this catabolic effect of cortisol coincides with the ability of this class of hormones to promote gluconeogenesis.
• Thus, the insulin:cortisol ratio is an important determinant of net protein turnover.
• In the fed state of children (high ratio of insulin:cortisol), net protein formation occurs. In fasting, insulin falls to lower the ratio so that protein is mobilized via proteolysis.
• In trauma, the concentration of cortisol increases to markedly lower the ratio.

Question 8

Transamination Reactions

Answer 8

• The first step in the degradation of most amino acids is removal of their amino nitrogen group by transferring it to alpha-ketoglutarate to produce glutamate. (Recall that alpha-ketoglutarate is also an intermediate in the citric acid cycle.) This group of reactions is catalyzed by specific aminotransferases also called transaminases.
• The transaminases depend on pyridoxal phosphate as a cofactor. Pyridoxal phosphate is derived from pyridoxine (vitamin B6).
• Transaminases are freely reversible. The alpha-ketoacid accepts an amino group from glutamate to produce a new amino acid.
• The most common aminotransferases are for alanine and aspartate, with the respective alpha-ketoacids being pyruvate and oxaloacetate. These aminotransferases are often measured in the blood to test for liver damage due to diseases.
• Transaminases generally transfer nitrogen to glutamate in non-hepatic tissues, such as muscle, as a way of getting rid of excess nitrogen from those tissues.
• In contrast, in liver nitrogen is dumped onto glutamate as an initial step in the conversion of nitrogen to a form that can be readily excreted; that is urea.

Question 9

Nitrogen Removal From Non-hepatic Tissues

Answer 9

• Glutamate dehydrogenase serves different functions depending on the tissue in which it is found. In one direction, the reaction involves the addition of nitrogen to alpha-ketoglutarate as ammonia. The reaction proceeds in this direction in non-hepatic tissues.
• This pathway for glutamate production is an important way to remove harmful ammonia from these tissues.
• Glutamate is not readily transported across the plasma membrane, but glutamine does easily leave cells.
• Glutamine is formed through the addition of a second ammonia molecule by glutamine synthetase to produce glutamine.
• The glutamine is processed by the kidney, which contains large amounts of the enzyme glutaminase that, in combination with glutamate dehydrogenase, removes the amino groups from glutamine. The ammonia released in this manner is then excreted in the urine.

Question 10

Glutamine in the kidney…

Answer 10

•The glutamine is processed by the kidney, which contains large amounts of the enzyme glutaminase that, in combination with glutamate dehydrogenase, removes the amino groups from glutamine. The ammonia released in this manner is then excreted in the urine.

Question 11

Glutamine and amino acids in the liver…

Answer 11

•In liver, the glutamate produced by transamination gives up its nitrogen as free ammonia via glutamate dehydrogenase for the eventual synthesis of urea, which can be excreted.

Question 12

Answer 12

• 5 Steps
• The urea cycle is found primarily in the liver and to a lesser extent in the kidney. It provides a means of ridding the body of nitrogen waste as urea. Ammonia is derived from amino acids by the combined actions of transamination and glutamate dehydrogenase.

Question 13

Urea Cycle Step 1

Answer 13

• The urea cycle is found primarily in the liver and to a lesser extent in the kidney. It provides a means of ridding the body of nitrogen waste as urea.
• Ammonia is derived from amino acids by the combined actions of transamination and glutamate dehydrogenase.
• In the mitochondria, ammonia is incorporated into carbamoyl phosphate via carbamoyl phosphate synthetase-I (CPS-I). This enzyme is not directly part of the cycle but instead the reaction product, carbamoyl phosphate, provides a substrate for the cycle.

-The reaction is energy-requiring with one ATP molecule providing the phosphate that combines with carbon dioxide and ammonia and the other ATP providing the driving force for the reaction.

• Carbamoyl phosphate directly introduces the first source of nitrogen for the cycle.
• Carbamoyl phosphate synthetase-I is allosterically activated by Nacetylglutamate that is produced by the enzyme-catalyzed reaction of acetyl CoA with glutamate.

