ROS

Question 1

How are ROS synthesised/explain the key sources of ROS in the body? (4)

Answer 1

Enzymatic
○ NADPH oxidase - generates superoxide - antimicrobial defence
○ NO - converts arginine and oxygen into nitric oxide
○ Superoxide Dismutase - converts superoxide into hydrogen peroxide

Organelles
○ E.g. mitochondrial electron transport
§ Leak of electrons from electron transport chain to form superoxide

Transition metals
○ Fenton chemistry - transition metals e.g. copper or iron

Chemical reactions
○ Haber-Weiss reaction - interaction between two reactive oxygen species

Question 2

What is the reaction for Fenton chemistry - transition metals (iron)

Answer 2

Fe(II) + H2O2 – intermediate oxidising species -> Fe(III) + *OH (ROS) + OH-

Question 3

What is the Haber-Weiss reaction

Answer 3

O2*- +H2O2 -> O2 + *OH +OH-

Question 4

Explain the difference between physiological (5) and pathophysiological (3) roles of ROS (and give examples)

Answer 4

Physiological role - normal, healthy function of a biological process or component in the body
○ Cell defence
○ Immune response
○ Cell signalling
○ Hormone synthesis
○ Gene expression

Pathophysiological role - role a process or component plays in disease or abnormal conditions
○ Protein oxidation - damage to essential cellular proteins e.g. enzymes
○ Oxidation of DNA - causes cellular mutations, cell disruption and cell death
○ Lipid peroxidation - damage to cell membrane leading to altered cell function and cell death

Question 5

What is the relationship between ROS/antioxidants and oxidative stress

Answer 5

Concentration and location are key

The body needs ROS to function, but if unchecked they are damaging. The body therefore uses antioxidants to maintain a balance
○ -> oxidative stress

Antioxidants scavenge ROS and convert them into a less reactive compound e.g. SOD, catalase, Glutathione, Ascorbic acid, alpha-tocopherol plus many more

oxidative stress = more ROS than we can manage

Question 6

What are the 5 key antioxidant mechanisms of action

Think causes of ROS synthesis

Answer 6

1. Chelate transition metals - bind to metals so therefore cannot react
2. Inhibit oxidative stress
3. Reduce electron formation from organelles
4. Neutralise ROS by donating electron
5. Repair damaged cellular components

Question 7

List the criteria for establishing a role of ROS in disease (4)

Answer 7

1. The ROS (or oxidative damage caused) should always be demonstratable at the site of injury
2. The time course of formation of the ROS (or oxidative damage caused) should be consistent with the time-course of tissue injury
3. Direct application of the ROS over the appropriate time course to the tissue at concentrations within the range found in-vivo should reproduce the tissue injury and oxidative damage observed
4. Removing the ROS or inhibiting its formation should diminish the tissue injury

Question 8

What is the definition of an antioxidant

Answer 8

Molecules that protect the body against reactive oxygen species

Question 9

What are the differences between exogenous (dietary) and endogenous antioxidants (with examples)

Answer 9

Endogenous - produced by the body
○ Superoxide dismutase (SOD)
○ Catalase
Exogenous - dietary
○ Vitamin’s C and E
○ Flavonoids
○ Polyphenols

Question 10

What evidence is there to support the benefits of an antioxidant rich diet

Answer 10

A diet rich in fruit and vegetables reduces the risk of developing a range of diseases including cancer and cardiovascular disease

Fruit and veg reduce the risk of certain diseases -> fruit and veg are rich in antioxidants -> could antioxidant supplements reduce risk of disease

Question 11

Explain the key findings of the Physicians Health Study with regards to supporting or disproving the role of antioxidant supplements in the treatment of disease

Answer 11

14,000 male physicians > 50 or older

Neither vitamin E or vitamin C supplements reduced the risk of major cardiovascular events, cancer or cataracts

Vitamin E supplements were associated with an increased risk of haemorrhagic stroke in this study

