Session 1: Alcohol Metabolism and Oxidative Stress Flashcards
• Describe how alcohol is metabolised and explain how alcohol can cause liver damage. • Explain the mechanism of action of Disulfiram • Describe the production of superoxide radicals • Discuss other reactive oxygen (ROS) and reactive nitrogen (RNS) species. • Outline defences against reactive oxygen species • Explain the role of oxidative stress in disease state examples
1
Q
Where is alcohol mainly metabolised?
A
In the liver. 90%
2
Q
Where does the remaining alcohol go?
A
It is excreted passively in urine and on breath.
3
Q
What is the recommended limit of alcohol intake per week?
A
14 units/week spread over at least 3 days for both men and women.
4
Q
What is a unit of alcohol?
A
8 g or 10 ml of pure alcohol
5
Q
Describe the rate of elimination of alcohol.
A
It occurs at a constant rate. Around 7 gram per hour.
6
Q
Smaller amount of alcohol can also be oxidised by a number of enzymes. Which and where?
A
Cytochrome P450 2E1 enzyme (CYP2E1)
Catalase in the brain
7
Q
Outline the metabolism of alcohol.
A
Happens mainly in liver.
Alcohol -> Acetaldehyde -> Acetate
8
Q
What enzyme converts alcohol to acetaldehyde?
A
Alcohol dehydrogenase
9
Q
What enzyme converts acetaldehyde to acetate?
A
Aldehyde dehydrogenase
10
Q
What are the products of alcohol metabolism?
A
Acetate and two NADH from NAD+.
11
Q
Briefly outline acetaldehyde.
A
It is a toxic metabolite which in a build up of it causes hangover.
12
Q
Briefly outline acetate.
A
It can react with coenzyme A in order to form acetyl-CoA. This can in its turn be used in TCA cycle or go on to fatty acid synthesis.
13
Q
How is acetaldehyde toxic?
A
In excessive amounts it can accumulate in the liver and cause liver damage.
14
Q
What are the metabolic responses to chronic alcohol consumptions?
A
There will be a decrease in the NAD+/NADH ratio as NAD+ is used up.
There will be an increase in acetyl-CoA.
15
Q
What are the consequences of a decreased NAD+/NADH ratio?
A
- Inadequate NAD+ for conversion of lactate to pyruvate
- Inadequate NAD+ for glycerol metabolism
- Inadequate NAD+ for fatty acid oxidation
16
Q
What is a consequence of inadequate NAD+ for conversion of lactate to pyruvate?
A
Lactate will build up in the blood. This can cause lactic acidosis.
17
Q
Apart from lactic acidosis, what else can lactate accumulation cause?
A
A reduction in the kidney’s ability to excrete uric acid. This causes an accumulation of urate crystals in tissues producing gout.
18
Q
What is a consequence of inadequate NAD+ for glycerol metabolism?
A
A deficit in gluconeogenesis which can lead to hypoglycaemia.
19
Q
What is a consequence of inadequate NAD+ for fatty acid oxidation?
A
An increased synthesis of Triacylglycerol.
20
Q
What is a consequence of increased acetyl-CoA?
A
Increased synthesis of fatty acids and ketone bodies.
21
Q
What is a consequence of increased synthesis of fatty acids and ketone bodies?
A
Increased synthesis of triacylglycerol.
22
Q
What is a consequence of increased synthesis of triacylglycerol?
A
A fatty liver.
23
Q
Give an example of a drug used to treat alcohol dependence.
A
Disulfiram
24
Q
Explain the mechanism of Disulfiram.
A
Disulfiram acts on aldehyde dehydrogenase. This means that there will be an accumulation of acetaldehyde. It prevents the conversion to acetate.
25
Acetylaldehyde is toxic in its accumulation in the liver. How can then Disulfiram be a drug used to treat chronic alcohol dependence.
Acetaldehyde will accumulate yes, however it can't be turned into acetate which means the patient will get a much worse hangover. This is not a biological approach of treating CAD but rather a psychological. A much worse hangover might give you an incentive to stop as every time you drink you feel sick.
26
What are free radicals?
An atom or molecule that contains one or more unpaired electrons. This means that they want another electron and will get it from other atoms, molecules or ions.
