Protein Breakdown and Urea Formation

Question 1

What is nitrogen balance?

Answer 1

Nitrogen balance is a measure of nitrogen input minus nitrogen output.

Question 2

What are the two parts of an amino acid?

Answer 2

• the carbon skeleton
• nitrogen

The carbon skeleton is broken down by energy metabolism and biosynthetic pathways.

Question 3

Why do we need to remove nitrogen, and what is it converted into to be removed?

Answer 3

Nitrogen is toxic, so it has to be removed safely.

In mammals, the nitrogen is converted to the non-toxic, neutral compound urea and excreted in urine.

Question 4

What are the three steps in which amino acid nitrogen is transferred to urea?

Answer 4

• transamination
• formation of ammonia
• formation of urea

Question 5

Describe transamination.

Answer 5

• The nitrogen group of one amino acid is transferred to a particular keto acid to give us a second amino acid. (the original amino acid becomes a keto acid now).
• Transaminase (aka aminotransferases) is the enzyme involved in this reaction. It catalyses a transamination reaction between an amino acid and a α-keto acid.
• The synthesised molecules can be metabolised more readily.

Question 6

Give some examples of α-keto acids.

Answer 6

• α-ketoglutarate
• pyruvate
• oxaloacetate
  • α-keto acids are important metabolic intermediates. They can be oxidised or converted to glucose.

Question 7

Name two important aminotransferases and the chemical reaction that they catalyse.

Answer 7

• ALANINE (ALT)
  Alanine will react with α-ketoglutarate to give pyruvate and glutamate. In the context of urea formation, this reaction predominates.
• ASPARTATE (AST)
  Aspartate will react with α-ketoglutarate to give oxaloacetate and glutamate. In the context of urea formation, the opposite of this reaction predominates.

Both generate glutamate, and both reactions are fully reversible.
- Requires pyridoxal phosphate derived from vitamin B6

Question 8

How can the levels of transaminases be used diagnostically?

Answer 8

Transaminases are primarily liver enzymes, so high levels of ALT and AST in the blood can be indicative of liver damage (since they’re not meant to be released into circulation).

Question 9

What happens during ammonia formation?

Answer 9

What happens during ammonia formation?
• Glutamate, produced from transamination, can release ammonia directly by the action of Glutamate Dehydrogenase.– glutamate is converted to α-ketogluterate.
• This reaction is called Oxidative deamination and it takes place in the mitochondrial matrix.
• The reaction is fully reversible and can use either NAD or NADP; however, it is usual for NAD to be used for degradation and NADPH for synthesis.

Glutamate is a very useful molecule because it is freely interchangeable with the α-keto acids as well as the ability to donate and accept ammonium ions.

Question 10

What is the significance of having the transamination to glutamate and then the oxidative deamination back to α-ketoglutarate of amino acids?

Answer 10

The reason it’s very important is because it allows conversion of many amino acids from their original state into glutamate, which can be transported (note it is not often transported as glutamate) and then re-converted back into something the body can use for energy (or transamination again) while re-synthesising the ammonia which can be fed into the urea cycle.

Question 11

How do we eliminate free ammonia?

Answer 11

Free ammonia generated in tissue combines with Glutamate to give Glutamine.
Glutamate + NH4+ + ATP –> Glutamine + ADP
This reaction is catalysed by Glutamine Synthase.

Question 12

What is the importance of glutamine in the transport of nitrogen?

Answer 12

• Glutamine is readily soluble and the main transporter of nitrogen.
• It’s formed from glutamate which, in addition to having already accepted amino-groups to α-ketoglutarate, accepted more nitrogen to form glutamine.
• Glutamine can donate nitrogen for the biosynthesis of amino acids, nucleotides, amino sugars and NAD+.

Question 13

Describe the structure of urea.

Answer 13

Urea is made up of two amine groups joined to a C=O. One amine group is donated from aspartate, while the other comes from glutamine/glutamate.
The carbon C=O comes from the carbon skeleton, through using CO2 that has been produced from its breakdown.
Hence, the detrimental products of amino acid degradation can be used to combine to form urea, a non-toxic, soluble compound that can be readily excreted.

Question 14

The urea cycle and the TCA are interlinked. With that in mind, describe the urea cycle.

