Reactive Species and LDL Oxidation

Question 1

What are ROS?

Answer 1

ROS are not necessarily radicals, those with an unpaired electron, but can also just be highly reactive chemical species.

Question 2

What are superoxide anions?

Answer 2

Superoxide anions are the most abundant form of ROS, with 1.5kg produced by the body per year. They are however, relatively weak in terms of reactivity, especially compared to the potent hydroxyl radicals.

Question 3

How are superoxide anions produced?

Answer 3

ROS are produced as a by product of various metabolic processes – ultimately due to the high proportion of oxygen in our atmosphere.

Superoxide anions can be produced by three mechanisms:
• In mitochondria by leakage from the ETC
• NADPH Oxidase
• Xanthine Oxidase

Question 4

How are superoxide anions degraded?

Answer 4

These are converted by Superoxide Dismutase (SOD) into hydrogen peroxide, which is also produced by amino acid oxidases. Since this too is a ROS, albeit not a radical one, this is broken down to water via various enzymes including catalase and glutathione peroxidase (GPX).

Question 5

What can be a by product of superoxide degradation?

Answer 5

However, the hydrogen peroxide produced can also be, in rare circumstances, converted to hydroxyl radicals in the Fenton Reaction, a process catalysed by the presence of free transition metal ions; primarily Fe2+ and Cu2+.

Question 6

Describe the scheme of the fenton reaction.

Answer 6

Fe2+ + H2O2 -> Fe3+ + OH- + OH*

Question 7

How is the fenton reaction inhibited?

Answer 7

To prevent this from occurring, the required free metal ions are kept rare in the body through their chelation within proteins, including haemoglobin, transferrin, ferritin and caeruloplasmin.

The free ion ‘sponges’ can be overwhelmed in circumstances such as after serial blood transfusions (eg in treatment of thalassaemia), requiring co-treatment with ion chelators.

Question 8

What mechanisms other than SOD are used to prevent damage by superoxide?

Answer 8

As well as SOD and the various enzymes used to directly degrade ROS, antioxidants (commonly Vitamin C) are used to counteract them by participating in termination reactions – acting as an acceptor for the unpaired electron.

Question 9

How can ROS damage DNA? what does this lead to?

Answer 9

• Scission of the deoxyribose ring
• Base damage
• Single or double strand breaks
• Cross-linkage

This can lead to heritable mutations, translational errors and inhibition of protein synthesis. While there are many DNA repair mechanisms that restore the DNA code, they can be overwhelmed or themselves damaged by ROS.

Question 10

What non-mutagenic effect do ROS have on DNA?

Answer 10

Free radicals have been implicated in ageing and telomerase shortening, (see 2008 – ageing and 3013 – replicative senescence). In degenerative diseases such as CVD there is a tendency towards shortened telomeres.

Question 11

What effect do ROS have on proteins?

Answer 11

ROS induce aggregation in proteins by causing cross-linking and fragmentation, and is also responsible for breakdown modifications of thiol groups which may impair function. This can of course have a wide variety of effects, but the most common is modified ion transport – particularly increased calcium influx.

Question 12

What are ROS used for in the body?

Answer 12

ROS also serve several useful functions in the body. They are important in the first line of immune defence, neutrophils forming large amounts of hydrogen peroxide (via increased activity of NADPH oxidase and SOD) which is then converted by myeloperoxidase to hypochlorite (HOCl) using chloride ions. This hypochlorite is used to destroy the targeted pathogens or host cells.

Question 13

Give the reaction scheme that produces hypochlorite.

Answer 13

H2O2 + Cl- + H+ -> HOCl + H2O

Catalysed by myeloperoxidase

Question 14

What does myeloperoxidase deficiency cause?

Answer 14

Chronic granulomatous disease is caused by a genetic deficiency of myeloperoxidase, and results in increased fungal and bacterial infection susceptibility.

Question 15

How are ROS thought to regulate the endothelium?

Answer 15

Low levels of hydrogen peroxide is thought to be a beneficial vasodilator via its regulation of K+ channels.
Some speculate that it is in fact the thus-far unattributed endothelium dependent hyper-polarising factor (EDHF).

Even the more dangerous hydroxyl radicals may have a role in vasoactivity through regulation of cyclo-oxygenase, and subsequently on the formation of prostaglandins.

Nitric oxide is also a free radical – though an RNS rather than ROS – and has a multitude of beneficial effects in the vasculature which will be examined later.

Question 16

What risk factor are ROS thought to be a component of?

