prepared during lens session

1. Biochemistry and
BiophysicsBIPIN BISTA
RESIDENT
OPHTHALMOLOGY

2. Introduction
 ATP is the principle source of energy by hydrolysis of its high energy
phosphate bonds.
 ATP produced within the lens come from anaerobic mechanism of glucose.
 Other components include reduced form nicotinamide adenine
diphosphate ,produced by pentose phosphate pathway
 Acts as source of readily available reducing power used in biosynthesis of
many associated cellular components,such as fatty acid and glutathione
 For preventing oxidative damage,maintain sufficient antioxidant defences
to protect against damage and development of cataract.

3. Sugar Metabolism
 90-95% of glucose inside lens is phosphorylated to G-6-P catalysed by
hexokinase.
 Type 1 to 3 isomers of hexokinase has been found , 1 has greater affinity
for glucose and are found where the levels of glucose in nucleus is low,
whereas isomer 2 accounts for 70% total lens hexokinase but has lower
affinity found in epithelium and cortex where glucose are higher.
 G-6-P is used in glycolytic pathway (80%) or PPP(HMP;10%)
 Hexokinase is inhibited by higher level of G-6-P, H+ , citrate & ATP.
 Also regulated by PFK
 Pyruvate kinase : inhibited by higher level of ATP, alanine and F-1,6
Bisphosphonate.

4. Sugar Metabolism
 d/t avascularity and humor location, lens exists in hypoxic environment
with partial pressure of O2 as low as 5mmHg. Resulting in derivation of
lens ATP from anaerobic glycolysis – Relatively inefficient for ATP
production : 2 ATP from 1 molecule.
 Only 3% molecule glucose goes through TCA where 36 ATP is generated
from 1 glucose molecule.
 Glycolysis and TCA generate 2 energy rich molecules, reduced form of
NADH & FADH2 which donate their e- to O2 , which releases large amount
of free energy to generate ATP. Restricted to epithelial layer, provides
carbon skeletal intermediates for biosynthesis of AA & porphyrins.

5. Sugar Metabolism
 Bulk of pyruvate produced by glycolytic pathway is reduced to lactate in a
reaction catalysed by LDH. Formation of lactate results in reoxidation of
cofactor NADH to NAD+ . Glyceraldehyde -3- phosphate dehydrogenase
used in glycolytic pathway regulates activity of LDH by controlling the rate
of conversion of G-3-P into 1,3-diphosphoglycerate and thus availability of
NADH.
 Although PPP only utilises 10% of total lens , its activity increases if
glucose levels are increased above normal.
 Activity of PFK , NADP+ & ATP determine whether G-6-P follows glycotic or
PPP . Primary function of PPP is to produce reduced form of NADPH,
used in FA synthesis, maintainence of reduced glutathione, conversion of
glucose into sorbitol.

6. Sugar Metabolism
 5-10% of unphosphorylated glucose enters sorbitol pathway/or gluconic
acid .
 Glucose is convereted into sorbitol by aldose reductase, localised in
epithelial layer, this enzyme uses NADPH supplied by P-P-P as a cofactor.
Sorbitol is then converted into fructose by polyol dehydrogenase.
 Since both sorbitol and fructose have potential to increase the osmotic
pressure.

7. Protein Metabolism
 Concentration of protein is higher than other tissues
 Mostly concerned with production of crystalline and Major Intrinsic Protein
(MIP 26)
 Protein synthesis occurs in epithelial cells and surface cortical fibers.
 Most protein undergoes post-translational modification.
 Lens protein are stable d/t inhibition of degenerative enzymes.
 Lens controls the breakdown of protein by marking those to be degraded
with small 8.5kDA protein –Ubiquitin, which are ATP dependent and active
in epithelial layer.

8. Protein Metabolism
 Lens protein are broken down into peptides by endopeptidases and the
peptides are broken down into AA by exopeptidases.
 These neutral endopeptidases are activated by Ca 2+ & Mg2+ and is
optimally active at pH 7.5
 Principle substrate is α – crystalline.
 Calpain (I & II) are present in epithelial cells and cortex used for degrading
crystallins and cytoskeletal proteins.
 These enzymes are inhibited by calpastatin,a natural inhibitor found at
higher concentration than calpains.

9. Protein Metabolism
 Main exopeptidase : Leucine aminopeptidase, an enzyme optimally active,
which catalyses removal of AA from N-terminal of peptide controlled by
activity of metal ions binding at 2 binding sites found on each subunit , one
site must contain zinc, another zinc, magnesium or manganese can confer
activity.
 Endogenous inhibitor of leucine aminotransferase is present in the lens.

10. Protein Metabolism
 Glutathione (Glutamyl-1-cysteine glyceine) is found at higher concentration
in lens (3.5-5.5 µmol/gwt) in (epithelial >> nucleus, cortex).