Answer 14

1. CO2 comes from the bicarbonate and reacts with the ammonium ion that has come from glutamine/glutamate (formed by transamination of α-ketoglutarate and α-amino acid). They form carbamoyl phosphate (in the mitochondria).
2. Carbamoyl phosphate then reacts with Ornithine to produce Citrulline.
3. Citrulline reacts with Aspartate to form Argininosuccinate.
4. Argininosuccinate then is metabolised to Arginine (urea cycle) and Fumarate (TCA).
5. The Arginine is acted upon by the enzyme arginase which is how, ultimately, urea is formed. The urea cycle continues.
6. The Fumarate is converted to Malate which is transported back into the mitochondria and converted into oxaloacetate. The TCA then continues.

Question 15

How are muscles involved in the breakdown of amino acids?

Answer 15

Muscles don’t have the enzymes needed to form urea, so the urea cycle doesn’t take place in muscles. However, muscles do break down amino acids for energy during prolonged exercise or starvation.

Question 16

What are the two ways in which remaining amino acids are dealt with in the muscle?

Answer 16

1) Nitrogen is transferred to alanine (via glutamate and pyruvate)
2) Circulating/intracellular glutamate can be made into glutamine (and return to the liver)

Question 17

How do muscles come into the removal of nitrogen?

Answer 17

The muscle can export alanine, as it is one of the major exports of muscle that is actively being broken down (due to exercise or starvation).

Question 18

Describe the glucose-alanine cycle between the muscle and the liver.

Answer 18

1. In the muscle, branched amino acids are taken and broken down.
2. The carbon skeleton is used for energy production. Then the NH4 can be used to convert from pyruvate to Alanine.
3. Alanine is then exported into the blood and travels to the liver.
4. The alanine is then converted to glutamate via transamination (reacting with α-ketoglutarate) also producing a pyruvate.
5. The pyruvate can enter the gluconeogenic pathway to form glucose, and the glucose can be transported in the blood back to the muscle where it can be used for energy.
6. The glutamate will then be used along with the CO2 generated to produce urea in the liver.

Question 19

What is the growth principle?

Answer 19

GROWTH = SYNTHESIS – BREAKDOWN

• Applies to individual proteins, single cells, while tissues or the entire body.

Question 20

Where are proteins from and where are they stored?

Answer 20

• Amino acids are supplied in the diet
• There are no specific protein stores
• Proteins are structural or functional
• Excess protein is broken down and excreted

Question 21

What is nitrogen balance?

Answer 21

Nitrogen balance is a measure of nitrogen input minus nitrogen output.

Question 22

Factors to include when measuring nitrogen balance.

Answer 22

1) Body Protein
2) Amino acid pool
3) Dietary protein
4) Urea, other products

Question 23

What happens when balanced nitrogen balance?

Answer 23

Input=output

Normal healthy individual.

Question 24

What happens when positive nitrogen balance?

Answer 24

• Input>output
• Exercise and tissue hypertrophy (tissue enlargement)
• In response to anabolic hormones
e.g. Growth, Pregnancy

Question 25

What happens when negative nitrogen balance?

Answer 25

• Input

Question 26

Fate of the carbon skeleton

Answer 26

• Broken down amino acids form carbon skeleton, which can make α-ketoacids.
• Other amino acids can feed into different components of TCA cycle.
• For transferase reaction, all amino acids can become α-ketoacids, but 2:
1. Lysine
2. Proline
- The body deals with these by other metabolisms.

• Carbon skeletons can be used for the production of glucose, ketone bodies and/or energy.

Question 27

Protein and amino acid metabolism after a meal.

Answer 27

• Normal metabolism = high insulin, low glucagon
• In a normal individual most amino acids from a protein meal will be used for protein synthesis in peripheral tissues such as skeletal muscle.
• Excess amino acids can also be used as sources of energy, and the nitrogen derived from their oxidation will be incorporated into urea in the liver and excreted

Question 28

Protein and amino acid metabolism during starvation

Answer 28

• In a normal individual: insulin is low, glucagon is high
• During short-term starvation there will be a net flow of amino acids from muscle to the liver, with increased production of glucose and urea.
• During long-term starvation, tissue protein is “spared” because ketone bodies replace glucose as a major energy fuel for the brain.

Question 29

Protein and amino acid metabolism in untreated diabetes.

Answer 29

• Here the negative nitrogen balance associated with short term starvation persists even though the subject is fed, leading to muscle wasting.
• Negative nitrogen balance due to decreased protein synthesis and/or increased protein breakdown is also seen in conditions of chronic infections, late stage cancer or trauma, including that following surgery or burns injury.
• Some of these effects are mediated by cytokines.