I. GENERAL INTRODUCTION INTRODUCTION
The World Health Organization (WHO), 1983, has estimated the blind population throughout the world at as many as 28 million people. More than two thirds of these live in developing countries. Poor living conditions, environmental hazards and the lack of health services and education contribute to this. In fact, the main causes of visual loss, such as trachoma and vitamin A deficiency, need to be dealt with, on the basis of a community as a whole, rather than individual cases requiring technical intervention. Similarly, the present enormous problem of 17 million unoperated patients with cataract calls for health services to be made available for all, rather than for technological expertise.
We know that the number of blinds in the world will probably be double in number in the next century, unless rapid action is taken against the prevention and control of cataract. The population growth and the increasing number of the elderly, particularly in developing countries, will result in a pronounced increase of age related eye disease as cataract.
Though effective surgical techniques for cataract removal are available, there is no known way of preventing cataract. At the same time, the medical costs associated with cataract surgery are very high. Thus cataract has a personal as well as a financial impact that is substantial and far reaching. It would, therefore, be most desirable to develop alternative methods of treating, preventing or retarding the development of cataracts without resorting to surgery. This is just one example of the many areas where research is needed, the results of which, if applied at the primary level, could have a great impact on prevention of blindness. There is a tremendous difference in the frequency of cataract in different geographic areas. Senile cataract in certain areas in Africa and Asia appears to have an earlier onset thart elsewhere, its incidence becoming significant as early as the 40 to 50 age groups.
Ultraviolet light and/or sunlight have long been felt to be a factor leading to cataract formation, and there seems to be a positive correlation between the prevalence of senile cataract and the duration of sunlight but other studies have revealed other possible causes.
The present WHO strategy to combat blindness due to cataract is based on simple ocular surgery, but this is not sufficiently available to cope with new cases and to take care' of the existing backlog of cases. The statistics show that every year about 600 ophthalmologists emerge out of various medical institutes in our country and today we have got about 7000 eye specialists i.e. at a ratio of 1 doctor to 1.5 lakh population which is quite an inadequate situation. One approach to the problem, which is being used in several Asian countries, is to organize mobile eye camps, where many patients can be assembled and operated on, in a relatively short time. Indeed, the cataract camps in our country, and Pakistan demonstrate how such surgery can be carried out at a minimum cost. These mobile eye clinics, may permit efficient intervention against blinding diseases in certain areas. However, it is important to ensure the continuity and follow up activities initiated by such mobile teams, and to have close collaboration with the local health personnel and the community in such matters as initiating health education activities.
There is no medical treatment for cataract. Lens extraction is indicated when visual impairment interferes with the patients' normal activities. Surgery definitely improves visual acuity in well over 90% of the cases.
But, in light of the limited resources and capacity to reach populations in need, specially in developing countries like ours where majority of the populations are in rural areas the western models of systems of health care or of educating and rehabilitating the blind are not necessarily appropriate. Therefore research needs to be directed in improving systems of intervention into control and reduction of blindness, and especially towards prevention of blindness. There is thus a need for further investigations into the etiology of the blinding disease and for possible measures for its prevention. Research in cataractogenesis has naturally been recognised as a primary area for intervention in the field of blindness.
At the present time no complete and satisfying description can be given of the etiology of cataract. Our knowledge of the mjtabolism of the lens is still so incomplete that an analysis of still more complex problem of its derangement is impossible. Fundamentally the loss of transparency is due to disturbance of intimate structure of the lens, the fibers of which form a colloid system into which a large amount of water is found.
We know numerous causes for the development of lens opacities, including (a) physical factors, (b) chemical factors, (c) predisposing systemic conditions, (d) predisposing eye diseases, (e) genetic factors and developmental deformities, (f) virus infection during embryonic life and (g) ageing. Thus many cataractogenic factors have been detected. The biochemical background of cataractogenesis, however, is still unknown. In general, only the initial steps of the involved pathogenetic mechanism and the resulting morphologic changes due to hydration and/or protein denaturation are known. A better understanding of the pathological mechanisms involved in cataract formation has been derived from animal research. Some experimental opacities, such as those due to galactose, naphthalene, and ionizing radiation, have been studied extensively and theories have been evolved, which satisfactorily explain the responsible morphological and biological changes.
Experimental investigations of cataracts, however, are still of limited value in our efforts to understand the etiology of human cataracts. Experimentally it is possible by selecting suitable conditions to implicate individual factors as the causes of opacities. Clinical cataracts can only in certain instances be traced to such a simple mechanism (human galactosemia, cataracta diabetica vera, hypocalcemia). The causes of senile cataract are presently still unknown.There are many hypotheses which are as contradictory to one another as are the general theories on ageing. Quite surely, every ageing person develops morphologic lens changes. However, it is remarkable that only in some patients do these circumscribed opacities progress to mature cataracts and that the time of occurrence varies so widely. Furthermore, it is even more remarkable that these age related lens changes can have such different morphologic manifestations, and it is difficult to understand why opacification in some patients is more intense within the anterior cortex (water clefts, spokes, cuneiform opacities), whereas in others, within the nucleus, or within the posterior subcapsular region. These modifications of senile cataract are not inevitably concurrent with our fate of ageing, but are caused by other, superimposed influences.
Despite intensive research going on throughout the world, we know little about the fundamental changes which determine cataractogenesis.
Protein metabolism of the lens has generated an unparalleled interest and the changes occurring in the soluble and insoluble lens proteins have been studied in great details. We know the changes taking place in the proteins but a cause - effect relationship cannot be established, and the observed changes occur not in the early but in the later phase of development of cataract.
The study of enzymes is a subject which has a special interest because it lies just on the borderline where the biological and the physical sciences meet. On one hand, enzymes are of supreme importance in biology, life depends on a complex network of chemical reactions brought about by special enzymes and any modifications of enzyme pattern may have far reaching consequences for the living organisms, on the other hand, enzymes as catalysts, are receiving increasing attention.
The lens contains a great number of enzymes, usually at a very low concentration, but very little is known of their nature apart from their metabolic activity; whether they are distinct species existing independently of structural proteins, whether they are in some sort of conjugation with the crystallins or whether any of the crystallins bear enzymatically active sites is not known.
It seems very likely that oxidative mechanisms play a major role in the etiology and pathogenesis of senile nuclear cataract. The lens like other tissues, contains a series of defence mechanisms that may protect it from the deleterious effects of oxidation. Very little is known about such defence mechanisms. Reduced glutathione (GSH) and Ascorbic acid, possibly maintain the thiol (SH) groups of proteins in the reduced state, thus preventing formation of high molecular weight (HMW) protein aggregates. The formation of HMW proteins in X-ray induced cataracts through disulphide bond formation and the involvement of SH oxidation in HMW proteins isolated from human cataractous lenses suggest a role for GSH in protecting protein SH groups. It is essential to find out some means of preventing or delaying cataract formation. It has been estimated that delaying the development of cataract by ten years would reduce the number of cataract by 45%. Such a breakthrough to delay the onset of cataract is only possible by intense research in this area and will be considered a significant landmark, if achieved. HISTORICAL ASPECTS (Groni,1975)
The word 'Cataract' stems from Latin "Cataracta" which in turn is traced back to the Greek "Kataraktes" which means waterfall (breakdown, downrushing). The history of cataract goes back some 4000 years and probably further. In ancient 'Hindu Medicine', however it occupied a prominent place, being interpreted as a type of corrupt humor and the teaching of Susruta on its relief by coaching was remarkably precise and detailed. In Hindu Medicine cataract was defined by Susruta as an opacity due to a derangement of the intra-ocular fluid, subsequent history is full of fantasies and prejudices concerning its nature. In ancient Greece, ocular operations were performed; indicated by the findings of ocular instruments belonging to that period.
