3. Protein / amino acid metabolism

Question 1

Define the term ‘nitrogen metabolism’.

Answer 1

Metabolic processes relating to the metabolism of all N-containing compounds.

Question 2

How much nitrogen is present in the body? In which compounds is N found (N-compounds)?

Answer 2

• 3% of body weight (~2kg)
• Major N-compounds:
  
  • proteins (>90% of N)
  • amino acids
  • DNA/RNA (purines and pyrimidines)
• Smaller amounts in:
  
  • some hormones (eg adenaline)
  • neurotransmitters (eg dopamine)
  • porphyrins (haem)
  • creatine

Question 3

What are the main pathways for N intake and output?

Answer 3

Main N source (>90%) = dietary protein

N loss in urine (some in sweat and faeces) mainly as urea, and a little as creatinine, ammonia and uric acid.
Some direct loss of protein (skin, hair, nails…) from the body.

Question 4

Explain the term ‘N-balance’. In which situations might this change?

Answer 4

N-balance: steady state in which N intake = N ouput - no change in total body protein.

Positive N-balance: N intake > N output - increase in total body protein. Normal state in growth and pregnancy, tissue repair and malnutrition convalescence.

Negative N-balance: N intake < N output - net loss of body protein. Abnormal state caused by trauma, infection or malnutrition/starvation.

Question 5

What is ‘protein turnover’?

Answer 5

All body proteins undergo continuous breakdown and resynthesis, contributing to the free amino acid pool.
Involves:
- digestion of dietary protein
- de novo amino acid synthesis
- synthesis and proteolysis of cellular proteins
- breakdown of amino acids to carbon skeleton and amino group

Question 6

What are the main functions of free amino acids?

Answer 6

1. Protein synthesis (~75% of aa released in protein breakdown are reutilised, requires all 20 aa)
2. Synthesis of other N-compounds (requires specific aa)
   - 2 important signalling molecules: nitric oxide (from L-arginine) and hydrogen sulphide (from L-cysteine)
   - neurotransmitters, e.g. 5-HT (from tryprophan)
   - local mediators, e.g. histamine (from histidine)

Question 7

What is unusual about the role of glycine and tyrosine?

Answer 7

Role in synthesis of several N-compounds:

• tyrosine = melanin, thyroid hormones and catcholamines
• glycine = purines, glutathione, prophyrins and creatine

Question 8

Which components are required for non-essential amino acid synthesis?

Answer 8

• C atoms from intermediates of glycolysis (C3), pentose phosphate pathway (C4 and C5) and TCA cycle (C4 and C5).
• Amino group from other amino acids by transamination, or from ammonia.

Question 9

What happens to amino acids available in excess of those needed for protein and N-compound synthesis?

Answer 9

Cannot be stored or excreted so broken down into smaller molecules, mainly in the liver.
Each amino acid has its own pathway of breakdown but pathways share common features:
1. C-atoms converted to intermediates of carbohydrate and lipid metabolism
2. Usually start with removal of NH2-group (transamination/deamination)
3. N-atoms are usually converted to urea

Question 10

What is the difference between glucogenic and ketogenic amino acids?

Answer 10

(i) C-atom of glucogenic amino acids converted to:
- pyruvate
- oxaloacetate
- fumarate
- alpha-ketoglutarate
- succinyl~CoA
Can be used to synthesise glucose or glycogen

(ii) C-atom of ketogenic amino acids converted to:
- acetyl CoA
- acetoacetyl CoA
Can be used to synthesise fatty acids or ketone bodies.

Some amino acids are both glucogenic and ketogenic.

Question 11

Give examples of amino acids that are glucogenic, ketogenic or both.

Answer 11

Glucogenic: alanine, glycine
Ketogenic: lysine, leucine
Both: threonine, tryptophan

Question 12

What happens to the C-atom products of amino acid degradation? How is this different during starvation/diabetes?

Answer 12

• Oxidised to CO2 and H2O with released energy used by the cell.
• In starvation and diabetes, products can be used to produce glucose and ketone bodies.

Question 13

Describe the control of the mobilisation of protein reserves during starvation.

Answer 13

Under hormonal control:

1. Insulin and growth hormone
   
   • increase protein synthesis
   • decrease protein degradation
2. Glucocorticoids (e.g. cortisol)
   
   • decrease protein synthesis
   • increase protein degradation

Question 14

Why is striae formation a symptom of Cushing’s syndrome?

Answer 14

Excess cortisol… excessive protein breakdown… weakens skin structure… striae formation.

Question 15

Which 2 pathways facilitate removal of nitrogen from amino acids?

Answer 15

Transamination (major mechanism) and deamination

Question 16

Describe the mechanism of transamination.

Answer 16

Is the transfer of an amino group from the amino acid to a keto acid, catalysed by an aminotransferase.

• Amino acid thus becomes a keto acid, such as pyruvic acid.
• Keto acid used is usually alpha-ketoglutarate, which becomes glutamate.

Exception being the aspartate aminotransferase which transfer amino group to oxaloacetate, producing aspartate.

Question 17

Which coenzyme do all aminotransferases require?