This reaction of a radical with a molecule will typically generate a second radical and thereby propagating damage.
27
What are reactive oxygen species?
ROS are either free radicals or non free radicals with oxygen.
28
Give examples of free radical and non-free radical with oxygen that are ROS.
```
Free radical:
Hydroxyl radical OH•
Superoxide O2•-
Nonfree radical:
Hydrogen peroxide H2O2 (oxidant)
```
29
What are reactive nitrogen species? Give examples.
Free radicals or oxidants that have nitrogen in them.
Nitric oxide NO• (free radical)
Peroxynitrite ONOO- (powerful oxidant)
30
What do ROS and RNS do to cells?
They can damage nucleic acids, proteins and lipids.
31
What are the sources of ROS and RNS?
They can be both external and internal sources.
32
How do you defend from ROS and RNS?
By using antioxidants which reach with the free radicals or oxidants.
33
Outline the reactive oxygen species.
Oxygen is a biradical mean it has 2 unpaired electrons in different orbitals. It can gain an electron to form a free radical called Superoxide (O2•-).
Superoxide can gain 2 protons and an electron to form the oxidant hydrogen peroxide (H2O2)
Hydrogen peroxide can react with iron (Fe2+) in order to produce free radicals.
Hydrogen peroxide can gain an electron and a protein in order to form water (H2O) and a hydroxyl radical OH• which is the most reactive and damaging free radical as it reacts with anything. As this then gains an electron and a proton it will form water.
34
Outline the reactive nitrogen species.
Nitric oxide (NO•) reacts with superoxide (O2•-) to form peroxynitrite (ONOO-). Peroxynitrite is not itself a free radical but a powerful oxidant that can damage cells.
35
How can ROS damage DNA? What main types of damage are there?
ROS can react with a base which can lead to misfiring and mutation.
ROS can react with a sugar (deoxyribose or ribose) which can cause strand break and mutation on repair.
This can lead to cancer.
36
How can we measure oxidative damage?
By the amount of 8-oxo-dG present in cells. 8-oxo-dG is an oxidised Deoxyguanosine.
37
How can ROS damage proteins?
It can either react with the back bone of the protein or the side chain.
If it reacts with the back bone it can cause fragmentation and protein degradation.
If it reacts with the side chain it can for example create inappropriate disulphide bonds from cysteine which can lead to a change in protein structure. This can cause loss of function, gain of function or protein degradation.
38
What are disulphide bonds and how are they formed?
They are bonds between two sulphides. They play an important role in folding and protein stability of some proteins.
They are formed between thiol groups of cysteine residues.
39
What are thiol groups?
Groups containing -SH.
40
What happens if inappropriate disulphide bonds form?
This can occur if ROS takes electrons from cysteines. This can cause misfolding, cross linking and disruption of function.
41
Explain how ROS can damage lipids.
Free radicals such as OH• can gain a hydrogen atom from a polyunsaturated fatty acid in membrane lipid. This causes the lipid to form a lipid radical. This lipid radical in its turn can react with oxygen to form a lipid peroxyl radical. This is a chain reaction occurring as the lipid peroxyl then continues to extract hydrogen from nearby fatty acids and so the reaction continues.
42
How can this be damaging to the body?
This can cause the hydrophobic environment of the bilayer to be disrupted and the membrane integrity might fail.
43
What are some endogenous sources of biological oxidants?
```
Electron transport chain*
Peroxidases
Nitric oxide synthases*
Lipooxygenases
NADPH oxidases*
Xanthine oxidase
Monoamine oxidase
* most important (ENN)
```
44
Give some examples of exogenous biological oxidants.
Radiation
Pollutants
Drugs (e.g. primaquine)
Toxins (e.g. herbicides)
45
How can the electron transport chain be a source of exogenous biological oxidants?
Electrons are used to power the complexes used to transport H+ from the matrix to the intermembrane space. Occasionally these electrons can accidentally escape the chain and react with dissolved O2 to create superoxide.
46
What are nitric oxide synthases?
They are enzyme which converts Arginine to Citrulline and nitric oxide. They produce nitric oxide.
47
What is needed for the nitric oxide to convert arginine into citrulline?
NADPH and O2 which is converted into NADP+ and H2O.