Knowledge of the anatomy of the lens advanced at a very slow pace, as did knowledge about cataracts and their exact location.
Guy de Ghauliac (1300-1368) from Avignon, stated in his book 'Chirurgia Magna', that the cataract is a spot similar to skin placed in front of the pupil which interferes with sight due to excessive humidity that gradually penetrates into the eye and coagulates.
Girolamo Fabricio of Acquaendente in his book De Oculo (1600), located the lens in its proper place, that is, behind the iris.
About the midsixteenth century, Caspar Stromayr wrote a study on medicine in which he included the concept that cataracts may be caused by a ray (ful-guration). Jacob Schalling first stated that cataracts involve the lens. Werner Rolfinck (1599-1673) first published the fact that the cataract is nothing but the opacification of the lens.
At the end of the seventeenth century, Antoine Maitre-Jan (1650-1730) realized that by coaching the cataract the lens itself is displaced. Michel Pierre Brisseau (1676-1743) presented his belief (17th Nov 1705) that the opaque lens forms the cataract, before the Academic Royale de Sciences in Paris. Maitre-Jan (1705) also published his observations on cataract.
Moreover, Maitre-Jan made his observations in the period 1680 to 1690 so he rated priority over Brisseau. As very seldom happens in history, no dispute over the question of priority took place between these two authors. It is worthwhile mentioning that Brisseau's teacher, Duverney, advised him not to annojance his observation. However, Jean Mery backed this concept along with Brisseau and Maitre Jan.
However, there were several oculists who attacked on this discovery severely. Christian Lange in 1706, and Thomas Woolhouse (1650-1730), opposed this theory. In 1719 an article was published under the pen name of Sincerus Fidelis, who called the concepts held by Woolhouse and Le Cref 'Harmful lies'. Similarly, during the same year Jacob Hovius and Johann Heinrich Freytag opposed the new concept regarding cataracts.
However, the medical authority represented in the person of Hermann Boerhave (1668-1738) in Holland, spread the new theory all over Europe. Giovanni Battista Morgagni (1682-1771), the famous Italian anatomist, was in favour of it. Doubts continued until Jacques Daviel published his work in 1753 rejgarding the removal of cataracts. Ji * The beginning of the era of cataract extraction is usually identified with the work of Jacques Daviel (1696-1762). The first cataract operation performed by Daviel took place on April 2,1745. In Memoires de "Academie Royale de Chirurgie Daviel" wrote that upto November 16, 1752 he had performed 200 extractions, of which 180 produced satisfactory results. Daviel performed a demonstration of cataract extraction on a female stag. CO
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ANATOMY OF LENS
The crystalline lens is a lentil shaped transparent structure in the anterior segment of the eye. It is positioned behind the iris. It is completely free of vessels and has no nerve supply. It obtains nutrition from the surrounding fluids : the aqueous and the vitreous.
The main functions of the lens are as follows :
1. To refract the light entering the eye through the pupil and focus it on the retina. 2. To maintain its own clarity. 3. To provide accomodation which allows the eye to clearly focus objects placed within a six metre range. 4. To absorb ultraviolet light..
The lens has the shape of a flattened globe (Fig. 1). Its various parts are given geodesic terms. The most anterior part of the lens is the anterior pole, the peripheral area is the equator and the most posterior area, the posterior pole. The lens is entirely surrounded by a capsule, and under the anterior capsule, there is a single layer of epithelial cells. The innermost portion of the lens is the nucleus and outer part is the cortex.
The lens is suspended from the ciliary process by the zonules. When the ciliary muscle contracts and the zonules relax, the lens becomes thicker and more convex, increasing its refractive power. The lens becomes thinner following relaxation of the ciliary muscle.
Capsule : The capsule is secreted at the embryonic stage by the lens epithelium. The capsule appears to be an anatomical structure unchanged throughout the life. It surrounds the lens and maintains its structural integrity. The capsule is 2.2 mm 11
thick and homogenous by light microscopy. With the electron microscope many lamellae measuring 300-400 A° in thickness are found. Each lamella contains fine filaments (Hogan et al,1971). The lens capsule depends on contact with lens epithelium and fibres for metabolic supplies. It is tough and resistant to traction. It restricts penetration into the lens of various molecules based on size, charge and lipoid solubility.
Epithelium : The cuboidal cells of the lens epithelium form a monolayer. The epithelial cells are firmly attached to the anterior capsule and loosely attached to the underlying fibers. In the course of terminal differentiation, epithelial cells lose their nuclei as well as other cytoplasmic elements, such as mitochondria and microsomes. The epithelium is the area of the lens with the highest metabolic rate.
Nucleus and cortex : The fibers make up the bulk of the lens cortex and nucleus. Each fiber, hexagonal in cross section, represents an elongated cell with a membrane. The areas in which the fibers meet anteriorly and posteriorly are the sutures of the lens. The lens fibers lose their nuclei as well as other cytoplasmic components like mitochondria as they grow old. The morphological structure: of the lens is arranged so that the dedifferentiated fiber cells are not eliminated but are pushed in towards the centre of the tissue. Thus oldest fiber cells are in the centre of the lens and the youngest cells are at the periphery. Fiber cells produced at all stages of life from fetus to old age are present. The spaces between the lens fibers, the extracellular spaces of the lens, are very small, accounting for only about 5% of the lens volume.
The difference between the cortex and the nucleus lies in integrity of lens fibers. As one approaches the more central region 12
of the adult lens the cell walls become less recognizable and the lens substance in the nucleus comes to have a homogenous structure. The thickness of the nucleus of the human lens was found to remain unchanged after the age of 20 years.
