Session 4 ILO's - Oxidative stress and Protein and amino acid metabolism

Question 1

Describe the metabolism of alcohol

Answer 1

Technically, it is the ethanol that gets metabolised

Location:

• Most (>90%) alcohol is metabolised by liver
• Remainder excreted passively in urine and on breath.

Process:

• Alcohol oxidised to acetaldehyde by ALCOHOL DEHYDROGENASE
• That reaction converts NAD+ ——-> NADH
• And then acetaldehyde is oxidised to acetate by ALDEHYDE DEHYDROGENASE
• That reaction converts NAD+ ——-> NADH
• Conjugated to coenzyme A to form acetyl-CoA and metabolised in TCA cycle or utilised for fatty acid synthesis

Other:

• Smaller amounts of alcohol can also be oxidized by the cytochrome P450 (for detoxifying drugs, ect) 2E1 enzyme (CYP2E1), or by catalase in brain.

Question 2

Draw out alcohol metabolism

Answer 2

Question 3

Explain how the metabolism of alcohol can cause damage to the liver

Answer 3

Self assessment ‘model answer’ to this question:

• The intermediate metabolite of alcohol metabolism, acetaldehyde, is toxic to liver cells.

Acetaldehyde toxicity normally kept to a minimum by aldehyde dehydrogenase (low Km for acetaldehyde)
* Prolonged and excessive alcohol consumption can cause sufficient acetaldehyde accumulation to cause
liver damage
* Excess NADH and Acetyl-CoA lead to
changes in liver metabolism:

This can lead to
* “Fatty liver”
* Alcoholic hepatitis
* Alcoholic cirrhosis

Question 4

What are the recommended limits for alcohol ?

Answer 4

14 units per week spread over at least 3 days for both men and women

Question 5

What causes hangover?

Answer 5

• Accumulation of acetaldehyde
• Ethanol at high concentrations inhibits ADH, so you become dehydrated

Both of these contribute to hangover feeling

Question 6

Describe Acetaldehyde

Answer 6

• Toxic Metabolite

Question 7

What is the significance of acetate?

Answer 7

• Can be conjugated to coenzyme A
• To form acetyl-CoA and metabolised in TCA cycle
• Or used for fatty acid synthesis

Question 8

Aside from damage to the liver, what other metabolic responses/consequences occur due to chronic alcohol consumption

Answer 8

Watch videos to understand explanations

Question 9

Explain the mechanism of action of Disulfiram in the treatment of alcohol dependence.

Answer 9

• Disulfiram is a drug
• Can be used as an adjunct in the treatment of chronic alcohol dependence
• It is an inhibitor of aldehyde dehydrogenase leading to a build up of the metabolite acetaldehyde
• If patient drinks alcohol, acetaldehyde will accumulate causing symptoms of a ‘hangover’ and they will feel very sick
• This creates an immediate negative association between drinking and being hungover - classical conditioning.

Question 10

Describe the concept of oxidative stress

Answer 10

• We have evolved to live in an oxygen rich environment, so we have lots of protective mechanisms against damaging oxidative reactions
• We have defensive mechanisms which can generally cope with any production of reactive oxygen species and reactive nitrogen species
• The oxidative stress occurs if those defence systems become compromised or there is too much oxidative damage for those defence systems to cope with and the protective mechanisms get completely swamped. The system is then out of balance.

Question 11

Why is oxidative stress such an important topic to understand? (Give 10 disease states relevant to this)

Answer 11

• Over recent years, it has become apparent that oxidative stress has played a role in a very wide range of disease states

(Cellular damage caused by ROS & RNS is a significant component in a wide range of disease states)

Question 12

Describe how reactive oxygen species come about

Answer 12

• Oxygen itself is a free radical, but because it has 2 unpaired electrons in different orbitals (biradical), molecular oxygen is quite stable
• Buț molecular oxygen can gain an electron, to produce a superoxide (which is a free radical that can cause damage, and lead to the generation of other free radicals)

Question 13

Describe the production of superoxide radicals by mitochondria

Answer 13

Model answer from self assesment:

‘During oxidative phosphorylation about 0.1 - 2% of electrons do not reach the end of the electron transport chain and they prematurely reduce oxygen to from superoxide radicals(O2 -).’