48
Give three examples of Nitric oxide synthases.
iNOS (inducible nitric oxide synthase)
eNOS (endothelial nitric oxide synthase)
nNOS (neuronal nitric oxide synthase)
49
What is nitric oxide used for?
It is a signalling molecule that is important in neurotransmission, it's a vasodilator and also important for S-nitrosylation.
50
What is respiratory burst?
When there is a rapid release of superoxide and hydrogen peroxide from phagocytic cells like neutrophils and monocytes.
51
Why would respiratory burst happen if ROS are damaging?
They are in fact not always damaging and play an important part in the immune system.
52
How do respiratory burst, NADPH oxidase and iNOS work together in antimicrobial defence system?
NADPH oxidase turns NADPH into NADP+ which causes O2 to form superoxide. Superoxide is then converted into hydrogen peroxide which in its turn reacts with chloride ion to form HOCl• which is also called Hypochlorite by an enzyme called myeloperoxidase. Hypochlorite then kills bacteria.
iNOS also produce nitric oxides which can react with superoxide and create peroxynitrite which can also kill bacteria.
53
What is chronic granulamatous disease?
A genetic defect in NADPH oxidase which leads to an increased susceptibility to bacterial infections.
54
Give two examples of cellular defences against ROS.
```
Superoxide dismutase (SOD)
Catalase
```
55
What does superoxide dismutase do?
SOD is an antioxidant that come in different forms. There are three isoenzymes which are Cu+-Zn2+ (cytosolic) Cu+Zn2+ (extracellular) and Mn2+ (mitochondrial)
They connect superoxide to hydrogen peroxide and oxygen.
56
But hydrogen peroxide is still an oxidant. How does this even help?
Catalase will then convert hydrogen peroxide into water and oxygen. This is a widespread enzyme important in immune cells to protect against oxidative burst/respiratory.
57
Give another example of a cellular defence mechanism against ROS.
Glutathione
58
What is glutathione?
It is a tripeptide consisting of glycine, cysteine and glutamate. In its reduced form it has a SH-group and called GSH (glutathione-SH).
59
How does glutathione work?
The thiol group (-SH) donates an electron to ROS. This causes GSH to react with another GSH to form disulphide GSSG (glutathione-S-S-glutathione). This means that the ROS no longer works as it is saturated, it will no longer cause damage.
60
What enzymes are used in the glutathione redox?
Glutathione peroxidase and glutathione reductase.
61
What does glutathione peroxidase do?
It causes hydrogen peroxidase to convert into water as 2 GSH are converted into one GSSG.
62
What does glutathione reductase do?
Since GSSG can no longer be used to reduce ROS it is rendered 'useless' so it needs some way to be converted back. This is done by glutathione reductase. The enzyme converts NADPH into NADP+ and GSSG into 2 GSH.
63
How is the pentose phosphate pathway essential for protection against free radical damage?
The NADPH that is oxidised into NADP+ comes from the pentose phosphate pathway.
64
What specific cofactor is needed for glutathione peroxidase to work?
Selenium
65
What important enzyme is used in the pentose phosphate pathway?
Glucose 6-phosphate dehydrogenase.
66
Why is this pathway important?
It produces C5 ribose required for synthesis of nucleotides and DNA/RNA.
However maybe more importantly it is an important source of NADPH.
67
Why is NADPH important?
It is a reducing power for biosynthesis.
It reduces GSSG into GSH to protect against free radical damage.
It is an essential part of detoxification reactions.
68
What are free radical scavengers?
Different molecules which donate an electron and a proton to a free radical in order to protect damage done to cells. This is a nonenzymatic reaction!
69
Give examples of free radical scavengers.
Vitamin E, Vitamin C, Carotenoids, Flavonoids, Uric acid, Melatonin.
70
What is vitamin E?
A lipid soluble antioxidant that is important for protection against lipid per oxidation in for example the cell membrane.
71
Why is vitamin C important?
Not only for scurvy and a cofactor in propyl hydroxyls but it also plays an important role in 'regenerating' Vitamin E's reduced form in order for it to be able to react again.
72
What is oxidative stress?
When the oxidants outweigh the defences against them.
73
What is galactosaemia?