Zonules : The zonules are thin, delicate, filaments maintaining the lens suspended in position. The zonules stretch from the ciliary epithelium and peripheral retina to the lens capsule and form a thin pericapsular membrane at the lens equator. 0 Z E J 3 z < 3 0 3s a S u < 08 > Z CO of, D + 0 ""< u •"< 3 "X a X m ^ 0 P *? >o
LENS COMPOSITION
1. Lens water 2. Proteins and free amino acids 3. Enzymes 4. Carbohydrates 5. Lipids 6. Nucleic acids 7. Major inorganic constituents 8. Trace elements 9. Organic constituents of low molecular weight a) Phosphates b) Glutathione c) Ascorbic acid d) Myo-inositol e) Taurine f) Nicotinamide adenine nucleotides
The efficiency of accomodation in young normal lenses depends primarily on two factors : the ease with which the lens curvature can be altered by deformation and the difference in refractive index between the lens and the two humors that surround it. These two factors are determined by the gross composition of the lens, (Fig.2) i.e. the fraction of water and the fraction of highly refractile material, the protein. The lens is one of the unique tissues in the body. It is a dehydrated organ. It contains only 66% of water. It contains the highest concentration of protein (33%) of any biological tissue found in nature (Cotlier, 1981) (e.g. brain 10%, muscle 18%).
Composition of the lens may vary according to : a) The total age of the lens b) In a given lens; according to the region selected. 14
Lens Water
The low amount of water in the lens, about 66%, is a natural consequence of the need for a structure having a refractive index as far removed as possible from that of the watery fluids at the two optical interfaces of the lens.
The lens cortex is more hydrated than the lens nucleus. Lens hydration is maintained by an active Na ion-water pump that resides within the membranes of cells in the lens epithelium and each lens fiber. In the human lens there is a difference in water content between nucleus (63.4 ± 2.9%) and cortex (68.0 ± 4.3%). There seems to be no tendency for the water content to decrease with age. The water is chiefly intracellular but whether or not it is all in the same state, i.e. whether free or bound, is still open to question. Recent research on lens water using several techniques, including nuclear magnetic resonance, suggest that about 3% of total lens water is bound to surfaces within the lens. It is in a state of extremely rapid exchange with the free water. There does seem to be a small fraction of water which is extracellular. The amount • is probably less than 10% of the total lens volume, and the experimental value is dependent on the method used to determine it.
Proteins and Free Amino Acids The protein content of the lens, 33% of the total weight, is higher than that of any other organ in the body (e.g.brain 10%, muscle 18%). The high concentration cf protein is presumably to create a medium of high optical density and therefore high refractive index. Most of this protein is structural protein represented by the crystallins, whose only function is to contribute to the transparency and refractive power of the tissue.
Lens proteins can be divided into two fractions, soluble proteins 85% and insoluble proteins 15%. The insoluble proteins are attached to or constitute the membranes of the lens fibers. 15
This fraction is known as albuminoid and it increases with age. Most insoluble proteins are found in the lens nucleus, whereas most soluble proteins are in the lens cortex (Harding and Dilley, 1976). The soluble proteins of the lens were separated into three fractions by Morner in 1894. These fractions, now called alpha, beta and gamma crystallins, have since been prepared by many different procedures, and subjected to various methods of separation. These proteins were specially adapted for the specific purpose of maintaining transparency, otherwise they would impart to the lens a milkiness that would militate against the formation of distinct images. It is for this reason that the identification of the proteins and the determination of their structure have been investigated in great detail. The function of the lens is to provide a transparent structure which focuses light on the retina and which will continue to do so accurately as the eye increases in size; this will require a fall in the refractive index of the lens from centre to periphery which is associated with a parallel fall in protein concentration (Clayton, 1978). The earliest formed, central fibres eventually have the highest protein concentration, and in order to sustain this and maintain transparency it appears that they have a different array of proteins from those in the outer fibres formed later.
Some properties of Lens crystallins
Alpha Beta Gamma Crystallin Crystallin Crystallin
Molecular Weight High Intermediate Low Solubility at pH5 Insoluble Soluble Soluble Electrophoretic Medium Low mobility, at pfl8.6 High Thiol (SH) content Low High High N terminal Masked Masked Free glycine amino group (acetyl in man, cow, methionine) rabbit, alanine in ra 16
Minor proteins : Minor proteins of the lens include nucleoproteins, phosphoproteins, lipoproteins, proteolipids, and fluorescent proteins.
Amino acids : The concentration of amino acids in the lens is two to six times higher than in aqueous. Amino acids are actively transported into the lens. Once, in the lens, free amino acids are incorporated into RNA (Devi et al,1961) to form lens protein, or can be metabolized with formation of CO-, or can efflux from the lens; the turnover of free amino acids in the lens is very rapid, the renewal rate for lysing being 16% of the total in the lens per hour (Cotlier,1961). The transport of tryptophan by the lens is the most active among all amino acids and requires metabolic energy.
Enzymes
The lens contains a great number of enzymes, usually at very low concentration, but little is known of their nature apart from their metabolic activity whether they are distinct species existing independently of the structural proteins, or whether they are in some sort of conjugation with the crystallins, or whether any of the crystallins bear enzymatically active sites. The epithelium contains the respiratory enzymes - the cytochromes, and flavoproteins. The enzymes which have an important role in lens metabolism and are studied in lens are, Hexokinase, Phosphofructokinase, Lactate dehydrogenase. Aldose reductase, Glucose-6-phosphate dehydrogenase, Sorbitol dehydrogenase and Malate dehydrogenase etc.
Carbohydrates The free carbohydrates of the normal lens (Kuck,1965) occurring in any significant amount are few in number, viz. glucose, fructose and glycogen. Derivatives of sugars found in the lens are : sorbitol, inositol, ascorbic acid, gluconic acid 17 and glucosamine. The lenticular glucose has its source in the aqueous humour. The concentration of glucose within the lens is normally about one-tenth its concentration in the aqueous humour (Kuck,1965). Free glucose of the lens is in the extracellular space and is in practical equilibrium with the glucose of the aqueous humour.
Glycogen occurs in the mammalian lens in very small amounts. Fructose is present in all lenses (Kuck,1965). Its concentration is usually rather close to that of glucose. There is species variation in the fructose concentration, for instance the lens sugar in the rabbit is chiefly fructose with very little glucose. Fructose can hardly be confined to the extracellular space since it is produced within the lens fibers by operation of the sorbitol pathway.
Lipids
The lipids of the human lens are unique and differ, markedly from those of other animal species (Broekhuyse, 1969). Lipids represent 3% to 5% of the dry weight of the lens (Feldman,1968). In the human lens cholesterol represents approximately 50% of lipids, where as phospholipids account for 45% and glycosphingolipids and ceramides for 5%. The lipids are major components of the lens fiber membranes. The cholesterol-phospholipid ratio of human lens fiber membranes is the highest among cell or organelle membranes, thus conferring the lens resistance to deformation.
The existence of proteolipids (Feldman,1965 and Feldman,1968) in the lens was a factor leading to much confusion in the earlier work on lens lipids. 65% of the total lenticular lipid in man is in the form of a lipid-protein complex. They are not soluble in ordinary fat solvents and miss being extracted. The conversion of this bound to unbound forms is a characteristic change, which according to Feldman (1967) is a translocation of organized lipid in membranes to disorganized and perhaps intercellular deposits. 18
Nucleic Acids
Deoxy ribonucleic acid (DNA) : Because the len's' has so few cells with nuclei, the total amoung of DNA in a lens amounts to very little. Its synthesis is confined to the epithelium in the intact lens (Reeder and Bell, 1965). An analysis of Calf lens showed the following distribution : epithelium 30mg%, cortex 7mg%, with a trace in the nucleus (Mandel and Schmitt, 1957).