Important: Superoxide is an important source of the other reactive oxygen species (hydrogen peroxide, hydroxyl radical, nitric oxide and peroxynitrite)

Question 14

Discuss other reactive oxygen (ROS) and reactive nitrogen (RNS) species.

Answer 14

Hydrogen peroxide (H202):
- Formed from Superoxide
- Superoxide gains 2 hydrogens and an electron to form hydrogen peroxide
- Not actually a free radial but it is quite damaging to cells and can react with Fe2+ to produce free radicals (hydroxyl radial)
- Readily diffusible

Hydroxyl radical:
- Formed from Hydrogen peroxide gaining an electron and Hydrogen
- H2O is produced swell
- Most reactive and damaging free radical
- Reacts with anything (can pinch an electron from anything it wants to)
- The hydroxyl radical gains an electron and hydrogen, and is converted back to water

RNS:
Peroxynitrite:
- Superoxide can react with nitric oxide to produce peroxynitrite
* Peroxynitrite is not itself a free radical, but is a powerful oxidant that can damage cells

Question 15

Briefly explain how a respiratory burst is produced by some leucocytes

Answer 15

Some cells of the immune system, such as neutrophils and monocytes, when stimulated can rapidly produce a release of ROS which is known as a respiratory burst (sometimes called oxidative burst). The respiratory burst is produced by a membrane-bound enzyme complex termed NADPH oxidase. This enzyme is present in the cell membrane and it transfers electrons from NADPH across the membrane to couple these to molecular oxygen to generate superoxide radicals.

Question 16

Name the cellular defences against reactive oxygen species

Answer 16

1) Superoxide dismutase (SOD) and catalase
2)Glutathione
3) Vitamin C
4) Vitamin E

Question 17

List 6 agents in cells which protect against reactive oxygen species

Answer 17

Any of following:
* Superoxide dismutase
* Catalase
* Glutathione
* NADPH
* Antioxidant vitamins (e.g. C and E)
* Other antioxidants free radical scavengers in the
diet (e.g. carotenoids, Flavonoids)

Question 18

Outline cellular defences against reactive oxygen species

Describe the primary defence (Superoxide dismutase and Catalase)

Answer 18

Superoxide dismutase (SOD):
- Converts superoxide to hydrogen peroxide and oxygen
- Primary defence because superoxide is strong initiator of chain reactions
- 3 isoenzymes:
* Cu+-Zn2+ Cytosolic
* Cu+-Zn2+ Extracellular
* Mn2+ Mitochondria

Catalase:
- Converts the hydrogen peroxide to water and oxygen (superoxide dismutase and catalyse work together!!)
- Widespread enzyme. Important in immune cells to protect against oxidative burst
- Declining levels in hair follicles with age may explain grey hair!

Question 19

Briefly explain the relationship between NADPH and glutathione

Answer 19

There is a recycling system between NADPH and glutathione. NADPH reduces oxidised glutathione to its reduced form, via the enzyme GSH reductase. The reduced glutathione is then available to be oxidised by reactive oxygen species, thus removing ROS.

Question 20

Outline cellular defences against reactive oxygen species

Glutathione

Answer 20

Glutathione:

• Tripeptide synthesised by body to protects against oxidative damage
• Thiol group of Cys donates e− to ROS. GSH then reacts with another GSH to form disulphide (GSSG).
• Glutathione peroxidase requires Selenium
• GSSG reduced back to GSH by glutathione reductase which catalyses the transfer of electrons from NADPH to disulphide bond
• NADPH from pentose phosphate pathway is therefore essential for protection against free radical damage

Question 21

Outline cellular defences against reactive oxygen species

Vitamin E and C

Answer 21

They are free radical scanagers:

Free radical scavengers reduce free radical damage by donating hydrogen atom (and its electron) to free radicals in a nonenzymatic reaction