-Thus, N-acetylglutamate deficiency presents physiologically similar to CPS-I deficiency despite the latter being a functioning enzyme.

glutamate + acetyl CoA —> N-acetylglutamate + CoA

•The mitochondrial carbamoyl phosphate synthetase-I (CPS I: NH3) can be distinguished from the cytoplasmic carbamoyl phosphate synthetase-II (CPS-II: glutamine) in that the latter uses glutamine, rather than ammonia, as the nitrogen source and is involved in synthesis of the pyrimidine base.

Question 14

Urea Cycle Step 2

Answer 14

• Ornithine transcarbamoylase: In the first true reaction of the urea cycle, carbamoyl phosphate is combined with ornithine to form citrulline via ornithine transcarbamoylase.
• This reaction occurs in the mitochondrial matrix, putting it in the same compartment as the site of carbamoyl phosphate formation.
• Ornithine for the reaction is transported into the mitochondria from the cytoplasm. The citrulline product is released from the mitochondria to the cytoplasm in exchange for ornithine.

Question 15

Urea Cycle Step 3

Answer 15

• Argininosuccinate synthetase: In the cytoplasm, citrulline reacts with aspartate via argininosuccinate synthetase yielding argininosuccinate.
• Aspartate for this reaction is formed by transamination of glutamate with oxaloacetate. Thus aspartate is the second direct source of nitrogen for the cycle.
• As a synthetase, it is an energy requiring reaction that cleaves ATP to AMP + PPi thus costing two high-energy phosphate bonds (recall that PPi spontaneously splits to two Pi).

Question 16

Urea Cycle Step 4

Answer 16

• Argininosuccinate is then cleaved by argininosuccinase into fumarate and arginine.
• The fumarate can be reconverted to oxaloacetate in the citric acid cycle that in turn can be used to regenerate aspartate.
• Hence the carbons from aspartate are recycled with only the nitrogen claimed for the urea cycle.

Question 17

Urea Cycle Step 5

Answer 17

• This last enzyme of the cycle cleaves arginine into urea and ornithine.
• Urea is secreted from the liver into the blood to be cleared by the kidney for excretion.
• The ornithine is regenerated for another turn of the cycle.
• Under certain conditions, arginase is unable to keep up with the high rate of arginine formation leading to accumulation of arginine.
• When this occurs, arginine stimulates the formation of N-acetylglutamate to increase the formation of carbamoyl phosphate.
• Carbamoyl phosphate in turn reacts with ornithine to produce a mass action effect on the arginase reaction thus increasing the formation of urea

Question 18

Hyperammonemia

Answer 18

• Acquired
• Inherited

Question 19

Hyperammonemia - Acquired

Answer 19

• Acquired hyperammonemia results from development of collateral circulation of the portal system in response to severe liver damage (e.g., cirrhosis); the blood flow from the intestines thus bypasses the liver. It is the development of this collateral circulation, and not the cirrhosis itself, that is responsible for the hyperammonemia.
• This is because the microorganisms in the gastrointestinal tract produce a large amount of ammonia that is absorbed into the portal system and is sent to the liver for detoxification.
• The development of this alternate path of blood flow is known as portal-systemic shunting because the blood from the portal vein is diverted around the liver directly into the inferior vena cava.
• For this reason, the name portal-systemic encephalopathy (PSE) is used to describe the constellation of abnormalities associated with acquired hyperammonemia.
• The portal-systemic shunting leads to a drastic reduction in ammonia detoxification by the liver.
• Ammonia arising from amino acid or protein catabolism cannot be converted to urea to any significant extent causing blood ammonia to rise.
• During an episode of PSE, blood ammonia can increase to >500 uM, resulting in coma.
• Definitive treatment is by liver transplantation. Until this can be accomplished, treatment measures are aimed at a reduction of absorption of ammonia.
• One effective measure is the administration of the disaccharide lactulose (a molecule similar in structure to lactose).
• This carbohydrate is not digestible and thus moves to the colon intact.
• There it is fermented by the microorganisms to short chain organic acids that lower the pH of the intestinal lumen.
• This converts the ammonia (NH3) produced by the bacteria to ammonium ion (NH4 + ) that is not readily absorbed across the intestinal epithelium and is instead excreted in the stool.