11. Glutathione – Functions
 Maintenance of protein thiols in reduced state, which helps to maintain
lens transparency by preventing the formation of high molecular weight
crystallins aggregates.
 Protection of thiols groups critically involved in cation transport &
permeability. Eg : Oxidation of sulfhydryl group of Na+ - K+ ATPase pump
which results in increased permeability to these ions.
 Protection against oxidative damage
 Removal of xenobiotics

12. Glutathione
 Has half-life of 1-2 days, is recycled by γ-Glutamyl cycle, synthesis and
degradation occur at same rate.
 Glutamyl cysteine synthethase and I-cysteine are the rate limiting enzyme
and rate limiting substrate for the synthesis.
 Glutathione exists in both an oxidized form (GSSG) and a reduced form
(GSH). At least 95% of lens glutathione is found in the reduced GSH form.
 Glutathione reductase, an enzyme that uses NADPH as a cofactor
(provided by the pentose phosphate pathway) reduces GSSG into GSH .
Because this enzyme works at low substrate levels, any GSSG formed is
converted rapidly into GSH.

13. Antioxidant mechanism
 Reactive oxygen species is a collective term for highly reactive oxygen
radicals (including free radicals) that have the potential to damage lipids,
proteins,carbohydrates, and nucleic acids.
 Radicals : Superoxide anions, Hydoxyl free radical, hydroperoxyl radical,
lipid peroxyl radical, singlet oxygen & H2O2 .
 Reactive oxygen have 2 origins : cell metabolism & photochemical
reaction.

14. Photochemical reaction
 Photochemical damage occurs when light is absorbed by chromophore –
photosensitizer, which goes into photoexcitation goes to intersystem
crossing and forms a transient excited triplet state.
 Triplet state allows for interaction with other molecules producing free
radicals via electron transfer or singlet O2 via transfer of excitation energy
from the photsensitizer in the triplet to molecular oxygen.
 Continuous entry of shorter wavelength makes lens susceptible to
photochemical reaction
 Major UV absorbers are aromatic AA (Tryptophan), numerous pigments (3-
hydroxykynurenine) & fluorophores.
 Reactive O2 species enters lens from surrounding tissues as well.

15. How does it damage the lens ?
 Peroxidising membrane lipids results in formation of malondialdehyde ,
which in turn can form cross-links between membrane lipids and proteins.
 Damages bases of DNA, base modification , helical distortion of DNA
initiates DNA repair mechanisms.
 Polymerising and cross-linking protein : aggregation of crystalline and
inactivation of many essential enzymes including those with an antioxidant
role – catalase and glutathione reductase.

16. Protection
 Superoxide anion undergoes dismutation by superoxide dismutase or
interaction with ascorbate which results in formation of H2O2 which is
detoxified by glutathione peroxidase or catalase.
 Glutathione peroxidase >> catalase ( Epithelium>Fibers)
 Glutathione also provides important protection against lipid free radical chain
reaction by neutralising lipid peroxides.
 Ascorbic acid also plays important role as an antioxidant. (1.1 mmol/kg) Outer
layer > Nucleus.
 AA rapidly reacts with superoxide anions,peroxide radicals and hydroxyl
radicals to give dehydroascorbate.
 AA and glutathione are coupled in that dehydroascorbate reacts with reduced
form of glutathione to generate ascorbate and GSSG .

17. BIOPHYSICS
 Lens has 2 major functions : focusing visible rays on fovea, preventing
damage from UV radiation from retina.

18. Light Transmission
 Ability of absorbing more energetic wavelengths of magnetic spectrum .
 Cornea (<295 nm) while lens absorbs stronger (300-315 nm)
 With age there will be difficulty in transmission of more short wavelength
reaching retina.

19. Transparency
 During the early stage, lens is opaque, but as development continues &
hyaloid vascularity is lost, lens become transparent.
 Transparency : d/t absence of chromophores
 Minimal light scatters d/t minimal difference in Refractive Indices from
aqueous.
 There are newly formed cortical fibers which contains all organelle but they
don’t cause any scattering of light because they elongate and are not in
visual axis.
 Crystallins which contains 30% of lens fiber , but are minimal scattering as
there is high level of spatial order exists, scattered from 2 neighbouring
molecules tends to cancel out Refractive Indices fluctuation between 2
molecules.

20. Refractive Indices
 Although light is refracted at cornea, AH, lens & VH, major site of refraction
is cornea & lens .
 R.I is higher between air-cornea than aqueous-lens & lens-vitreous
interfaces, Refractive power of Cornea > Lens.
 R.I increases from 1.38 to 1.41 from peripheral cortex to nucleus.
 As curvature and R.I increases from periphery to center, light bends to
greater extent.
 Change in protein concentration, packing and hydration properties
 γ – crystallins are attracted , but α & β repulse each other, and γ –
crystallins are located primarily in nucleus which tends to increase in R.I

21. Optical Performance
 Visible light passes through lens and then scatters into spectrum of light.
 Yellow (570-595 nm) focused in retina whereas Blue ( 440-500 nm)
 Ref. Indices at Periphery > Center
 Curvature increases towards poles.
 Curvature anterior capsule > posterior capsule.

22. References
 Ophthalmology : yanoff and duker : 4th Edition
 American academy of ophthalmology : section 11 : 2014-2015

23. THANK YOU