Vitamin E:
- Vit E Donates an electron
- Lipid soluble antioxidant
- Important for protection against lipid peroxidation

Vitamin C:
- Vit C regenerates the reduced form of Vit E
- Water soluble antioxidant
- Important role in regenerating reduced form of Vitamin E

Question 22

Give 4 more examples of free radical scavengers

Answer 22

• Carotenoids
• Flavonoids
• Melatonin
• Uric acid

Question 23

Name 5 oxidants and 6 defences

Answer 23

Question 24

Define oxidative stress

Answer 24

Oxidative stress occurs when the production of ROS is excessive or antioxidant levels are low in a cell, and the balance is shifted in favour of ROS.
Usually, cells have sufficient antioxidant power to cope with the normal production of ROS.

Question 25

Explain the role of oxidative stress in disease states.

Galactosaemia

Answer 25

Galactosaemia:

Background info
- Deficiency in any of the 3 enzymes (galactokinase, uridyl transferase and UDP-galactose epimerise) favours the conversion of galactose to galactitol (resulting in galactosaemia)
- increased activity of aldose reductase consumes excess NADPH

Basically:
- Occurs when the NADPH gets used up (galactose is redirected down different pathway)
- Reduced defence against ROS
- Crystallin protein is denatured in the lens of the eye - causing cataracts

G6PDH deficiency:
- Occurs when there is a deficiency in G6PDH in the pentose phosphate pathway which is responsible for regenerating NADPH
- Unable to regenerate GSH from GSSG (NADPH not available for glutathione reductase)
- Lower GSH means less protection against damage from oxidative stress (ie infection, drugs like anti-malarial, and broad beans)

• Less H2O2 removed
  = lipid peroxidation and protein damage (HEINZ BODIES) which leads to haemolysis

Question 26

Explain the role of oxidative stress in disease states.

G6PDH deficiency

Answer 26

G6PDH deficiency:
- Occurs when there is a deficiency in G6PDH in the pentose phosphate pathway which is responsible for regenerating NADPH
- Unable to regenerate GSH from GSSG (NADPH not available for glutathione reductase)
- Lower GSH means less protection against damage from oxidative stress (ie infection, drugs like anti-malarial, and broad beans)

• Less H2O2 removed
  = lipid peroxidation and protein damage (HEINZ BODIES) which leads to haemolysis

Question 27

Explain lipid peroxidation and give an example of a disease where it is prevalent

Answer 27

Initiation
- Free radicals can extract hydrogen from lipids in the lipid membrane and this can form a lipid radical

Propagation
- The lipid radial reacts with oxygen to form a lipid peroxyl radial = results in a chain reaction

Result: membrane integrity fails (damage to lipid membrane)

Atherosclerosis - occurs in LDL particles which are engulfed by macrophages which settle in vessel walls

Question 28

Explain how metabolism of Paracetamol can be toxic (overdose)

What is the antidote to Paracetamol overdose?

Answer 28

Normally, Paracetamol is metabolised with conjugation with Glucuronide or Sulphate

However, in an overdose, these enzymes become saturated. Paracetamol is diveerted and results in toxic metabolite NAPQI (has direct toxic effects). It conjugates with Glutathione which leaves cells susceptible to ROS (lipid peroxidation, damage to proteins, damage to DNA)

Antidote: Acetylcysteine = helps to replenish glutathione levels. But needs to be given Acetylcysteine within 8 hours for good prognosis.

Question 29

What is a clinical sign of G6PDH Deficiency?

Answer 29

Heinz bodies

Question 30

What are Heinz bodies and what do they do?

Answer 30

• Dark staining within red blood cells resulting from precipitated haemoglobin
• Bind to cell membrane altering rigidity
• Increased mechanical stress when cells squeeze through small capillaries
• Spleen removes bound Heinz bodies resulting in “blister cells”

Question 31

Sources of endogenous source of biological oxidants

Answer 31

• Electron transport chain
• Nitric oxide synthase
• NADPH oxidase

Question 32

Describe ROS damage to DNA

Answer 32

Watch lecture to understand how this diagram works and be able to explain it

Question 33

Describe ROS damage to proteins

Answer 33

Watch lecture to understand how this diagram works and be able to explain it

Question 34

Describe the importance of disulphide bonds in the vein of disease

Answer 34

Watch lecture to understand how this diagram works and be able to explain it

Question 35

Describe ROS damage to lipids

Answer 35

Watch lecture to understand how this diagram works and be able to explain it

Question 36

What is the typical dietary intake of protein per day?

