Every component of the eye is vulnerable to damage from ROI, particularly retina. There are several reasons for the vulnerability of the retina, including high concentrations of polyunsaturated fatty acid (PUFA), constant exposure to visible light, high consumption of oxygen, an abundance of photosensitisers in the neurosensory retina and the RPE, the process of phagocytosis by the RPE which is known to generate hydrogen peroxide.

1. Unit: IV
Minerals and trace elements and eye
Carotenoids and eye
Oxidative stress and the eye
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2. Unit: IV
Oxidative stress and the eye
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3. Oxidative Processes
• oxidation refers to the removal of electrons, and reduction refers to the gain of
electrons
• TCA cycle is responsible for most of the oxidation of dietary carbohydrates, proteins
and lipids to CO2 and H2O
• The energy yielded is conserved in the form of the reduced electron accepting
coenzymes, nicotinamide adenine dinucleotide (NADH) and flavin adenine
dinucleotide (FADH2)
• The electrons of these coenzymes can be used to reduce O2 to H2O via the
electron transport chain, and this reaction releases energy for the conversion of
ADP and Pi to ATP in a process known as phosphorylation.

4. Oxidative Processes
• Oxidative phosphorylation occurs in the mitochondrion and is catalysed by ATP
synthase
• The electron transport chain accounts for approximately 90% of our total O2
consumption, the remainder being utilised by reactions involving oxidases or
oxygenases
• The majority of ROI are formed during energy generation from mitochondria, or
during the detoxifying reactions involving the liver cytochrome P450 enzyme
system

5. Reactive Oxygen Intermediates
• Most ROI are the inevitable byproducts of normal and essential metabolic reactions,
such as energy generation from mitochondria
• Pollution
• Asbestos
• fungal or viral infections
• Cigarette smoking
• Excess consumption of alcohol
• Irradiation (mainly blue wavelength light)
• Inflammation and ageing are all known to be associated with increased production
of ROI.

6. Reactive Oxygen Intermediates
• ROI can be classified according to their reactivity towards biological targets, their
site of production, their chemical nature, or their free radical or non-radical
subgroups

7. Reactive Oxygen Intermediates
• Free radicals are molecules that
contain one or more unpaired
electrons in their outer orbits
• In order to achieve a stable state, free
radicals extract electrons from other
molecules, which are themselves
rendered unstable by this interaction,
and a cytotoxic oxidative chain
reaction results
• Non-radical ROI contain their full
complement of electrons, but in an
unstable state
• The most important among them is
hydrogen peroxide and singlet oxygen
• Hydrogen peroxide can generate free
radicals through the Fenton reaction and
singlet oxygen can damage molecules as
it converts back to normal oxygen.

8. ROI and Cellular Damage
Every component of the eye is vulnerable to damage from ROI – particularly retina
There are several reasons for the vulnerability of the retina, including
1. high concentrations of polyunsaturated fatty acid (PUFA)
2. constant exposure to visible light
3. high consumption of oxygen
4. an abundance of photosensitisers in the neurosensory retina and the RPE
5. the process of phagocytosis by the RPE, which is known to generate hydrogen
peroxide.

9. ROI and Cellular Damage
• The high concentrations of PUFA (50%) are found in the lipid bilayer of the outer
segment of the rod photoreceptors, and DHA accounts for approx. 50% of the
vertebrate rod photoreceptor phospholipids
• The susceptibility of an unsaturated fatty acid to oxidation correlates directly with
the number of its double bonds
• PUFAs are particularly susceptible to free radical damage because their conjugated
double bonds are convenient sources of hydrogen atoms, which contain one
electron
• The lipid radical thus formed then combines with oxygen to form lipid peroxyl
radicals and lipid peroxides, which can only be stabilised by acquiring a quenching
electron, probably from an adjacent PUFA.

10. ROI and Cellular Damage
• A series of special conditions imposed upon photoreceptors puts them in what can
only be termed a high-risk, pro-oxidant environment
• Rod photoreceptor outer segments contain a high proportion of PUFAs which are
readily oxidised, while the inner segments contain a considerable number of
mitochondria, which leak a small but significant fraction of newly formed ROI
• Also, it has been shown that the partial pressure in this environment is higher than
found elsewhere in the body

11. ROI and Cellular Damage
• Under normal conditions, the production of ROI is met by an ample supply of the
detoxifying enzyme, SOD, whereas catalase and glutathione reductase are readily
available to cope with hydrogen peroxide
• These enzymes, in concert with antioxidant proteins and small molecular
reductants, form an effective antioxidant defence within the photoreceptor
• When overwhelmed, lipid peroxidation within the outer segment results

12. ROI and Cellular Damage
• Proteins make up the remaining 50% of the lipid bilayer of the rod outer segment
• Fragmentation, cross-linking and aggregation of proteins, as well as enhanced
vulnerability to proteolysis, can result from oxidation of their amino acids
• The oxidized bases of DNA, arising from interactions with ROI, are believed to
contribute significantly to ageing and age-related disorders involving many organ
systems, including the eye

13. Assessments of ROI activity
• There are various methods for assessing ROI activity; however, a consensus with
respect to which is the most valid and reliable technique is still lacking. The three
most commonly used techniques are:
(1) determination of endogenous antioxidant levels
(2) measurement of the products of oxidised macromolecules
(3) Direct detection of ROI.