Ribonucleic Acid (RNA) : The level of lenticular RNA is usually in the range of 30 to 200mg%, variations being due partly to species but mostly to age. Lerman(1965) has proposed 57.5 jug/lOOmg wet weight values for human lens. The lens RNA contains only a small proportion of mRNA, and characteristics of lens RNA is its long life. RNA is partitioned between the various parts of the lens and in each partition it exists in two forms, a soluble fraction and an insoluble fraction.
Major Inorganic Constituents
As expected in a tissue with high percentages of intracellular space and protein, the lens contains high concentration of potassium and a low concentration of sodium ions, chloride ions, and water than the aqueous and vitreous. Lens sodium occurs chiefly in the extracellular space. Depending on a number of factors such as species, age and viability of the lens, the lens sodium concentration is one-half to one-tenth that of potassium (Pirie and van Heyningen, 1956). Along with potassium it is involved primarily in osmotic balance. It appears that fractions of both sodium and potassium are not readily exchangeable but are bound in some way (Harris and Becker, 1965). The bulk of this non- diffusible, fraction probably lies in the nucleus.
Calcium occurs at a very low level in the young lens, but functionally it is highly significant. It is thought to be a most important factor in maintaining normal lens membrane permeability 19
(Thoft and Kinoshita, 1965).The lens is extremely rich in magnesium. The function of magnesium in the lens is almost certainly connected with its capacity to act as a co-factor in several enzymatic reactions. Normally the lenticular content of magnesium is adequate for its metabolic needs so that a lens incubated in a magnesium- free medium suffers no disadvantage.
Phosphate is the predominant anion in the lens, comprising nearly half the ash. Of the total phosphate only 10% exists as inorganic phosphate; the majority is in the form of various organic phosphates. Nevertheless, this small amount is important because it is undergoing rapid turnover. Older lenses with a decreasing rate of metabolism require less ATP, this lower need is reflected in lower levels of organic phosphates and a somewhat lower level of inorganic phosphate. There are no adequate analyses for inorganic sulfate in the lens; the older values for total sulfate included the larger part of organic sulfur which was converted to sulfate during the analytical procedure of ashing. Function of sulfate is not exactly known. Chloride in the lens is probably associated with sodium in the extracellular space. The lens fibre membranes appear relatively impermeable to chloride. Chloride has no apparent function outside of its osmotic effect and its ability to associate with cations in the maintenance of ionic neutrality. The remaining lens anion of major importance is bicarbonate, which functions to maintain the pH, especially in the extracellular space which contains little protein or phosphate and is in closer communication with the aqueous humor.
Trace Elements Lenses of various species have been analyzed for trace elements. The human lens contains 0.15mg/100gm wet weight of iron, 0.23mg% copper, less than 0.003mg% manganese, 0.30mg% zinc and 0.036mg% boron (Sizeland,1952). Of these metals, iron, 20
copper, zinc and manganese are known to be cofactors for enzymes.
Organic Constituents of Low Molecular Weight
Phosphates : Adenosine triphosphate (ATP), Adenosine diphosphate (ADP) and Adenosine monophosphate (AMP).
ATP is relatively abundant in the lens, probably as a result of several factors : Extremely low level of ATPase, low level of high energy phosphate acceptors, and the low activity of hexokinase and other enzymes which use ATP (Kuck, 1970a). For mammals the normal level is 40-50mg/100g. In fish it is nearer 30mg/100g, in birds over lOOmg/lOOg. Klethi and Mandel (1965) relate these differences to the structure and function of the different species of lenses with respect to visual accomodation. In fish no work is done within the lens itself, since focusing is accomplished by external muscles which move the lens along the optical axis. In birds there is no zonule and some of the burden of accomodation appears to be taken up by the annular pad. The mammalian lens lies between these two extremes.
ADP is one of the most abundant organic phosphates in the lens and, along with ATP, it makes up the bulk of the acid labile or high-energy phosphates of the lens. The level of ADP in the cow lens is only about 25% of that of ATP, about 7-8mg/100g; (Kuck, 1970b). AMP occurs at a concentration of about 4mg/100g, half that of ADP.
The major part of the non-nucleotide organic phosphate of the lens consists of alpha glycerophosphate, glycerophosphocholine, glycerophosphoethanolamine, phosphocholine phosphoethanolamine and phosphocreatine.
Glutathione : This tripeptide. glutamyl-cysteinyl-glycine, is of importance for the lens since its concentration is some 1000 times that in the aqueous humors. Most of lens glutathione is ir\ the 21
reduced form (GSH). Only 6.8% of all lens glutathione is in the oxidized form (GSSH) (Reddy, 1971).GSH and GSSG are in equilibrium.
2 GSH + i O2 ^ GSSG + HjO.
GSH lens concentrations (in micromoles per gram) are 2.80 in the monkey, 6.70 in the rat, 12.0 in the rabbit. GSH levels are much higher in the cortex than in the nucleus of the lens. In the human lens, GSH levels decrease slightly with age.
The main functions of lens GSH are :
1. To preserve the physico-chemical equilibrium of lens proteins by maintaining high levels of reduced sulfhydryl (SH) groups. 2. To maintain transport 'pumps' and the molecular integrity of lens fiber membranes.
Ascorbic Acid : In most animals the lenticular concentration of ascorbic acid is unusually high compared with other tissues but in the rat lens it is so low as to be almost undetectable. The usual level in most animals is 20 to 30mg/100g (Heath, 1962; Glick and Biskind, 1936). In a particular species, the level of ascorbic acid in the aqueous humor may be higher or lower than the level in the lens for no obvious reason, but the level in aqueous humor is many times that in blood plasma except in the cat (Pirie and van Heyningen, 1956). The ciliary processes have the capacity to concentrate ascorbic acid and to secrete it into the posterior chamber (Becker, 1967). The ascorbic acid concentration, in the posterior aqueous humor is significantly higher than that in anterior aqueous humor, a relationship which is not incompatible with uptake by the lens. Certainly the lens has no known mechanism for the synthesis of ascorbic acid, although it appears to reduce de-hydroascorbic acid. This reduction may be a major metabolic process in the lens. 22
Myo-Inositol : The high concentration of free myo-inositol in the lens (sometimes over 500mg/100g) has aroused much interest, but nothing definite is known about its metabolism or function. Inositol occurs in the lens in very small amounts as a component of certain complex lipids. Its structural resemblance to the sugars and acyclic polyols suggests a metabolic relationship, but none has been demonstrated. It is suggested that inositol has a function in the lens which depends on its shape as well as its chemical structure (Kuck, 1975a).