Question 12

Explain the key findings of the Women’s Health Study with regards to supporting or disproving the role of antioxidant supplements in the treatment of disease

Answer 12

40,000 healthy women > 45 years of age

Vitamin E supplements did not reduce of heart attack, stroke cancer, age-related macular degeneration, or cataracts

Question 13

Explain the key findings of the Women’s Antioxidant Cardiovascular Study with regards to supporting or disproving the role of antioxidant supplements in the treatment of disease

Answer 13

8000 female health professionals >40 at high risk of cardiovascular disease

Found no beneficial effects of vitamin C, vitamin E or beta-carotene supplements on cardiovascular events (heart attack, stroke, or death from cardiovascular diseases) or the likelihood of developing diabetes or cancer

Question 14

Summarise key findings in investigating the effectiveness of antioxidant supplements. Do they support or disprove their use?

Answer 14

Systematic review evidence
○ Study sizes: 78 randomised clinical trials
§ 296,707 participants randomised to antioxidant supplements e.g. vitamin A, vitamin C, vitamin E
○ Diseases included
§ Gastrointestinal
§ Cardiovascular
§ Dermatological
§ Rheumatoid
§ Renal
§ Endocrinological
○ Results
§ No difference in the number of deaths between treatment type:
□ Antioxidant: 11.7%
□ Placebo: 10.2%

Other studies disprove their use as there is no evidence to support antioxidant supplements for primary or secondary prevention

There is some but limited evidence for their effectiveness in age related macular degeneration

Question 15

What is the antioxidant paradox

Answer 15

The antioxidant paradox refers to the apparent contradiction where, despite the theoretical benefits of antioxidants in reducing oxidative stress and preventing disease, clinical studies have often failed to show consistent or significant health improvements from antioxidant supplementation

Question 16

What theories are used to explain the antioxidant paradox (9)

Answer 16

Role of ROS - not all ROS are detrimental

Nutrition - supplementation is uneccessary excess if diet is good

Distribution - not all reach site of action e.g. cross BBB

Combinations - isolated from fruit but prehaps needs whole fruit to be beneficial

Location - AOs meant for the eye may have no impact elsewhere

Time frame - may not be supplemented for long enough

Antioxidants tested - only a small proportion of dietary AOs have been investigated

Lifestyle - health benefits seen from fruit and veg could be a healthier lifestyle overall

Dose - supplements use a high dose prehaps too much?

Question 17

Why is the role of ROS used to explain the antioxidant paradox

Answer 17

Not all ROS are detrimental

Did the ROS cause the disease or where they a bi-product

Question 18

Why is nutrition used to explain the antioxidant paradox

Answer 18

A healthy adult with good nutrition will have sufficient levels of endogenous antioxidants

Supplementation just leads to an unecessary excess

Question 19

Why is Distribution used to explain the antioxidant paradox

Answer 19

Not all antioxidants are able to reach their site of action e.g. cross the BBB

Question 20

Why is combinations used to explain the antioxidant paradox

Answer 20

Purified antioxidants may work differently to antioxidants found in food which are found in combination with other plant compounds e.g. fibre, minerals etc.

Question 21

Why is location used to explain the antioxidant paradox

Answer 21

Antioxidants may have differing effects in different parts of the body

E.g. antioxidants that are present in the eye, such as lutein, might be more beneficial preventing diseases related to the eye than those that are not found in the eye, such as beta-carotene

Question 22

Why is time frame used to explain the antioxidant paradox

Answer 22

The antioxidant supplments may not have been given for a long enough time tp prevent chronic diseases, such as cardiovascular diseases or cancer, which develop over decades

Question 23

Why is antioxidants used used to explain the antioxidant paradox

Answer 23

Although dietary antioxidants are a large and diverse group of compounds, only a small proportion of candidate agents have been tested

E.g. 8 chemical forms of Vitamin E in food. However, supplements tend to only contain alpha-tocopherol