14. Assessments of ROI activity
Antioxidant levels
• The concentration of antioxidants in plasma and cells, and the cellular activities of
antioxidant enzymes, can be used as a reflection of ROI activity
Products of oxidised macromolecules
• ROI damage may be identified, indirectly, by the presence of degradation products
of lipids, such as malondialdehyde, in blood and/or urine
• For assessing ROI-induced protein oxidation, protein nitrotyrosine has been widely
used as a convenient and stable marker
• Finally, urinary excretion of 8-hydroxydeoxyguanosine represents a useful means of
assessing DNA base oxidation in humans.

15. Assessments of ROI activity
Direct detection of ROI
• Direct detection of ROI can be assessed using electron spin resonance and spin-
trapping techniques
• The electron spin resonance technique is suitable for detecting ROI in vitro, but it
has limited application in vivo
• The spin-trapping technique involves the conversion of ROI to relatively inert
radicals, which are then detected by electron spin resonance analysis.

16. Defense Mechanisms Against Oxidative Stress

17. Endogenous Antioxidants
Glutathione
• abundant in cytoplasm, nuclei and mitochondria of cells, and is dependent on selenium as a
cofactor
• The concentration of cellular glutathione has a major effect on its antioxidant capacity, and
it varies considerably as a result of nutrient limitation, exercise and oxidative stress
• Glutathione exists in the following forms: the antioxidant reduced glutathione, known as GSH
and the oxidised form, known as glutathione disulphide (GSSG)
• The GSSG/GSH ratio in a living cell is believed to reflect oxidative stress.

18. Endogenous Antioxidants
Glutathione

19. Endogenous Antioxidants
Superoxide dismutases
• SODs are metalloproteins, some of which contain manganese, whereas others contain
copper or zinc
• SOD catalyses the quenching of the superoxide anion to produce hydrogen peroxide and
oxygen

20. Endogenous Antioxidants
Catalase
• Catalase is an iron-dependent enzyme that scavenges H2O2, either catalytically or
peroxidatively
• It has been demonstrated in human retina and RPE, where its activity has been shown to
decline with increasing age.
• Also, a reduction in retinal catalase activity has been demonstrated in eyes with AMD.

21. Exogenous Antioxidents
Vitamin A
• It is believed that vitamin A may protect photoreceptor membranes against oxidative
damage by breaking the chain reaction during lipid peroxidation
• In addition, vitamin A is also involved in the repair of cells that have been injured by
oxidation.

22. Exogenous Antioxidents
Vitamin C
• Vitamin C is a major water-soluble antioxidant
• It is an ideal scavenger because of its water solubility, stability and mobility, and because it
can be transported, reabsorbed and recycled
• Compared to plasma, all ocular tissues have very high concentrations of vitamin C.

23. Exogenous Antioxidents
Vitamin E
• Vitamin E acts synergistically with carotenoids in scavenging free radicals
• In the presence of vitamin E deficiency, various changes indicative of oxidative damage are
seen in the rod outer segments and RPE, suggesting that this vitamin protects the retina
against such injury

24. Exogenous Antioxidents
Carotenoids
• Carotenoids act as antioxidants by virtue of their free radical scavenging and singlet-oxygen
quenching capacity
• The macular carotenoids are increasingly believed to protect the macula against oxidative
damage by at least one of the following two mechanisms: filtering blue light at a
prereceptorial level, thus limiting photochemical reactions; quenching free radicals

25. Exogenous Antioxidents
Bioflavonoids
• Bioflavonoids are large polyphenolic molecules, which are derived from the peel and
coverings of teas, berries, grapes and bark
• They exhibit a myriad of properties, such as anti-inflammatory, antibacterial and antioxidant
activity.
• Of the sources of bioflavonoids, such as red wine, green tea and English blueberry, green tea
produces the most potent antioxidants known to humans
• Numerous studies have shown their unique role in protecting vitamin C from oxidation in the
body, thereby allowing the body to reap more benefits from vitamin C.

