LENS OF EYE Diabetic Cataract PSC role of AGEs2025.pptx

Chemistry of lens protein , alteration in chemical compositions, hyperactivity of polyol pathway, MGO pathway ,AGEs formation ,glycation process , mechanism of Diabetic & corticosteroid induced cataract , role of crystallin glycation in cataract

1. Lens Chemistry Satya Bhushan Sharma

2. 34% Protein 65% Water Biochemical composition of lens Water constitutes about 65% of the lens wet weight Of the solids, the highest is protein which constitutes about 34% of the total weight of an adult lens lipids, inorganic ions, carbohydrates, ascorbic acid, glutathione and amino acids 09-10-2025 SBS 2

3. } 65% Water Biochemical composition of lens: Lens water  Lens is a relatively dehydrated organ, cortex being more hydrated than nucleus  Fischer suggested that out of this about 80% is free while remaining is bound water  A small portion of the lens water is located in the extracellular space  Low amount of water in the lens is a natural consequence of the need for having a refractive index quite different from that of the watery fluids at the two optical interfaces of the lens  The normal human lens does not show significant alteration in hydration with age 80% free water 09-10-2025 SBS 3

4. Biochemical composition of Lens: Proteins  Protein content of the lens is higher than that of any other organ in the body The physical state of proteins is an important factor for the maintenance of transparency  The soluble fraction has 3 components namely alpha- , beta- and gamma crystallin The 3 crystallins can be separated by precipitation at Different pH, Salting out, Electrophoresis or by running through cellulose column  Morner for the first time classically divided the proteins of crystalline lens of cattle into an insoluble fraction called albumin and the soluble fraction called crystallins 09-10-2025 SBS 4

5. Biochemical composition of lens: Proteins Krause studied the various protein fractions in the lens as follows: Proteins Insoluble albumin 12.5% Alpha – Crystallin 31.7% Beta – Crystallin 53.4% Gamma – Crystallin or albumin 1.5% Mucoproteins 0.8 % Nucleoproteins 0.07% Soluble fraction 09-10-2025 SBS 5

6. Biochemical composition of lens: Proteins Proteins  few minor proteins reported in the lens are glycoprotein, phosphoprotein, lipoprotein and fluorescent proteins  cortex contains more soluble proteins than nucleus which contains more insoluble proteins  The cortex of the young lens practically contains no albuminoid , whereas the nucleus of old lens is composed o f almost entirely of albuminoid alpha-crystallin and albuminoid are immunologically similar Alpha Crystallin (Soluble) Albuminoid protein (Insoluble) Age ↑ 09-10-2025 SBS 6

7. Biochemical composition of lens: Proteins Soluble proteins  The lens crystallins make up the bulk of refractive fibres of the lens and are considered structural proteins  Synthesis of soluble proteins takes place in the equatorial part and on the surface of lens  The newly formed fibres contain very little or no albuminoid  A part of the soluble lens proteins may be formed in deeper lens fibres, at least in those which contain nuclei  The high concentration of crystallins and the gradient of refractive index are responsible for the refractive properties of the lens  The short range order of these proteins ensures that the lens remains transparent 09-10-2025 SBS 7

8. Biochemical composition of lens Alpha- crystallin  T he highest molecular weight  at alkaline pH it has the greatest positive charge  Average molecular wt . of monomeric α -crystallin is 20kD  Alpha-crystallin is known to form heterogeneous oligomeric structures, meaning the size of these complexes can vary.  These large aggregates can consist of fewer than 15 subunits or over 50 subunits , leading to a wide range of molecular weights.  The overall mol. wt. of these native assemblies typically starts from around 200 kDa and can extend upwards to over a million Daltons 09-10-2025 SBS 8

9. Beta- crystallins . This fraction of lens proteins is a heterogenous group of proteins with molecular wt from 50 kD to 200kD The beta- crystallins contain many polypeptide chains, some of which appear to be present in aggregates Shapiro has reported that this protein group contains three different polypeptide chains of the molecular weights of 21 kD , 23 kD and 29kD They also have a relatively high thiol content and might have disulphide linkages Biochemical composition of lens 09-10-2025 SBS 9