Answer 16

While ROS and RNS are not considered a primary risk factor for CVD directly, they are linked through smoking, a habit which has the most influence on early death from CVD and associates with low plasma ascorbate and endothelial damage. Each cigarette puff contains an estimated 1015 free radicals.

Question 17

How does LDL oxidation occur?

Answer 17

LDLs carry cholesterol, phospholipids, cholesteryl esters and PUFAs. PUFAs, the molecules required for cell membrane synthesis and eicosanoid formation, are highly susceptible to oxidation.

LDL oxidation is caused not just by ROS and RNS, but also exposure to transition metal ions, again primarily copper and iron.

Question 18

What protects LDLs from oxidation?

Answer 18

A degree of protection from oxidation is provided to the LDLs by their carrying 3-6 vitamin E molecules – usually preventing ocidation for the 1-2 day lifestime of the LDL.

Question 19

What is the first step in PUFA oxidation?

Answer 19

PUFAs contain a series of non-conjugated double bonds, the carbon atoms between which are targeted by reactive species. The reactive species abstract a hydrogen from the saturated carbon atom, producing a radical.

Question 20

What is the second step in PUFA oxidation?

Answer 20

The PUFA then undergoes rearrangement, with one of the adjacent double bonds moving towards the radicalised base to produce a conjugated diene with the other adjacent double bond, the radical electron being transferred two carbons down.

Question 21

What is the final step in PUFA oxidation?

Answer 21

The radical associated with a conjugated diene can react with O2, which is then hydrogenated by a species which accepts the unpaired electron and produces the final lipid peroxide.

Question 22

How can LDLs be oxidised other than PUFA oxidation?

Answer 22

ApoB can also be modified directly by oxidising agents, predominantly peroxynitrite and hypochlorite. This is thought due to the high level of chlorinated and nitrated tyrosine residues found in proteins within atherosclerotic plaques.

Cholesterol can be non-enzymatically oxidised also, being converted to 7-hydroperoxycholesterol and on to 7α-hydroperoxycholesterol, 7β-hydroperoxycholesterol or 7-ketocholesterol.

Question 23

What are the three phases of LDL oxidation?

Answer 23

Oxidation occurs in three phases: the lag phase, propagation phase and decomposition phase.

Question 24

What is the lag phase of LDL oxidation?

Answer 24

In the lag phase, depletion of endogenous LDL antioxidants (e.g. vitamin E, carotenoids, α-tocopherol, lycopines, retinylstearate) leads to oxidation.

Question 25

What is the propagation phase of LDL oxidation?

Answer 25

In the propagation phase there is rapid PUFA oxidation to lipid peroxides, but with no damage to ApoB100. These LDLs are called minimally modified (mm)LDLs.

Question 26

What is the decomposition phase of LDL oxidation?

Answer 26

The decomposition phase is characterised by the reactive aldehydes produced by PUFA/phospholipid oxidation reacting with lysine residues in ApoB100 to form Schiff’s Bases.

This electronegativity causes them to be recognised as damaged protein, resulting in oxidized LDL that can be recognised by SR-A and CD36. These recognise highly negative ligands in order to clear the negative products of apoptosis and necrosis.

Question 27

How can LDL oxidation be measured?

Answer 27

Susceptibility of LDL to oxidation can be evaluated by incubation in vitro with copper to measure the lag phase, and to determine the effects of pro-and antioxidants (e.g. water soluble ascorbate).

Many of the lipid oxidation products have direct biological effects (immunogenic, genotoxic and cytotoxic) whose measurement can be used to estimate the bodily oxLDL levels.

The level of isoprostane, a vasodilator, in the blood and urine is an excellent marker.

Question 28

What evidence is there that LDL oxidation occurs as part of atherosclerosis pathology?

Answer 28

• Presence of oxLDL in Atherosclerotic plaques
• Trace amounts of mmLDL found in plasma
• Antibodies raised against LDLs found in plasma
• Lipid oxidation products found in blood, urine and tissue samples

• HDLs are atheroprotective and prevent oxidation
o HDLs allow themselves to be oxidised but unlike LDLs contain paraoxonase to remove lipid peroxides

• CVD patients tend to have lower plasma levels of antioxidant vitamins

Question 29

How are Oxidised LDLs are linked to Infections?

Answer 29

The presence of oxLDL has been linked to the immune response that responds to infections such as those linked with atherosclerosis (including chlamydia and helicobacter). They activate expression of Toll-like Receptor 4 in macrophages which recognise LPS, a key component of gram-negative bacterial surfaces, to initiate the inflammatory immune response.