Question 24

Why is lifestyle used to explain the antioxidant paradox

Answer 24

The health benefits seen in people who eat fruit and vegetables may be linked to healthier lifestyles in general e.g. greater physical activity

Question 25

Why is dose used to explain the antioxidant paradox

Answer 25

Antioxidant supplements tend to use a higher dose than that seen in fruit and vegetables

Question 26

Very briefly explain the role of electrons in the electron transport chain

Answer 26

The role of electrons in the electron transport chain is to transfer energy by moving through protein complexes driving the pumping of protons (H⁺) across the mitochondrial membrane. This creates a proton gradient, which powers ATP synthesis through ATP synthase

Question 27

How does electron leak lead to mitochondrial damage and disease

Answer 27

Electron leak in the electron transport chain can lead to the production of reactive oxygen species (ROS). The leaked electron can combine with O2 to form superoxide (O2*-) Excessive ROS can damage mitochondrial DNA, proteins, and lipids, impairing mitochondrial function and contributing to diseases like neurodegeneration, aging, and metabolic disorders

Question 28

How do ROS lead to mitochondrial dysfunction and eventual apoptotic or necrotic cell death

Answer 28

Mitochondrial damage leads to reduced energy production and increases ROS production which can lead to mitochondrial disease - O2*- from leaky complexes is broken down by SOD to hydrogen peroxode (H2O2) - H2O2 broken down by catalase to H2O - if there is too much mitochondrial damage this leads to more leaky electrons - too many ROS formed for catalase to keep up - superoxides (O2*-) can react with H2O2 to form *OH - leads to oxidative stress

Question 29

What is the key difference between acquired and genetic mitochondrial disease

Answer 29

Acquired diseases ○ Linked to environmental factors or normal ageing process ○ Affects organs with high energy demands e.g. heart, skeletal muscle, kidney, eye and brain ○ Diseases include: heart failure, age-related macular degeneration, neurodegeneration, certain skeletal muscle disorders Genetic diseases ○ Inherited: characterised by known genetic defects ○ >200 types of these

Question 30

What in vitro evidence is there to support a direct antioxidant effect of SS-31 against ROS

Answer 30

SS peptides originally developed as a highly potent and effective opioid analgesic by Hanzel Szeto and Peter Schiller SS-peptides later found to act as small cell-permeable antioxidants that can cross the BBB Original SS-peptides re-designed to remove opioid activity but maintain antioxidant properties SS-31 : D-Arg-Dmt-Lys-Phe-NH2

Question 31

What is the uptake of SS-31

Answer 31

Cross BBB Localise on the inner mitochondrial membrane

Question 32

What is the toxicity of SS-31

Answer 32

Limited side effects SS-31 is essentially devoid of opioid activity Exposure of a variety of cell-types to SS-31 did not result in cytotoxicity even at concentrations of 1mM

Question 33

What is the MoA of SS-31

Answer 33

Good antioxidant profile Stabilises cardiolipin ○ Scavenges a range of ROS and inhibits oxidation of biological molecules

Question 34

What is the pharmacokinetics of SS-31

Answer 34

Highly favourable Water soluble Stable Relatively long elimination half lives

Question 35

What role does cardiolipin play in maintaining an efficient electron transport chain

Answer 35

Cardiolipin promotes curvature in lipid membranes due to its conical structure It ensures an efficient ETC Ensures correct IMM structure encouraging super complex formation Anchors cytochrome C to complexes for efficient electron transfer Cyt c is loosely attached to the ETC via an electrostatic interaction with cardiolipin This is supported by high ATP levels

Question 36

How do elevated levels of ROS lead to oxidation of cardiolipin have on the electron transport chain and overall mitochondrial function