10. Biochemical composition of lens G amma- crystallin  These are composed of monomers only  Gamma-crystallins constitute about 11% of the total protein in a neonatal lens.  As the lens ages, there is a progressive and significant decrease in the relative concentration  Approximately 7% of the total crystallin protein mass in adult human lens.   Gamma- crystallin level is high in nucleus and low in cortex , especially in young cortex  These are the smallest crystallins. 09-10-2025 SBS 10

11. Biochemical composition of lens Insoluble proteins  The main insoluble protein is albuminoid - 12.5% of total protein  Molecular wt . – 60kD  Found as a mixture - it is only partly digested by urea - extracted by soidic (acidic ) aqueous solution  The amino acid composition of this protein is similar to alpha crystallin  In human lens, most of the albuminoid is urea Soluble and appears to be derived from the alpha-crystallins 09-10-2025 SBS 11

12. Biochemical composition of lens : Other lens proteins  Glycoproteins are a group of proteins to which sugars are covalently bound  They are primarily associated with the lens cell membrane and are of considerable importance  They also contribute to the intercellular ground substance .  The lens cortex contains considerably more glycoproteins than the nucleus  Some other rare lens proteins include nucleoproteins, phosphoproteins, lipoproteins and fluorescent proteins 09-10-2025 SBS 12

13. Biochemical composition of lens: Amino acids Amino Acids Proteinogenic A lanine , L eucine , G lutamic acid, A spartic acid, G lycine , valine, phenylalanine, tyrosine, serine, isoleucine, lysine, histidine, methionine, proline, threonine and arginine Non - proteinogenic taurine, alpha- amino butyric acid, ornithine, 1- methyl- histidine, 3- methyl- histidine and homo-carnosine  lens contains all the amino acids present in any other tissue except tryptophan, cysteine and possibly hydroxy- proline  Concentration of each amino acid is higher in the lens as compared to aqueous humour , vitreous humour leading to a conclusion that they are actively transported into the lens 09-10-2025 SBS 13

14. Biochemical composition of lens: Amino acids  An active process is necessary to ensure that protein synthesis is not limited by the availability of amino Acids  The free amino acid pool is quite characteristic and constant for similar animals  Amino acid concentration of lens is not appreciably affected by ageing , fasting or feeding a protein- free diet  U n - altered level of amino acids in lens may be a result of balance of protein synthesis and catabolism on one hand and amino acid excretion, uptake by lens and the synthesis and breakdown of amino acids 09-10-2025 SBS 14

15. Biochemical composition of lens: Carbohydrates Carbohydrates Free glucose, fructose, and glycogen Derivatives S orbitol , inositol, ascorbic acid, gluconic acid and glucosamine 09-10-2025 SBS 15

16. Biochemical composition of lens: Carbohydrates Glucose The glucose level - vary from 20-120 mg% The lenticular glucose has its source in aqueous humour The level of glucose in lens is 1/10th of aqueous , where glucose concentration has been found to be 100 mg%. 09-10-2025 SBS 16

17. Biochemical composition of lens: Carbohydrates Fructose It is produced from glucose in the crystalline lens.  Concentration of fructose is very low in normal human lens ( 0.0018 to 0.2522 mg/gm) Its concentration also varies with age higher concentration of fructose in the lens, is implicated in the development of diabetic cataracts .  Fructose 3 phosphate   ( Fru-3-P ), A metabolite of fructose, is a potent glycating agent, Fru-3-P can directly modify proteins and contribute to the cross-linking and loss of transparency seen in diabetic cataracts 09-10-2025 SBS 17

18. Biochemical composition of lens: Carbohydrates Glycogen  Only traces of glycogen have been found in mammalian lenses  Concentration varies with age and the region of lens  Localized principally in the nucleus where it appears to replace γ -crystallin  normally present there to functionally to increase refractive index  It is located as a thin layer of intracellular granules, surrounding the nuclei of epithelial cells 09-10-2025 SBS 18