Taurine : On analysis of lens amino acids by automatic amino acid analyzer, it has been realized that free taurine is as ubiquitous a§ inositol and that its concentration in lens, (6.7mg/100gm in human eye bank lens) (Barber,1968) places it among the major free amino acid constituents, being exceeded only by glutamic acid, alanine and the tripeptide glutathione. The function of taurine in the lens is completely unknown but the existence of an active pump suggests that its occurrence is not fortuitous.
Nicotinamide Adenine Nucleotides : The importance of Nicotinamide Adenine Nucleotides lies in their function as co-factors in various dehydrogenation reactions. In most tissues NAD is the major one, followed by NADH, then NAIJPH and finally NADP (Kuck, 1970c). There is lack of agreement as to whether or not this pattern is followed in the lens; the use of various methods of extraction probably accounts for the difference. 23
LENS METABOLISM
1. Carbohydrate Metabolism 2. Protein and Amino Acid Metabolism 3. Lipid Metabolism 4. Nucleic Acid Metabolism 5. Glutathione Metabolism
The lens, in comparison with other organs, is considered to be metaboUcally rather inactive. The metabolism of the lens has been the subject of a great deal of research. Because of the slow turnover of lens protein (about 5%/day) the need for ATP in the protein biosynthesis process is low, probably about 10% of the energy generated by anaerobic glycolysis. Most of the work accomplished by the lens is accounted for by the operation of the cation and amino acid pumps (Kuck, 1975b). The immediate needs of the lens for available energy are met by a store of high energy phosphate in the form of ATP and creatine phosphate whose concentration is roughly inversely proportional to that of ATP (Frohman and Kinsey, 1952). The lens normally obtains most of the energy from glucose metabolized anaerobically to lactic acid, fixing it in the usable form of ATP which is expended chiefly for the transport of cations. Much less is needed for amino acid transport and actual synthesis of the peptide bonds in lenticular protein.
Carbohydrate Metabolism Glucose from the aqueous and vitreous diffuses into the lens by facilitated diffusion and is rapidly metabolized through four main pathways as shown in table (Kinoshita, 1963 and Kinoshita, 1965a). 24
Glucose metabolism by lens
Pathway End Products Glucose Mole ATP through gained/mole pathway (%) glucose metabolised
Glycolytic Lactic acid 80 2 Krebs cycle CO2 : H^O 5 36 (Oxidative) +O2 Pentose shunt CO2 : NADPH 15 Sorbitol pathway Lactic acid Unknown
Anaerobic Glycolysis : The energy needs of the lens are met chiefly by the conversion of glucose to lactic acid via anaerobic glycolysis. This pathway is anaerobic because 0, tension of the aqueous humor is low, and Respiratory quotient of Oxygen being only 0.1. Lactic acid escapes into the aqueous humor by diffusion and is eliminated via circulating fluid. The enzymes involved are soluble, distributed in the cytoplasm throughout the lens. 80% of the glucose is metabolized by this pathway. Anaerobic glycolysis is an inefficient mode of glucose utilization because the energy available is obtained not by the combustion of hydrogen to water but rather from the re-arrangement of a glucose mol'ecule to give two molecules of lactic acid. The free energy change in this conversion is very small but for the avascular lens it has the considerable advantage of enabling the lens to maintain itself under completely anoxic conditions.
The Hexose Monophosphate Shunt Kinoshita and Wachtl (1958) have shown that the pentose phosphate shunt contributes significantly to the oxidative breakdown pathway in addition to the ordinary glycolytic pathway. About 15% of glucose is metabolized by this pathway in the lens. In most tissues in which it is active (e.g. mammary gland) the function of the shunt is biosynthetic 25
and reductive, rather than oxidative for the production of energy. The C0„ is a by-product. The useful product is the hydrogen extracted from the molecule and transported in an active form as NADPH. The usefulness of this hydrogen depends upon the fact that it is not generally available for oxidation (as is the hydrogen of NADH) by respiratory enzymes, but rather it is conserved for synthetic processes which have the necessary NADP specific enzymes. The function of the shunt as an energy producer in the lens is minor or nonexistent. Its function in biosynthetic reduction is more certain but still not extensive. The NADPH is involved in the sorbitol pathway, in effect setting up a trans- hydrogenase system, but a reaction which appears more useful is its capacity to serve as a • cofactor for glutathione reductase.
Citric Acid Cycle : Very little, that is, about 5% of glucose is used by the lens via this cycle. The operation of this pathway is confined to cells having the mitochondrial apparatus needed to utilize molecular oxygen. This restriction eliminates most of the lens except the single layer of epithelial cells on the anterior inner surface of the capsule. This layer is in most intimate contact with the aqueous humor which carries the major part of what little oxygen is available to the lens. The importance of the citric acid cycle to the lens lies in its efficiency in generating ATP from a small amount of glucose and oxygen at the very zone where, the substrates are most available and where the need is the greatest for the ATP produced.
Sorbitol Pathway : van Heyningen (1969) has shown that the lens contains sorbitol and fructose together with enzymes capable of converting glucose to fructose by way of sorbitol. Its importance in the lens derives from the fact that its operation becomes cataractogenic when the concentration of the initial substrate rises to abnormally high levels. The pathway consists of two consecutive reactions which are both reversible dehydrogenations. 26
+ Aldose reductase , Glucose + NADPH + H > Sorbitol + NADP
n w. 1 a,.,^+ Polyol dehydrogenase + Sorbitol + NAD < '- > Fructose + NADH + H
Normally, at the most about 5% of glucose used by the lens is funnelled through this pathway and the fructose resulting, although theoretically subject to hexokinase action and subsequent glycolysis, is not used appreciably in the presence of normal levels of glucose (Kuck,1962K
Alpha-Glycerophosphate Cycle : This organic phosphate is the second most abundant one in the lens after ATP. It is derived by splitting of the hexose-diphosphate formed in the course of glycolysis and subsequent reduction by NADH of the dihydroxyacetone resulting from the split. Its utilization by the lens appears to be slower than its formation, accounting for its accumulation. Pirie (1962) showed that the operation of the alpha-glycerophosphate cycle in the lens is essentially like that in other tissues except for a failure of the diffusion of alpha-glycerophosphate into the mitochondria where its further metabolism is linked with electron transport systems. It is the absence of this link in the lens which prevents the cycle from serving as an important energy producer.
Protein and Amino Acid Metabolism Protein synthesis in the lens is generally similar to that in other tissues. Protein synthesis is not confined to the epithelium, but the rate varies in different parts of the lens. The nucleus, for instance incorporates leucine so slowly that Wannemacher and Specter (1968) concluded that in the core, only 2 to 3 percent of new protein is laid down during the lifetime of the animal. The initial step in protein synthesis is carried out mainly by a fraction (Specter et al, 1968) which exhibits about the same level 27
of amino acid incorporating capacity of whole calf lens, equatorial region, and anterior cortex. The high label protein fraction appears to be an alpha crystallin subunit. It is known that both epithelial and cortical cells are specialized for the synthesis of alpha crystallin.