Question 30

What effect on the immune system do oxLDLs have?

Answer 30

Due to their foreign nature, the body raises IgM antibodies against oxLDLs. Their role remains unclear, though some suggest a protective role, but the level of anti-oxLDL antibodies has been shown to rise with CVD progression.

Question 31

How are oxLDLs recognised by the immune system?

Answer 31

Oxidised LDLs can have many modifications, and thus many epitopes to be targeted by antibodies.

One common epitope is phosphorylcholine, which although present in non-oxidised LDLs is exposed by the structural changes associated with oxidation of the fatty acid chain. Antibodies against this epitope are named EO6.

Oxidised LDLs activate this immune response indirectly, stimulating splenocytes to activate B-cells via IL-5.

Question 32

What shares the phosphorylcholine epitope found on oxLDLs?

Answer 32

The same antigen is found on bacteria such as Staphylococcus pneumoniae, and on apoptotic cells.

Antibodies against this, and hence the same as EO6, appear to belong to the antibacterial B-1 lymphocyte clonotype T15, which comprises part of the innate immune system.

Exposure to S. pneumoniae has been shown to reduce atherosclerosis in mouse models.

Question 33

How does oxLDL immune response affect atherosclerosis?

Answer 33

There is also evidence that the adaptive immune system responds to atherosclerosis via T-cells and T-helper cells, which are thought to have both protective and pro-atherogenic roles.

Question 34

How do oxLDLs affect CVD directly/obviously?

Answer 34

They stimulate monocyte differentiation into macrophages, and increase tissue factor expression increasing thrombosis.

Lipid oxidation products in general also increase cytotoxicity/necrosis, and stimulate expression of protective enzymes.

Question 35

How do lipid oxidation products other than oxLDLs affect arterial function?

Answer 35

• Stimulation of isoprostanes
o Increased vasodilation – remodelling

• Cholesterol oxides increase endothelial adhesion molecule expression
o Increased monocyte invasion

• Lysophosphatidic acid
o enhances platelet aggregation

• Lysophosphatidylcholine
o Impairs endothelium-dependent vasodilation

• Cholesterol oxides
o many effects

Question 36

What is caused by high levels of cholesterol oxides?

Answer 36

High concentrations of oxidised cholesterol impairs cholesterol efflux from macrophages, 7-ketocholesterol preventing ApoA1/HDL binding to the cell.

Question 37

What is caused by lower levels of cholesterol oxides?

Answer 37

At lower concentrations the cholesterol oxides still impact gene expression, in a way that is in fact atheroprotective.

Genes upregulated are thought to include oxidative stress response genes such as SOD and haemoxygenase (though this is also described as genotoxicity) as well as those involved in cholesterol efflux.

Question 38

How do cholesterol effect their gene expression responses?

Answer 38

This occurs due to the oxysterols stimulating PPARγ (as well as LXR-α directly) to increase LXR-RXR transcription factor response. These enhance efflux by activating genes including ABCA1, ABCG1, ApoE, SREBP1c and CD36.

Oxysterols also affect SREBP signalling by activating insig.

Question 39

What results have been seen in clinical trials of antioxidants as a CVD therapy?

Answer 39

Many trials of the effects of antioxidants on CVD have been undertaken, most showing no results. Some benefit was identified by two of the trials of tocopherol and β-carotene, but overall results have been disappointing.

Many of these trials involved doses of vitamin E or other mixtures around 10-20x greater than normal physiological requirement. However, some evidence suggests that even larger doses of vitamin E may be required for the inhibition of oxidation.

Question 40

What provides evidence that increasing antioxidant levels is not a viable treatment for CVD?

Answer 40

Their inefficacy is reinforced by the abundance of tocopherol and vitamin E within the atherosclerotic plaque, where they fail to properly inhibit LDL oxidation.

Tocopherol treatment is thought to be ineffective due to the cyclic depletion and repletion of tocopherol not accessible to oxidants, and it has in some cases been shown to promote lipid oxidation.

Question 41

What genetic mutation suggests that one antioxidant may have a role in CVD?

Answer 41

Natural ascorbate levels have been shown to vary due to genetic influences, specifically SNPs in the SVCT1 and SVCT2 L-ascorbate transporter genes, which cotransport the antioxidant with sodium in the intestines and liver.

Mutations that reduce the transport efficacy lower ascorbate levels significantly, as this is responsible for a large proportion of ascorbate level regulation.

Recent studies indicate that such genetically low ascorbate levels may increase blood pressure and risk of heart failure after myocardial infarction.