When there is a deficiency in one of the three enzymes that converts galactose into the pathway for either glycolysis or glycogenesis (uridyl transferase, UDP epimerase, galactokinase). This causes an increase in galactose which can go into another pathway required the enzyme aldose reductase. Aldose reductase converts galactose into galactitol by using NADPH and turning it into NADH+. Galactitol can accumulate in lens of eye and cause osmotic pressure which leads to cataracts. Also the decrease of NADPH means that there will be a compromised defence against free radical which can lead to inappropriate disulphide bridge formations in the lens of the eye as GSSG can't be reduced back into GSH.
74
What are the consequences of G6PDH deficiency?
G6PDH converts NADP+ into NADPH. This means that if there is a deficiency the levels of NADPH will decrease meaning less GSSG can be reduced back into 2 GSH meaning less protection against free radicals and oxidants. This can therefore cause oxidative stress. This leads to an increase in for example hydrogen peroxide leading to lipid peroxidation and protein damage.
75
What is the protein damage associated with G6PDH deficiency?
Since GSSG can't reduce back into GSSG there are aggregates of cross-linked haemoglobin formed.
76
What are the aggregates of cross-linked haemoglobin called?
Heinz bodies.
77
What is the consequence of Heinz bodies forming?
The Heinz bodies bind to cell membrane and alter the rigidity. This causes increased mechanical stress when cells squeeze through small capillaries. Due to this Heinz bodies are removed by the spleen causing a decrease in haemoglobin and red blood cells. This can cause anaemia.
78
Explain the metabolism of paracetamol.
This is done in hepatocytes. In small doses it can safely be metabolised into glucuronide or sulphate.
79
What happens if there is an overdose of paracetamol?
It can't be metabolised into glucuronie or sulphate anymore. It instead converts into NAPQI which is directly toxic to cells.
80
What are the toxic effects of NAPQI?
Lipid per oxidation
Damage to proteins
Damage to DNA
81
How is NAPQI treated?
Glutathione is an anti-oxidant which reduces NAPQI into a non-toxic form. Acetylcysteine is used to replenish or increase the levels of glutathione in order to lower the levels of NAPQI.
82
Workbook examples:
Follows
83
What is glucose 6-phopshate dehydrogenase?
A rate limiting enzyme of the pentose phosphate pathway.
84
What does an enzyme activity of 13 U/g tissue mean?
It means that 13 units convert 13 micro molar substrate per minute in 1 gram of tissue. It is essentially a measure of density of enzymes.
85
Why is there an increased G6PD activity in red blood cells?
Because RBCs have iron and oxygen which means they are targets for ROS. The NADPH formed by pentose phosphate pathway is then needed for glutathiones in order for GSSG to be reduced back into 2 GSH for example.
86
What is evidence of anaemia in a patient with:
Haemoglobin : 90 : ref - 130-180
RBCs : 3.5 : ref - 5
Reticulocytes : 3 : ref - 0.5-1.5
Unconjugated bilirubin : 300 : ref - 2-14
Low Hb count
High reticulocyte count
Low RBCs
A lot of unconjugated bilirubin.
87
How can an increased level of unconjugated bilirubin be a sign of anaemia?
Bilirubin is formed from breakdown of red blood cells. This usually means that there is a decrease in number of red blood cells and usually something wrong with the RBCs as well.
88
What are indications of jaundice?
Yellow sclerae
| High unconjugated bilirubin count and therefore dark urine.
89
Why does the absence of G6PDH produce sensitivity to anti-malarial medicine primaquine?
Primaquine is a free radical. Since the G6PDH levels are already low this means that primaquine cannot be reduced by GSH since all are stuck in GSSG as the NADPH levels are low. This causes aggregation of cross-linked haemoglobin - Heinz bodies.
90
What could cause an absence of G6PDH in red blood cells?
An x-linked recessive mutation is common. Also increased enzymatic decay due to malformation, conformational change, just less of it due to mutation.
91
Where else than the RBCs can you find a high level of G6PDH?
In the lens of the eye as it also has a lot of free radicals.
92
Why might a patient's G6PDH levels go up and down a lot?
Reticulocytes are immature red blood cells. As they mature they will still have the same G6PDH content. However as they become mature their G6PDH might degrade quickly, this means that initially the count can be high initially to go down.
