Biochemistry - Nitrogen Metabolism Notes

Nitrogen Metabolism

Learning Outcomes

• Explain the concept of nitrogen balance and the causes of positive and negative nitrogen balance.
• Outline the pathways of protein degradation.
• Explain how amino acids are classified as essential or nonessential and the significance of this classification.
• Explain the terms transamination, deamination, and transdeamination.
• Explain the importance of glutamate, glutamine, aspartate, and alanine in amino acid metabolism.

Nitrogen Balance

• Dietary protein intake is balanced against nitrogen excretion.
• Nitrogen excretion occurs through:
  • Urea
  • Uric acid
  • Ammonia
  • Creatinine
• The balance affects the amino acid pool and body proteins.

Amino Acid Pool

• Composed of free amino acids.
• Amino acids exist in very low concentrations inside cells or in the bloodstream.
• Mixing and exchange occur with other free amino acids throughout the body.

Protein Requirements

• There's no storage form of protein in the body to replace proteins and other nitrogen-containing compounds.
• Protein is needed in the diet to replace lost amino acids and allow for tissue repair.
• Recommendation: 50–70 g protein per day.
• High protein intake in a well-fed individual is wasteful because surplus amino acids are rapidly catabolized, and the nitrogen is excreted as urea in the urine.

Nitrogen Balance Types

• Positive nitrogen balance:
  • Nitrogen intake > Nitrogen excretion.
  • Protein synthesis exceeds the rate of breakdown.
  • Occurs during normal growth in children, in convalescence after serious illness, after immobilization after an accident, and in pregnancy.
• Negative nitrogen balance:
  • Nitrogen intake < Nitrogen excretion
  • Protein breakdown exceeds the rate of synthesis.
  • Occurs in starvation, during serious illness, in late stages of some cancers, and in injury and trauma.
  • If not corrected and becomes prolonged, there will be irreversible loss of essential body tissue which will ultimately lead to death.

Protein Degradation Pathways

• Most cellular proteins:
  • Recognized as ‘old’ or damaged.
  • Removed by the ubiquitin breakdown system.
  • Yield a mixture of the 20 amino acids.
• Foreign ‘exogenous’ proteins and ‘old’ or damaged subcellular organelles:
  • Taken into vesicles by endocytosis or autophagocytosis.
  • The vesicle fuses with lysosomes.
  • Proteolytic enzymes degrade proteins into amino acids.
• Starvation and hormones (e.g., cortisol) increase rates of protein breakdown in muscle.

Key Questions Addressed

• How are excess amino acids removed from the body?
• How are amino acids broken down?
• How is nitrogen safely removed from the body?
• How is this breakdown used to generate energy?

Deamination

• Deamination is the removal of the α-amino group.
• Glutamate is converted to α-Ketoglutarate + NH_4+
• Takes place in the liver mitochondrial matrix.
• Catalyzed by glutamate dehydrogenase.
• (Almost) only happens to Glutamate in humans.
• Ammonia is toxic!
• Brain particularly sensitive where ammonia toxicity causes cognitive impairment, ataxia, seizures
• Ammonia is converted to a non-toxic compound in the Urea Cycle.
• Urea is transported via the blood to the kidney for excretion.

Transamination

• Transamination is the conversion of one amino acid to another.
• Amino Acid + \alpha-KG \rightarrow Glutamate + Keto Acid
• α-amino group is transferred from an amino acid to α-ketoglutarate to produce Glutamate and a Keto Acid (depends on the side chain of the original amino acid).
• Catalyzed by aminotransferases (transaminases), which are specific for one amino acid.

Examples of Transamination

• Aspartate + \alpha-KG \rightarrow Glutamate + Oxaloacetate
  • Catalyzed by aspartate aminotransferase.
• Alanine + \alpha-KG \rightarrow Glutamate + Pyruvate
  • Catalyzed by alanine aminotransferase.

Significance

• Carbon skeletons produced are easily metabolized in the TCA cycle or Gluconeogenesis.

Transdeamination

• Combined action of aminotransferases & glutamate dehydrogenase allows α-amino groups from different amino acids to enter the Urea Cycle, using glutamate as an intermediate.
• For example, transdeamination of Alanine in the liver:
  • Alanine + \alpha-Ketoglutarate \rightarrow Glutamate + Pyruvate
  • Glutamate \rightarrow \alpha-Ketoglutarate + NH_4 +

Ammonia Transport

• Free ammonia is generated elsewhere in the body and needs to be transported to the liver
• Processes in other tissues generate ammonia (e.g., nucleotide degradation).
• This ammonia needs to be transported to the liver for safe disposal as Urea
• It is too toxic to transport in the blood as ammonium.

Glutamine's Role

• Glutamine – transport of ammonia in the blood
• Glutamate has 1N, Glutamine has 2N
• In extra-hepatic tissues, ammonia is added to glutamate to produce glutamine.
• Glutamine is safely transported in the bloodstream to the liver.
• Glutamine is converted back to glutamate, and the ammonia released enters the Urea Cycle for safe disposal.

Alanine's Role

• Alanine – generated in skeletal muscle during exercise.
• Vigorously exercising muscle uses protein as well as carbohydrate for energy.
• The carbon skeletons of amino acids can be used in the TCA cycle to generate ATP, but the ammonia must be safely removed.
• Skeletal muscle generates lots of pyruvate (and lactate).
• Transamination of pyruvate to alanine allows the ammonia to be safely transported to the liver.
• The glucose-alanine cycle allows the liver to regenerate glucose from this alanine.

Amino Acid Catabolism in the Liver - Integration

• Glutamine transports ammonia to the liver where it is converted to Glutamate + Ammonia
• Alanine from skeletal muscle is converted to Glutamate + Pyruvate.
• Excess amino acids are converted to Glutamate by transamination.
• Glutamate is deaminated to generate α-Ketoglutarate and Ammonia
• Ammonia is converted to Urea (in humans) in the Urea Cycle.
• Urea is transported to the kidneys for excretion

Fate of Carbon Skeletons

• Degradation of all 20 amino acids yields 7 major products (carbon skeletons).
• Pyruvate, Oxaloacetate, Fumarate, Succinyl-CoA & α-Ketoglutarate can:
  • Enter TCA cycle to generate ATP
  • Enter gluconeogenesis to release glucose into the blood
  • Amino acids producing these carbon skeletons are termed Glucogenic because they can produce glucose via gluconeogenesis
• Acetoacetayl-Coa & Acetyl-CoA can: produce ketone bodies
  • Amino acids producing these carbon skeletons are termed Ketogenic
  • Only Lysine and Leucine are strictly ketogenic.

Essential vs. Non-Essential Amino Acids

• Essential amino acids cannot be synthesized in humans – these must be obtained via the diet.
• Glutamate, Glutamine, Alanine & Aspartate are readily generated and degraded in humans and play important roles in metabolism:
  • Glutamate: central to amino acid degradation
  • Glutamine: transport ammonia to the liver
  • Alanine: transport ammonia from skeletal muscle to the liver
  • Aspartate: other roles e.g., Malate-Aspartate Shuttle