The most important facet of protein metabolism in the lens is the formation of albuminoid, whose accumulation is a characteristic of the ageing lens. Albuminoid is generated chiefly from alpha crystallin in the bovine lens by a process which is probably not enzymatic since new covalent bonds are not formed. Bracchi et al (1971) have shown that soluble lens proteins bind to lens fiber-membranes to give an insoluble complex. It appears that the aged alpha crystallin from the lens nucleus is the component most suitable for insolubilization.
The mechanism of protein degradation involves proteases and peptidases but the search for active enzymes of this type in the normal lens has been largely unrewarding.Neutral proteinase has been described whose properties might enable it to fulfil the purpose of a natural agent for protein breakdown in vivo (Waley and van Heyningen,1962). A number of investigators have studied proteases and peptidases. The most important peptidase of the lens is leucine aminopeptidase, which has been studied extensively by Wolff and Resnik (1963), Spector (1963) and Hanson (1968). An interesting speculation concerning this enzyme is that, the acetylation of N-terminal end groups in the subunits of alpha crystallin protects against the cleavage by this peptidase, an action which may serve the function of allowing newly synthesized subunits to be aggregated to a nonsusceptible form before they can be destroyed by peptidase action. Since the activity of leucine aminopeptidase is highest in that part of the lens where the latest synthesis is taking 28
place, it may play a role in the dismantling of the synthetic apparatus in the course of normal development.
Amino Acid Metabolism : The amino acids to be used for protein synthesis are for the most part transported in, from the aqueous humor, although the lens has some capacity for the conversion frcm keto acids by transmination. The aqueous humor amino acids are accumulated in the lens by a most efficient active transport system (pump) which in many respects resembles the cation pump (Kern, 1962). The result of this pump action is that the lens itself exhibits appreciable levels of free amino acids more than sufficient for its normal need. The lens like the brain may synthesize certain amino acids from glucose, namely glutamic acid and alanine (van Heyningen, 1965a). Dardenne and Kirsten (1962) also showed that the lens has the complement of enzymes necessary for the conversion of methionine to cysteine and subsequently to taurine or inorganic sulfate. Degradation of most amino acids occurs in the lens. Dardenne and Kirsten (1962) found that glutamic acid and arginine were rapidly deaminated, the latter giving rise to ammonia, urea, and ornithine. They concluded that the Krebs-Henseleit urea cycle was active in the lens because urea production was markedly accelerated by ornithine. Trayhurn (1972) has shown that, of the amino acids alanine, glutamic acid, and aspartic acid, the latter two undergo significant oxidative metabolism in the absence of glucose.
Lipid Metabolism Less is known about the synthesis and breakdown of lipids than almost any other area of lens metabolism. This could be due to lack of quantitative importance of lipids in the lens. Although fatty acids are rapidly transported into the rabbit lens and incorporated into lens phospholipids, cholesterol is 29
not transported into the lens (Broekhuyse and Bogemann,1978) and is derived primarily frpm acetate synthesis. Esterification of cholesterol takes place in the human lens, where 25% of total cholesterol is in the ester form (Cotlier and Apple, 1973). Anderson et al (1969) reported that both rabbit and bovine lens phospholipids contain no detectable polyunsaturated fatty acids. They present this point as evidence that all lenticular fatty acids are synthesized within the lens itself. Little is known of lipid catabolism in the lens. Werner and Cotlier (1968) reported that the lens can utilize palmitate aerobically with the release of CO,.
Nucleic Acid Metabolism
Deoxyribonucleic Acid : The lens has relatively little DNA, since this genetic material is confined to the cell nuclei which are essentially absent from the lens fibres in the main body of the lens. The DNA in the lens is synthesized in the epithelial cells all over the anterior face of the embryonic mouse lens, its synthesis being a preparatory stage for mitosis (Hanna,1965). In young mice before the eye opens, the synthesis of DNA drops precipitously, and still later when cell multiplication is confined chiefly to the equatorial zone, the rate of the synthesis of new DNA is so low that the total amount in the lens becomes relatively constant for the remaining life of the lens. Little is known of DNA biosynthesis except that in the young growing lens undergoing frequent mitosis the uptake of thymidine is rapid. The degradation of DNA is a very slow process although it obviously must occur when epithelial cells begin the process of transformation to elongated fiber cells, during which period there is a loss of nuclei. 30
Ribonucleic Acid : The synthesis of RNA has been detected by the incorporation of tracer phosphate (Lerman and Fontaine, 1962) and adenine and uridine (Lerman, 1965). Hanna (1965) showed that there was active turnover of RNA in the lenses of young animals but even in the adult rat lens there was an incorporation of labelled uridine, detectable autoradiographically. The ageing lens normally exhibits a gradual but marked decline in RNA synthesis but the metabolism of RNA may be sharply altered in response to an insult. Ortwerth and Byrnes (1972) showed the presence of a ribonuclease inhibitor which forms an inactivated complex with RNA. Actually, RNAase activity, which is localized chiefly in the epithelial cells is present in normal lenses at about the same level as in cataractous lenses, but it is easily detected only in cataracts because the inhibitor concentration is markedly depressed in cataractous lenses.
Glutathione Metabolism
Glutathione is made up of three amino acids, glycine, cysteine and glutamic acid. -Since there is no GSH in aqueous humor, the lens supply can come only from synthesis, a process which requires 11 percent of the ATP produced by anaerobic glycolysis. The synthesis of GSH proceeds via gamma glutamyl cysteine synthetase. Failure to detect either GSH or GSSG in aqueous humor means that the GSH lost by the lens muSt be by degradation to metabolites. Barber (1971) suggested that a metabolite which would be difficult lo find, is the pyrrolidone carboxylic acid derivative of GSH, produced by the action of gamma glutamyl cytotransferase, which is very active in the rabbit lens (Cliffe and Waley,1961).
While the function of glutathione is not clearly understood, it is believed to be a major contributor to the reducing potential 31
of the tissue. Kinoshita (1964) suggested that one of the functions of glutathione in the lens could be to maintain protein sulphydryls in the reduced form. Reduction of protein disulphides (P - S - S - P) by glutathione (GSH) could take place through the following reactions : (Augusteyn,1979)
1. P-S-S-P + G-SH ^ 5» P-S-S-G + P-SH 2. P-S-S~G + G-SH ^ > P-SH +G-S-S-G 3. P-S-S-P + 2G-SH ^ > 2P-SH +G-S-S-G
The reduced form of the peptide (GSH) is maintained via the generation of NADPH which converts oxidized glutathione (GSSG) to the reduced form with the enzyme glutathione reductase.