Answer 36

Elevated levels of ROS damage mitochondria thus reducing ATP production This alters the interaction between cardiolipin and Cyt c converting Cyt c into a peroxidase As a peroxidase, Cyt c oxidises cardiolipin (CLOOH) Cardiolipin peroxidation disrupts the ETC -> resulta in uncoupling of the ETC via: ○ Disruption of the IMM ○ Leak of electrons from the ETC ○ Detachment of Cyt c from IMM which triggers apoptosis

Question 37

How does SS-31 directly and indirectly protect the mitochondria against ROS-mediated damage

Answer 37

SS-31 inhibits cytochrome C peroxidase activity INDIRECT: antioxidant activity ○ Some evidence that SS-31 scavenges ROS thus protecting ATP levels DIRECT: SS-31 stabilises cardiolipin ○ This protects the structure of the ETC Stabilises cytochrome c in electron carrier form and prevents conversion to a peroxidase

Question 38

Explain how the MTPT-model of Parkinson's disease has been used to show the mitochondrial protective action of SS-31 ## Footnote how it is used, MoA

Answer 38

Neurotoxin ○ Studies carried out using neurotoxin MPTP Mechanism of action ○ Used to induce artificial state of parkinsonism in animal models: § Inhibits complex I (decreased ATP) § Alters mitochondrial membrane potential § Increases ROS § Apoptotic death of DA neurons SS-31 reduces MPP+ induced inhibition of mitochondrial oxygen consumption and ATP production SS-31 protects dopaminergic neurons in substantia nigra against MPTP damage SS-31 reduces MPP+ induced cell death in SN4741 dopamine cells

Question 39

Explain the key findings of the MMPOWER I clinical trial investigating the use of elamipretide in adults with primary mitochondrial myopathy (aim, study design, primary efficacy measure, conclusion)

Answer 39

Aim: to evaluate the safety and efficacy of I.V. elamipretide in adults with PMM Study design: phase I/II RCT. Participants randomised to I.V. elamipretide (0.01, 0.1 and 0.25 mg/kg/h) or placebo for 2 hours for 5 day. N = 36 The primary efficacy measure: change in distance walked in the 6 minute walk test (6MWT) after 5 days of treatment Conclusion: trial provides evidence that I.V. elamipretide in adults with PMM is well tolerated and improves exercise performance in a dose-dependent manner compared to placebo post-hoc analysis concluded a significant difference between placebo and the highest concentration of elamipretide

Question 40

Explain the key findings of the MMPOWER II clinical trial investigating the use of elamipretide in adults with primary mitochondrial myopathy (aim, study design, primary efficacy measure, conclusion)

Answer 40

Aim: to evaluate subcutaneous (SC) formulation of elamipretide comparable with highest dose from MMPOWER-1 Study design: cross-over RCT. Open to individuals from MMPOWER-1. 40mg SC once daily vs placebo for 4 weeks. N=30 Primary efficacy measure: change in distance walked in the 6-minute walk test Conclusion: clinically important observation based as it falls in the MCID (minimum clinically important differences) range

Question 41

Explain the key findings of the MMPOWER III clinical trial investigating the use of elamipretide in adults with primary mitochondrial myopathy (aim, study design, primary efficacy measure, conclusion)

Answer 41

Aim: to evaluate the safety and efficacy of SC elamipretide in adults with genetically confirmed PMD Study design: Phase III RCT. Participants randomised to SC 40mg/day elamipretide or placebo for 24 weeks. N=218 Primary efficacy measure: change in distance walked in the 6MWT after 24 weeks of treatment. Change in total fatigue on primary mitochondrial myopathy symptom assessment Conclusion: no significant change between elamipretide and placebo in the 6MWT or PMMSA

Question 42

List the current therapeutic opportunities being investigated with regards to elamipretide

Answer 42

Ophthalmic ○ Geographic atrophy (GA) in dry age-related macular degeneration (AMD) Neurology ○ Primary mitochondrial myopathy due to nDNA mutations (nPMM) Cardiology ○ Duchenne muscular dystrophy (DMD) ○ Barth syndrome