Answer 36

100g per day, which averages about 16 grams of nitrogen going into your body each day

Question 37

Describe what this image is showing

Answer 37

• We take in nitrogen in the form of dietary protein
• That nitrogen is entering the amino acid pool, and that’s in equilibrium with the proteins in our body and also we use some of that nitrogen for nitrogen containing compounds
• Our losses of nitrogen, are our nitrogen waste products, mainly urea in urine, faeces and everyday wear and tear of living, e.g hair, fingernails, skin cells
• These losses are in balance with the nitrogen that we take in from our diet

Question 38

Explain nitrogen balance

Answer 38

Question 39

Explain protein turnover

Answer 39

• The proteins in our body are constantly being recycled and remade by free amino acids in the body
• This amino acid pool can arise from:
• Synthesis of amino acids from other compounds in the body
• Dietary proteins, absorbed from GI Tract
• From recycling of proteins in our body
• We can break down the free amino acids and use their carbon skeleton for energy production
• When that occurs, we need t deal with the amine group of the amino acid
• The problem with metabolising amino acids, is that we potentially liberate ammonia, which is really toxic to cells
• Humans deal with that toxicity by converting those amine groups into urea, and we exert that urea in the urine.
• If you are going to use the carbon skeleton for energy, there are 2 types of amino acids:

1) Glucogenic amino acids can be used to synthesise glucose by gluconeogenesis

2) ketogenic amino acids, the carbon skeletons can form ketone bodies

Question 40

Briefly summarise the concept of protein turnover

Answer 40

• The recycling of our constituent proteins of our cells, resynthesisng them, using those amino acids that are released from degradation to synthesise new proteins.

Question 41

Describe how amino acids are catabolised in the body.

Answer 41

Need to remove nitrogen from amino acids which can then be by directing it to the liver, where it can be converted to urea, and that urea can be safely excreted in urine, without ammonia toxicity forming in the body.

There are 2 pathways which the body uses to facilitate the removal of nitrogen from the amine groups of amino acids:
Transamination and deamination

Transamination:
- Involves the transfer of amino group from one compound to another
Most aminotransferases use alpha-ketoglutarate to move amino group to glutamate (AST uses oxaloacetate to move amino group to glutamate) - requires Vit B6 derivative
- Key: Alanine aminotransferase (ALT) converts alanine to glutamate using alpha-ketoglutarate
- Key: Aspartate aminotransferase (AST) converts glutamate to aspartate using oxaloacetate

Deamination:
- Liberates the amino acid group as ammonia (in liver and kidneys)
- Involves D (and L) amino acid oxidases which are enzymes that convert amino acids to keto acids and NH3
- Ammonia is converted to urea in the urea cycle or excreted directly in urine

Question 42

Define the terms glucogenic and ketogenic amino acids.

Answer 42

Glucogenic amino acids - can be used to make glucose in gluconeogensis
e.g. Alanine

Ketogenic amino acids - can be used to make ketone bodies
e.g. Leucine

Both glucogenic and ketogenic:
e.g. Phenylalanine

Question 43

Explain the clinical consequences of a defect in phenylalanine metabolism.

Answer 43

• Phenylketonuria is an inborn error of metabolism (PKU)
• Caused by deficiency in phenylalanine hydroxylase
• Accumulation of phenylalanine in tissue, plasma & urine
• Phenylketones in urine
• Musty smell

Question 44

Describe the treatment of PKU

Answer 44

• Strictly controlled low phenylalanine diet enriched with tyrosine
• Avoid artificial sweeteners (contain phenylalanine)
• Avoid high protein foods such as meat, milk, and eg

Question 45

Give 5 symptoms of PKU

Answer 45

• Severe intellectual disability
• Developmental delay
• Microcephaly (small head)
• Seizures
• Hypopigmentation

Question 46

What pathways are affected in PKU?