_; Glutathione Reductase GSSG + NADPH + H =^ 2GSH + NADP"^ •%
Glutathione of extralenticular tissues acts as a substrate in two enzyme systems : glutathione peroxidase and glutathione S - transferase, each system representing a protective mechanism. These mechanisms are, respectively, destruction of hydroperoxides, especially those intracellularly produced, membrane lipid hydroperoxides which can impair membrane function,and conjugation of damaging electrophiles from extracellular sources. A nearly unlimited variety of electrophilic compounds can conjugate with glutathione through the action of glutathione S - transferases. It is reasonable to assume that these two enzymes may also serve the same functions in the lens (Rathbun and Hansan,1979). 32
CHEMISTRY OF ENZYMES
1. Lactate dehydrogenase and it's isoenzymes 2. Glucose-6-Phosphate dehydrogenase 3. Isocitrate dehydrogenase 4. Malate dehydrogenase and Malic enzyme
Lactate Dehydrogenase and It's Isoenzymes (EC 1.1.1.27; L-lactate : NAD Oxidoreductase)
Lactate dehydrogenase (LDH) catalyzes the following reaction:
coo" coo" I . . I HO - C - H + NAD ^ C = 0 + NADH + H LDH i:H.3 . CH3 L Lactate Pyruvate
The reaction is reversible, with the equilibrium strongly favoring formation of lactic acid. Both pyruvate and lactate in excess inhibit enzyme activity, although the effect of pyruvate is greater. LDH is a zinc containing enzyme. It is a tetramer with a molecular weight of about 1,35,000.
LDH plays an important role in several metabolic pathways: It forms the centre of delicately balanced equilibrium between the catabolism and anabolism of carbohydrates. In anaerobic glycolysis, LDH is the terminative enzyme in the sequence of reactions that promote the breakdown of glucose to lactate and therefore, it is essential for the production of ATP. LDH is also involved in gluconeogenesis in tissues in which lactate is converted to glycogen. Furthermore, in aerobic tissues such as the heart, lactate is used as a fuel, which is oxidized through the citric acid cycle and generates NADH and ATP. In the tissues like lens where oxygen tension is very low, 33
LDH provides the means of reoxidation of NADH and hence it is essential for the production of ATP.
It is known that lactate dehydrogenase exists as five isoenzymes. They are each designated LDHl, LDH2, LDH3, LDH4 and LDH5 in accordance with their mobility during electrophoresis in the pH range of 7-9. LDHl is the most negatively charged under these conditions and so moves most rapidly towards the anode. The other suffix numbers increase as the mobility of the isoenzymes towards the anode decreases. Each isoenzyme of LDH is a tetramer containing varying combinations of two monomer types, H (Heart) and M (Muscle), which may combine to form a series of five tetramers: H4 (LDHl), H3M (LDH2), H2M2 (LDH3),HM3 (LDH4) and M4 (LDH5) (Cahn et al,1962).
The LDH of adult human lens generally consists of 3 isoenzymes. LDH3 (H2M2), LDH4 (H1M3) and LDH5 (M4). The isoenzyme spectrum of LDH in the lens, indicates the predominance of anaerobic metabolism.
Glucose-6-Phosphate Dehydrogenase (EC 1,1.1.49; D glucose-6-phosphate : NAD? Oxidoreductase) Glucose-6-phosphate dehydrogenase (G6PD) is a hydrogen transfer enzyme which mediates the reversible transfer of hydrogen from /3-D-glucose-6-phosphate to NADP. The products of the reaction are 6-phosphogluconolactone and the reduced form of the coenzyme, NADPH. 34
The following reaction is involved.
H 0 I HO - f c- H - C - OH H - C - OH I , G6PD I HO - C - H 0 + NADP — > HO - C - H 0 + NADPH + H I H - C - OH H OH I I H - C H - C- HjC - OPO3''" OPO,
Beta - D-glucose-6- D-Glucono-alpha-lactone-6- Phosphate Phosphate
Glucose-6-phosphate dehydrogenase is present in practically all mammalian cells. Normally, the highest levels are present in the adrenal glands and in adipose tissue, although even higher concentrations are found in the mammary glands during lactation.
G6PD is highly specific for the Beta-anomer of glucose-6- phosphate. The pH optimum range is 8-9. The molecular weight of G6PD is variously stated to be 1,05,000-2,40.000. It is believed that the dimer contains the essential requirements for catalytic function (subunit molecular .weight of 53,000) and that the native protein is a tetramer.
Glucose-6-phosphate dehydrogenase plays an important physiologic role as the initiator of the hexose monophosphate shunt, which results in production of 2 mols of NADPH per mol of glucose-6-phosphate. The resulting NADPH has a number of important functions including methemoglobin reduction, reduction of oxidant compounds and drugs, and involvement in lipid synthesis, maintenance of reduced glutathione via glutathione 35
reductase and thus possible maintenance of the red blood cell membrane.
In the lens also G6PD has got an important function of maintenance of reduced glutathione via glutathione reductase. Thus it helps to prevent oxidation of lens proteins.
Isocitrate Dehydrogenase (EC 1.1.1.42;threo-Ds-isocitrate:NADP oxidoreductase[decarboxylating] )
The enzyme isocitrate dehydrogenase (ICD) is known to occur in two forms. NAD - linked isocitrate dehydrogenase (NAD - ICD; EC 1.1.1.41), present only in mitochondrial fraction, and NADP- linked isocitrate dehydrogenase (NADP-ICD; EC 1.1.1.42), present in both mitochondrial and cytoplasmic fractions of tissues. Both mitochondrial and cytoplasmic NADP-ICD are isoenzymes and are regulated by different genes. Despite the occurrence of three forms of ICD, it is generally accepted that NAD-ICD is mainly involved in mitochondrial isocitrate oxidation for the production of energy and mitochondrial NADP-ICD, directs Kreb's TCA cycle intermediates toward amino acid metabolism and lipid biosynthesis. The role of cytoplasmic NADP-ICD is to generate NADPH for biosynthetic purposes.
Isocitrate dehydrogenase (ICD) (EC 1.1.1.42), catalyzes the oxidative decarboxylation of isocitrate to alpha-oxoglutarate. coo" coo" H - C - OH ICD C = 0 "OOC - C - H + NADP"^_ ""NADPH -e H-C - H + CO2 ^ Mn^^ I CH„ Ctij COO COO Ds - Isocitrate alpha-oxoglutarate 36
ICD is found in high concentrations not only in the liver but also in heart, skeletal muscle, kidney and adrenal tissue, as well as in platelets and red cells. It has a relative molecular mass of 64,000 and requires Mn"^"*^ as an activator, the ion being essential for the decarboxylating reaction.
The function of NADP-ICD in the lens is to generate NADPH, which is an important coenzyme of Glutathione reductase.
Malate Dehydrogenase (EG 1.1.1.37; L-Malate NAD oxidoreductase) And Malic Enzyme (EC 1.1.1.40; L-Malate : NADP oxidoreductase [decarboxylating])
The oxidation of L-malate in most living organisms is catalyzed by two distinct types of pyridine nucleotide - dependent enzymes. In one case the principle product is oxaloacetate, while in the other it is pyruvate and CO,. The enzymes of the malate oxaloacetate class which utilize NAD have been referred to as 'simple' dehydrogenases,while enzymes of the malate-pyruvate type, which in contrast, use NADP have been designated 'decarboxylating' dehydrogenases and are commonly known as malic enzyme. Malate dehydrogenase (MDH) catalyzes the following reaction.
yO" MDH I ^ C = 0 + NADH ^ HO - C - H + NAD I ^^ 1
COO" coo" Oxaloacetate L Malate 37
Malic enzyme catalyzes the following reaction.
coo" coo" HO - C - H > C = 0 + NADPH + C0„ j Malic Enzyme I *
coo" + NADP"^ L malate Pyruvate
The 'simple' malate dehydrogenase occurs in virtually all eukaryotic cells in at least two unique forms identified as mitochondrial (m MDH) and soluble or cytoplasmic (s MDH) according to their cellular location. In addition, there have been numerous reports suggesting that both the cytoplasmic and mitochondrial enzymes can occur in multiple subtorms.