19. Biochemical composition of lens: Lipids 2.5% of wet weight human lens had lipids in two forms , a free form and a bound form as lipoproteins  Cholesterol , phospholipids ( Cephalin , lecithin, sphingomyelin and glycerides )  The proteolipids constitute 2% of the wet w t of lens and that 65% of lenticular lipids are bound to proteins  Lipids are most abundant in epithelial cells in children and in the cortex in adults  it may function as a lubricating cement substance  lipid content (cholesterol) increases with age especially in nucleus while the glycerides decrease  Similar changes occur in cataract where lecithin is abundant and cholesterol is found as crystals  In cataracts the concentration of free lipids increases and lipoprotein decreases. 09-10-2025 SBS 19

20. Biochemical composition of lens: Electrolytes Potassium It is the predominant cation in lens ( 114 and 130 mEq/kg of lens water )  The levels are higher than in any other eye tissue due to unusually large proportion of intracellular space in lens  K + conc.is typically lower in cataractous lenses than in normal lenses, especially in cortical and mature cataracts 2. Sodium  10-50% of potassium concentration ( 14-26 mmol/kg of lens water ) Higher serum Na + conc. is a potential risk factor and indicator for senile cataract formation, possibly due to high-sodium diets 09-10-2025 SBS 20

21. Biochemical composition of lens: Electrolytes 3. Calcium A mean value of 0.14 mg/mg dry weight is reported for human lens Increased calcium concentration within the eye's lens correlates with cataract development Abnormal elevations in both serum calcium and phosphorus may also be markers for age-related cataracts , potentially due to intracellular calcium concentrations and   Ca 2+ -ATPase activity The distribution of Ca ++ within the lens varies with age, and these changes are correlated with age-related cataracts.  09-10-2025 SBS 21

22. Biochemical composition of lens: Glutathione  3.5 to 5.5 mm/g wet weight of the lens  Produced from the interaction between glutamate and cysteine in lens cell  Glutathione being a tripeptide contains of three amino acids -Glycine , Cysteine & Glutamic acid Glutathione exists in two forms oxidized glutathione (GSSG) and reduced glutathione ( GSH ) More than 95% of glutathione is in the reduced state  By the virtue of presence of sulfa- hydryl group (-SH) in the cysteine fraction of glutathione it contributes to the redox systems in the lens microenvironment and buffers the effects of oxidants along with other enzymes 09-10-2025 SBS 22

23. Biochemical composition of lens: Glutathione Glutathione levels in the lens is also known to decrease with the age The lens contains high levels of glutathione with the highest concentration in the epithelium peroxidase activity also increases with age Several enzyme systems are available to minimize or buffer the effects of oxidants, including catalase, superoxide dismutase, glutathione reductase , Glutathione peroxidase and glutathione-S-transferase low levels of superoxide dismutase have also been identified in lens epithelium 09-10-2025 SBS 23

24. Biochemical composition of lens: Glutathione Glutathione peroxidase ( Selanium ) Glutathione reductase (Riboflavin) 09-10-2025 SBS 24

25. Polyol Pathway Glucose is utilized for energy by the cell Unused glucose enters the polyol pathway when aldose reductase reduces it to sorbitol The reduction of glucose to sorbitol is accompanied by the oxidation of NADPH to NADP + Sorbitol is oxidized to fructose by Sorbitol dehydrogenase The oxidation of sorbitol to fructose is paralleled by the reduction of NAD + to NADH HEXOKINASE can return the fructose molecule to glycolytic pathway by phosphorylating it to form FRUCTOSE-6-PHOSPHATE 09-10-2025 SBS 25

26. 09-10-2025 26 Aldose Reductase Sorbitol dehydrogenase SBS

27. Patho-physiology : In hyperglycemic state The affinity of aldose reductase for glucose rises. More of sorbitol is produced, using much more NADPH. Less NADPH for other processes of cellular metabolism Sorbitol accumulation starts NADPH acts to promote nitric oxide and glutathione production glutathione and nitric oxide Glutathione deficiency induces cell damage and oxidative stress. Nitric oxide will result in poor vasodilation further increasing the oxidative damage 09-10-2025 SBS 27