Answer 46

• Noradrenaline
• Adrenaline
• Dopamine
• Melanin
• Thyroid hormone
• Protein synthesis

Question 47

Explain the clinical relevance of measuring creatinine in blood and urine.

Answer 47

• Creatinine is a useful clinical marker
• It is the breakdown product of creatine & creatine phosphate in muscle
• Usually produced at constant rate depending on muscle mass (unless muscle is wasting)
• Filtered via kidneys into urine
• Creatinine urine excretion over 24h
  proportional to muscle mass
• Provides estimate of muscle mass
• ## Also commonly used as indicator of renal function (raised plasma level and low urine level on damage to nephrons)

Question 48

What is the reference range for men and women, for the amount of creatinine excreted per day?

Answer 48

• Men 14-26 mg/kg
• Women 11-20 mg/kg

Question 49

Describe how ammonia is metabolised in the body.

OR

Describe in general terms how amino acids are degraded in the body and list the products of their degradation.

Answer 49

Degraded to smaller molecules largely in the liver. Each amino acid has its own pathway of catabolism but the pathways share common features:
* The C-atoms are converted to intermediates of carbohydrate metabolism (glucogenic amino acids) or lipid metabolism (ketogenic amino acids)
* The N-atoms are usually converted to urea for excretion in the urine but some may be excreted directly as ammonia and some may be converted to glutamine and used for the synthesis of purines and pyrimidines.
* The first step in the various pathways usually involved the removal of the – NH2 group by transamination or deamination.
Products = urea, pyruvate, acetyl~CoA, α-ketoglutarate, oxaloacetate, succinate and fumarate.

Question 50

Pentose phosphate pathway

Question 51

Explain defects in amino acid metabolism

Answer 51

Excessive breakdown of proteins can occur in Cushing’s Syndrome (due to excess cortisol) - weakens skin structure leading to striae/stetch marks

Question 52

Explain how some amino acids can be converted to glucose (glucogenic), some can be converted to ketone bodies (ketogenic) and others can be converted to both glucose and ketone bodies (glucogenic & ketogenic).

Answer 52

An early step in the catabolism of an amino acid is the removal of the amino group (-NH2). This is converted to urea (CO(NH2)2) and excreted from the body in the urine. The remaining C-skeletons of the amino acids are converted to one or more of the following organic precursors: pyruvate, oxaloacetate, fumarate, a- ketoglutarate, succinate, acetyl~CoA.
Amino acids that produce acetyl~CoA (e.g. leucine, lysine) are described as ketogenic as acetyl~CoA can be used for the synthesis of ketone bodies.
Amino acids that give rise to the other products (glutamic, aspartic, serine) are described as glucogenic as they can be used for glucose synthesis by gluconeogenesis. Some of the larger amino acids (isoleucine, threonine, phenylalanine, tyrosine and tryptophan) are both ketogenic and glucogenic as they give rise to both acetyl~CoA and one of the other organic precursor molecules.

Question 53

Describe the processes that produce ammonia in the body.

Answer 53

Ammonia is produced in the body by the deamination of amino acids. It is also absorbed from the gut where it is produced by bacterial action.
Several deaminase enzymes of varying specificity are found in the liver and kidney that react with amino acids to remove the NH2-group as free NH3 (NH4+): L&D-amino acid oxidases are low specificity enzymes that convert amino acids to keto acids and NH3.
Glutaminase is a high specificity enzyme that converts glutamine to glutamate + NH3.
Glutamate dehydrogenase is a high specificity enzyme that catalyses the reaction: Glutamate + NAD+ + H2O ↔ α-ketoglutarate + NH4+ + NADH + H+
It is important in amino acid metabolism by the liver as it is involved in both the disposal of amino acids (glutamate → α-ketoglutarate + NH4+) and the synthesis of non-essential amino acids (α-ketoglutarate → glutamate).