26. Oxidative Stress and AMD
• The free radical theory of ageing proposes that ageing and age-related disorders are the
result of cumulative damage resulting from reactions involving ROI
• If this theory applies to the eye, an altered antioxidant/oxidant balance should be evident for
age-related eye disease such as AMD
• The evolutionary theory of ageing proposes that there is a decline in the force of natural
selection with increasing age, and that we may have evolved with genes which promote
senescence once we have passed our period of procreation
• In other words, we do not eliminate genes that have a detrimental effect in later life if they
have a beneficial effect, or no effect, in early life.

27. Oxidative Stress and AMD
• Evidence of oxidative stress can be seen in the RPE and in the neurosensory retina with
increasing age, and this damage is most prominent in the region of the retina where early
AMD changes are seen
• It has been shown that the concentration of lipofuscin in the RPE increases with increasing
age
• Lipofuscin consists of lipid/ protein byproducts resulting from oxidatively damaged
photoreceptor outer segments
• it has been shown that lipofuscin compromises RPE cellular function
• lipofuscin generates ROI in response to irradiation with blue light, and therefore contributes
further to oxidative stress in the local environment
• RPE dysfunction contributes to the pathogenesis of ARM/AMD, and that this dysfunction is
related to lipofuscin accumulation, which, in turn, is related to oxidative injury.

28. Macular Pigment and AMD
• MP is entirely of dietary origin, and is found in high concentrations in green leafy vegetables,
fruits and egg yolk, and dietary modification can augment the optical density of MP
• MP protects the retina from photochemical (oxidative) damage directly, by acting as a free
radical scavenger, and indirectly, by filtering damaging blue light
• it has been postulated that the optical and antioxidant properties of MP confer protection
against AMD, and that the augmentation of MP through dietary modification could delay, or
even avert, the onset of AMD
• Observational studies suggest that a lack of MP is associated with several known risk factors
for AMD, including female gender, smoking, light iris colour, increasing age

29. Antioxidant and AMD
• Several large studies have examined the role of antioxidant supplementation in ARM/AMD,
the largest of which was the AREDS
• AREDS was a multicentre, prospective study of 4757 individuals aged 55–80 years, designed
to assess the effect of dietary antioxidant supplements (vitamin E, vitamin C, b-carotene and
zinc) on the clinical course of AMD
• AREDS reported a beneficial effect of supplementation with this formulation, with reduced
risk for disease progression by 25% and vision loss by 19%

30. Oxidative Stress and Cataract
• ROI can be generated in the lens as a result of exogenous (UV light) or endogenous factors
• UV light represents an important factor, since the exposure of the lens to these wavelengths
renders it vulnerable to ROI production, with consequential protein modification, lipid
peroxidation and DNA fragmentation, all of which are believed to contribute to the genesis
of cataract.
• the most important insult is protein modification, which includes protein disulphide cross-
links and high-molecular-weight aggregation
• With increasing age, the protection and repair mechanism against oxidation in the lens, the
key component of which is GSH, slowly deteriorates and becomes ineffective
• As the lens ages, de novo synthesis and the recycling system of GSH become less efficient,
resulting in a net decline in its concentration
• Protein sulfhydryl groups can undergo oxidation, thus contributing to cataract formation

31. Oxidative Stress and Glaucoma
• It is believed that raised intraocular pressure in primary open-angle glaucoma is attributable
to malfunction of the trabecular meshwork (TM) – Schlemm’s canal outflow system
• The TM is believed to be exposed to chronic oxidative stress because of the presence of ROI
in the aqueous humour, and because of the generation of ROI by mechanical stress and
intracellular metabolism
• proteasome, which protects the TM from oxidative injury by eliminating the altered proteins
damaged by ROI
• function of proteasome can be impaired by excessive exposure to oxidative stress by at least
one of the following two mechanisms:
(1) saturation of the proteasome by the presence of an excessive number of altered proteins
(2) direct oxidation of proteasome components

32. Oxidative Stress and RP
• RP is a progressive degeneration of the retina, and is best described as a phenotypic
description of several related, yet distinct, dystrophies of the photoreceptors and the pigment
epithelium
• Apoptosis represents the final common pathway of cell death in RP, and it has been
demonstrated that ROI act as mediators of retinal cell apoptosis in this condition

33. Oxidative Stress and ROP
• there is a general consensus that ischaemia–reperfusion injury, with consequential
generation of ROI during the metabolism of ATP, results in Retinopathy of Prematurity
• ATP is degraded intracellularly via adenosine monophosphate to adenosine, which is further
degraded to inosine and hypoxanthine outside the cell
• Under normal conditions, hypoxanthine is metabolised to uric acid by the enzyme xanthine
dehydrogenase (XDH), but under conditions of ischaemia or anoxia XDH is converted to
xanthine oxidase (XO)
• Metabolism of hypoxanthine by XO results in release of superoxide anions, and
consequential oxidative injury