28. Polyol pathway and Diabetes mellitus Hyperglycemia-induced polyol pathway hyperactivity has an important role in the etiology of late-onset diabetic complications. Once sorbitol has been produced, it does not easily diffuse across cell membranes; this intracellular accumulation of sorbitol may be a factor in the etiology of diabetic complications Some of the complications include neuropathy, retinopathy, nephropathy, keratopathy, cataract-formation, possibly infection and atherosclerosis The inhibition of aldose reductase (AR), a rate-limiting enzyme of the pathway, could become a key element in the prevention and reversal of diabetic complications. Epalrestat ,  sorbinil ,  tolrestat & ranirestat are some examples of ARI .  09-10-2025 SBS 28

29. Diabetic Cataract : polyol pathway hyperactivity major cause of blindness in developed and developing countries. pathogenesis is still not fully understood Recent basic research studies have emphasized the role of the polyol pathway in the initiation of the disease process  Aldose reductase (AR) catalyzes the reduction of glucose to sorbitol through the polyol pathway a process linked to the development of diabetic cataract.  Intracellular accumulation of sorbitol leads to osmotic changes resulting in hydropic lens fibers that degenerate  In the lens, sorbitol is produced faster than it is converted to fructose by the sorbitol dehydrogenase .  Polar character of sorbitol prevents its intracellular removal through diffusion.  The increased accumulation of sorbitol creates a hyperosmotic effect that results in an infusion of fluid to countervail the osmotic gradient .  The osmotic stress in the lens caused by sorbitol accumulation induces apoptosis in lens epithelial cells (LEC) leading to the development of cataract. 09-10-2025 SBS 29

30. Diabetic Cataract : polyol pathway hyperactivity Aldolase Reductase Sorbitol dehydrogenase 09-10-2025 SBS 30

31. 09-10-2025 SBS 31

32. Diabetic Cataract : polyol pathway hyperactivity  The role of osmotic stress is particularly important for the rapid cataract formation in young patients with DM Type 1 due to the extensive swelling of cortical lens fibers.  Osmotic stress caused by the accumulation of sorbitol induces stress in the endoplasmic reticulum (ER), the principal site of protein synthesis, ultimately leading to the generation of free radicals .  ER stress may also result from fluctuations of glucose levels initiating an unfolded protein response (UPR) that generates reactive oxygen species (ROS) and causes oxidative stress damage to lens fibres. 09-10-2025 SBS 32

33. Corticosteroid induced CATARACT  Corticosteroids ( prednisolone for example) forms a Schiff base with crystallins. The C-20 carbonyl group of prednisolone reacts with ε-amino groups on the lysine residues of the crystallin to form an unstable Schiff base intermediate. C-21 -OH group on the prednisolone then participates in a Heyns rearrangement , converting Schiff base into a more stable amine-linked adduct. The covalent modification of crystallin increases the reactivity of protein thiols. This leads to the formation of high-molecular-weight, disulfide-linked protein aggregates. Aggregates cause opalescence (cloudiness) in the lens, which is the physical manifestation of a cataract ( Posterior subcapsular cataracts ) . 09-10-2025 SBS 33 Posterior subcapsular cataracts Posterior subcapsular cataracts

34. Advanced glycation end products (AGEs): diabetic cataract  Contribute to cataract formation by cross-linking and damaging lens proteins, leading to a loss of transparency.   AGEs form through a process of glycation and oxidation involving reducing sugars and proteins, a natural process that accelerates with age and is more pronounced in individuals with diabetes due to higher glucose levels.   The accumulation of AGEs, such as  N(ε)-carboxymethyl-lysine (CML) , alters the protein structure and function of the lens, a key factor in the development of cataracts.  09-10-2025 SBS 34