Answer 17

Pyridoxal phosphate (derivative of vitamin B6)

Question 18

Which transaminases are measured in serum to assess liver function?
When might levels of these enzymes be particularly high?

Answer 18

Alanine aminotransferase (ALT, converts alanine to glutamate) and aspartate aminotransferase (AST, converts glutamate to aspartate).

High ALT and AST in conditions that cause extensive cellular necrosis such as:

• viral hepatitis
• autoimmune liver diseases
• toxic injury

Question 19

What is amino acid deamination? Where does it occur?

Answer 19

The removal of the amino acid’s amino group as free ammonia (NH3), which is rapidly converted to ammonium (NH4+).
Reaction results in keto acid (can be used for energy) + NH3.
Mainly occurs in liver and kidney (as urea cycle on hand to prevent toxic effects).

Question 20

Which 2 types of non-specific enzymes catalyse amino acid deamination? Why is it important to have both types?

Answer 20

L- and D-amino acid oxidases

Important to also have D-amino acid oxidases as requried for deamination of D-amino acids found in plants and bacterial cells (so enter body through diet). Must not be used for protein synthesis as proteins would be structurally abnormal and non-functional.

Question 21

Give examples of enzymes that deaminate amino acids.

Answer 21

1. Amino acid oxidases
2. Glutaminase - high specificity enzymes that convert glutamine to glutamate + NH3.
3. Glutamate dehydrogenase - high specificity enzyme that converts glutamate to alpha-ketoglutarate + NH4+.

Question 22

What is the normal concentration of ammonia in the blood?

Answer 22

Very low: 25-40 umol/L

Question 23

Why is serum [ammonia] kept very low? What are the symptoms of hyperammonaemia?

Answer 23

Ammonia is readily diffusible and extremely toxic to cells. The CNS is very sensitive to ammonia; hyperammonaemia is associated with blurred vision, tremmors, slurred speech, coma and eventually death.

Question 24

Explain the molecular basis of ammonia toxicity.

Answer 24

May involve:

1. reaction with alpha-ketoglutarate to form glutamate in MT via glutamate dehydrogenase… removes alpha-ketoglutarate from TCA cycle which slows… disrupts energy supply to brain cells.
2. affects pH inside CNS cells (alkaline)
3. interferes with excitatory amino acid NT metabolism
4. interferes with amino acid transport and protein synthesis
5. disruption of cerebral blood flow and of blood-brain barrier

Question 25

Which 2 mechanisms are used for the safe removal of ammonia from tissues?

Answer 25

1. Ammonia combined with glutamate by glutamine synthetase to form glutamine… transported in blood to liver or kidneys… hydrolysed by glutaminase to reform glutamate and ammonia… ammonia fed into urea cycle in liver or excreted directly in urine in kidney.
2. Ammonia combined with pyruvate to form alanine… transported in blood to liver…. converted back to pyruvate and amino group by transmination… amino group fed via glutamate into urea cycle for disposal as urea and pyruvate used to synthesise glucose which can be fed back into tissues.

Question 26

What are the advantages of converting ammonia to urea?

Answer 26

Urea is:

i) very soluble in water… can be excreted in urine… performs useful osmotic role in kidney tubules
ii) non-toxic and metabolically inert
iii) high nitrogen content (47%)… effective for disposal of unwanted nitrogen

Question 27

Where is urea synthesised and excreted?

Answer 27

Synthesised in liver by urea cycle and transported via blood to kidneys for excretion in urine.

Question 28

Which organelle does the urea cycle involve?

Answer 28

3 reactions are cytoplasmic, 2 reactions are mitochondrial

Question 29

Which substrates are required for urea synthesis?

Answer 29

NH2 groups of urea come from ammonium and aspartate.

• ammonium comes from deamination of amino acids in liver, producing ammonia, and from ammonia produced by gut bacteria that enters the liver via the portal circulation.
• aspartate formed from oxaloacetate by transamination of glutamate

Question 30

How is the urea cycle controlled?

Answer 30

• Enzymes are not subjected to feedback inhibition by end-product of cycle as function of cycle is to dispose of ammonia as urea.
• But enzymes of cycle are inducible: high protein diet induces enzymes, low protein diet/starvation represses enzymes

Question 31

What is re-feeding syndrome?

Answer 31

Severely malnourished inidividuals have low levels of urea cycle enzymes. So are at risk of hyperammonaemia if given too much protein, as excess amino acids are degraded but the ammonia cannot be converted to urea.

Question 32

How should the diet of severely malnourished patients be managed to prevent refeeding syndrome?

Answer 32

Re-feed at 5-10 kcal/kg/day. Raise gradually to full needs within a week.

Question 33

Why might patients with kidney failure develop hyperammonaemia?

Answer 33

Urea diffuses from the liver cells to the blood and is carried to the kidney where it is filtered and excreted in the urine.
A small amount may diffuse across the intestinal wall and enter the intestine… broken down by bacteria, releasing ammonia that can be reabsorbed.

In kidney failure where the concentration of urea in the blood is high, ammonia production from urea by gut bacteria can contribute to hyperammonaemia.