Question 20

Hyperammonemia - Inherited

Answer 20

• Inherited hyperammonemia, caused by deficiencies of urea cycle enzymes, is almost exclusively seen in children.
• Elevation of ammonia is very, very toxic to the central nervous system. These disorders often present in the first week of life with overwhelming hyperammonemia.
• Symptoms may range from life-threatening to no symptoms at all, depending upon the degree of enzyme deficiency.
• A total or near total lack of activity of an enzyme is usually fatal to the newborn. If not fatal, the infants may suffer permanent neurologic damage if not treated by extreme measures (hemodialysis) to lower blood ammonia.
• The longer the duration of ammonia elevation, the greater is the likelihood of neurologic damage.
• The severity of inherited hyperammonemia depends on the proximity of the defect to the point of entry of ammonia in its processing to urea and correlates roughly to the severity of symptoms. Severe symptoms often include cerebral edema. Consequently defects of carbamoyl phosphate synthetase I [CPS] or ornithine transcarbamoylase [OTC] lead to the most severe hyperammonemia.

Question 21

Hyperammonemia - Inherited - Distinguishing Betwen CPS I Deficiency and OTC Deficiency

Answer 21

• Both of these deficiencies lead to low or absent citrulline.
• However these two defects can be distinguished by evaluating the appearance of orotic acid [a pyrimidine base] in the urine.
• With a defect of OTC, carbamoyl phosphate accumulates in the mitochondria. The excess carbamoyl phosphate can then leak into the cytoplasm where it increases the rate of pyrimidine synthesis.
• Normal excretion of orotic acid is 1-11 umol/mol creatinine while patients with OTC usually excrete more than 1000 umol/mol creatinine.
• Since the gene for ornithine transcarbamoylase is on the X-chromosome, a defect of this enzyme is primarily found in males.

Question 22

Possible Mechanisms for Hyperammonemia Causing Neurologic Damage

Answer 22

The exact reason for the neurologic damage is unknown but possible mechanisms include:

(1) ammonia reacting with alpha-ketoglutarate to form glutamate thus interfering with ATP production in the TCA cycle.
(2) excess glutamate undergoing successive amination to glutamine and then to alpha- ketoglutaramic acid, a neurotoxic compound
(3) elevated ammonia markedly increasing the blood levels of some amino acids, which in turn can compete with other amino acids for transport across the blood brain barrier; the predominant transport of one or a few amino acids would then limit the availability of other amino acids within the brain, thus reducing normal rates of protein synthesis.

Question 23

Treatment for Hyperammonemia

Answer 23

• The most important is restriction of dietary protein to minimal levels, thus reducing the amount of ammonia that must be detoxified.
• Detoxification requires hemodialysis with severe hyperammonemia.
• Alternative mechanisms of ammonia excretion are accomplished by taking advantage of the body’s detoxification of exogenous chemicals.
• In this instance, one of two organic acids, sodium benzoate or sodium phenylacetate, is given to patients.
• Benzoate conjugates with glycine to form hippuric acid, which is readily excreted in the urine taking with it the nitrogen from glycine.
• Glycine, itself, can be synthesized from CO2 and NH3.
• Phenylacetate conjugates with glutamine forming phenylacetylglutamine, which is excreted in the urine taking two nitrogens per molecule.
• In hyperammonemia, glutamine is continually synthesized in peripheral tissues, especially muscle, via the glutamate dehydrogenase and glutamine synthetase reactions.
• The combination of low protein diet and these medications remains the mainstay of treatment for the genetic urea cycle defects.
• More and more, however, liver transplantation is performed to provide the patient with a new liver that does not have the genetic defect. This is curative for the urea cycle defect, but has lots of complications related to the liver transplantation.

Question 24

Answer 24