35. AGEs formation in the Lens 1. Glycation:   Reducing sugars, like glucose, react with proteins in the lens, forming unstable intermediates called Schiff bases.  2. Oxidation:   These Schiff bases undergo further reactions, including oxidation and rearrangement, under conditions like oxidative stress.  3. AGE Formation:   This process forms stable, irreversible protein modifications known as Advanced Glycation End Products (AGEs).  4. Lens Protein Damage:   Lens proteins (crystallins) are long-lived, allowing for significant accumulation of AGEs over time, especially in the presence of high glucose levels.  Protein Cross-linking:   AGEs cause cross-linking of lens proteins, leading to protein aggregation, increased stiffness, and impaired transparency of the lens.  Lens Stiffness and Presbyopia:   The increased stiffness caused by AGEs correlates with presbyopia, an age-related hardening of the lens, and is an early symptom of age-related cataracts.   Accelerated by Diabetes:   Diabetic patients accumulate AGEs at a faster rate than non-diabetics due to chronic high blood sugar levels, leading to an earlier and more rapid onset of cataracts.  09-10-2025 SBS 35

36. Key AGE Indicators in Cataracts 1. N(ε)-carboxymethyl-lysine (CML):  A widely studied indicator of protein glycation.  2. N-(carboxyethyl) lysine (CEL):  Another significant AGE found in human cataractous lenses.  3. Methylglyoxal (MGO)-derived products :   MG-H1 (Methylglyoxal-derived hydroimidazolone ) are dominant forms of protein damage in aged lenses.  09-10-2025 SBS 36

37. Glycation of crystallin: Diabetic Cataract Non-enzymatic reaction of sugars, particularly glucose, with the lens protein crystallin, Causing protein cross-linking, aggregation, and the formation of advanced glycation end products (AGEs) Development of cataracts, especially in aging and diabetic individuals.  Sugars react with lysine amino groups on crystallins, form Schiff bases and later Amadori products through the Maillard reaction, eventually leading to structural changes and loss of protein function .  09-10-2025 SBS 37

38. Glycation of crystallin: Diabetic Cataract The Glycation Process 1. Initial Reaction:   Reducing sugars (like glucose & Fructose ) react with the free amino groups on proteins, forming a Schiff base .  2. Amadori Rearrangement:   The Schiff base undergoes an Amadori rearrangement to form a more stable Amadori product , which is a type of early glycation product.  e.g. Fructosamine, HbA1c (glycated Hemoglobin) and glycated albumin ( gHSA ).  3. Maillard Reaction:   Over time, these Amadori products undergo further chemical changes, including dehydration and rearrangement , a process called the Maillard reaction . Maillard reaction produce reactive dicarbonyl compounds such as methylglyoxal (MGO) and deoxyglucosones .    4.Advanced Glycation End Products (AGEs):   The end products are a diverse group of stable compounds that cause protein (Crystallin ) cross-linking and aggregation.  09-10-2025 SBS 38

39. Millard Reaction 09-10-2025 39 SBS

40. Methylglyoxal pathway  A bypass of the main glycolytic pathway.   Activated under certain conditions, during increased intercellular uptake of glucose or glycerol.  Methylglyoxal is an electrophile that can react with proteins and nucleic acids to form Advanced Glycation End products (AGEs).  Methylglyoxal synthase is activated by high substrate concentration (DHAP) due to accelerated glycolysis. Methylglyoxal synthase is inhibited in phosphorylated form. MGS is inhibited by feedback inhibition by Pi Low intracellular phosphates activates Methylglyoxal synthase which removes the phosphate-mediated allosteric inhibition  It is detoxified by the Glyoxalase system (Gly I and Gly II) into D-lactate, preventing its toxic accumulation.   Polyurea increases the phosphate excretion 09-10-2025 SBS 40

41. Methylglyoxal pathway : Consequences MGO a highly reactive and irreversibly modifying proteins, lipids, and DNA MGO increases reactive oxygen species (ROS) synthesis, which can damage cellular components and lead to inflammation. High levels of MGO damage the endothelium, the inner lining of blood vessels. MGO impairs insulin secretion by pancreatic beta-cells by inducing oxidative stress and mitochondrial dysfunction MGO can directly modify insulin and key components of the insulin signalling pathway, such as insulin receptor substrate-1 (IRS-1). This worsen the insulin resistance 09-10-2025 SBS 41

42. 09-10-2025 SBS 42 AMO AMO – Acetone monooxygenase Semicarbazide -sensitive AMINO-OXIDASE SSAO

43. 09-10-2025 SBS 43 Methylglyoxal synthase MGS