Malate dehydrogenase is a dimer of identical or nearly identical subunits. Molecular weight of Malate dehydrogenase is about 60,000 to 70,000.
The soluble isoenzyme is generally considered to take part in the cytoplasmic side of the 'malate shuttle' providing a means of transporting NADH equivalents in the form of malate across the mitochondrial membrane. The mitochondrial enzyme, in addition to its role in the other half of the malate shuttle is also a necessary component of the tricarboxylic acid cycle. The function of the Malic enzyme is to produce NADPH. In the lens the function of Malate dehydrogenase is to provide a means of reoxidation of NADH and the function of Malic enzyme is to produce NADPH. 38
PATHOLOGY OF CATARACT
Cataract is a lens opacification. A cataract disturbs the normal transparency of the lens, it reduces the visual acuity and in the advanced stage, causes blindness. Opacification of the lens, may be a highly localized 'spot' or a complete loss of transmission throughout the substance; it may occur as a senile change; as a result of trauma, which presumably injures the capsule and the underlying epithelium; as a result of metabolic or nutritional defects; or as a consequence of radiation damage.
Pierre Brisseau reported for the first time in 1706 that cataract is an opacity of the lens itself and not a clotted membrane in the anterior chamber. He was ridiculed by most contemporary ophthalmologists. Yet, without this observation the clinician Jaques Daviel in 1753 would not have been able to develop cataract extraction, which up to today is the most frequently performed eye operation in the world and has made cataract a treatable disease. -
Pathology of cataract can be described in two aspects. a) Process of lens opacification. b) Biochemical changes.
Process of Lens Opacification Many hypotheses have been presented for the inducing causes of lens opacification, and attempts have been made to prove them. The phenomenon that the crystalline lens opacifies probably consists of many stages and patterns. In any event, the opacification must have started already at the stage when the lens was still apparently transparent, and this start of opacification appears to be composed of a biochemically remote cause and a physiochemical immediate cause. It may be presented 39 as a model shown below which shows five stages (Iwata, 1972).
Postnatal stage
-Prenatal stage —>.
II III IV V Trigger Metabolic Protein Opaque Opacification reaction ageing reaction ageing
Biochemical process ^ Physico-chemical process-
Premature cataract
Diagram showing postulated process of opacification of the lens.
Trigger Reaction Stage : Cataract is roughly divided into experimental and pathological types; the former being artificially produced by administering a cataractogenic substance to' the animal and the latter being a disease clinically diagnosed as cataract. In these two types of cataract, there are various cataractogenic factors ranging from naphthalene to senile cataract. In any case, it is unquestionable that the lens which should be transparent follows the process of opacification due to some proximate cause.
Regarding the primary initiating mechanism as a 'trigger', it is obviously important to determine its nature. The trigger reaction stage, when one out of many events may initiate cataract development, is, in fact, the first stage in lens opacification, when biochemically remote causes commence to operate. 40
Metabolic Ageing Stage : Metabolic ageing is that stage when opacification of the lens advances following the initial triggering reaction inside or outside the crystalline lens. This phenomenon may be called the ageing lens, and means the start of the ageing phenomenon in a broad sense. Both the fall in metabolic activity due to senility and metabolic ageing may be attributed to the triggering mechanisms. Metabolic ageing in the lens entails not only the metabolism directly involved in the quantitative growth of the lens (such as biosynthesis of proteins and metabolism of carbohydrates) but also qualitative changes in the lens (such as variations in the protein pattern, the contents of various metabolites and metal ions, enzymes,coenzymes and their substrates), and in addition changes in the structure and function of the membranes concerned with transport. Swelling of the lens, which may be regarded as the initial reaction of cataract, and the accompanying disarrangement of the lens tissues also belong to this stage. ^ ^^^3
Protein Reaction Stage : The swelling of the lens indicates the stage at which soluble lens protein, an important constituent of the lens, changes to insoluble protein. This may be regarded as the start of macroscopic opacification, and as the physico- chemical, immediate cause of opacification.
This protein reaction involves not only the conversion of soluble to insoluble proteins, but also structural changes due to reactions of electrolytes with proteins, reaction with degeneration products, protein-water interaction, changes in the primary structure and reactions of proteins with aggregation promoting substances (Jedziniak et al,1973). The reactions in this stage are accomplished in a short period, and appear to be reversible. 41
Opaque Ageing Stage : In this stage, the aggregation of proteins advances, and there is increasing opacity due to degenerated protein. The amount of insoluble protein continues to increase and it is characteristic that the absolute amount of calcium increases linearly to 5 times the normal level.
Opacification Stage : At this stage the lens is completely opaque, and shows complete inversion of Na to K ratio, disappearance of the SH radicals, lack of amino acid uptake and macroscopically visible opacification.
In the foregoing account, in which the development of cataract is classified into five stages, those from the first trigger reaction stage to the latter half of the third protein reaction stage, are regarded as biochemical and those from the first half of the protein reaction stage to the final stage are taken to be physicochemical. Thus, process of development of cataract can grossly be divided into biochemical and physicochemical reaction stages which appear to overlap at the third stage. According to the publications available at present, most of the studies have been based on biochemical techniques, except for few optical studies, embryological studies and morphological studies. Studies of the opacification process in and after the third stage will, however, require physicochemical methods, especially from the stand point of steric structure in molecular colloid dimensions.
When the whole process is divided into acquired and congenital types,it is natural that the trigger is pulled postnatally in many cases of acquired cataract, while in congenital cataract, the trigger reaction may have already been taken place in the prenatal period. The opacity of the lens due to Rubella is an example of the latter. 42
Biochemical Changes
The most important change observed during cataract formation is in the concentration of protein. In all types of cataract which have been investigated, there is a decrease in the total proteins compared with a normal lens of the same age. In this change the insoluble protein (albuminoid) concentration is relatively increased, while the soluble proteins are markedly decreased and their synthesis diminished or arrested. No common or consistent pattern has yet been observed with respect to the various constituent proteins but the loss of the beta crystallin fraction is always conspicuous and this involves a diminution of protein sulphydryl groups. In consequence, the active soluble proteins give place to inactive substances, thus tending to metabolic inertia.This change may be caused to some extent by a transference of soluble to insoluble protein owing to denaturation and an alteration in the molecular configuration. The evidence suggests, however that all these changes occur not in the early but in the later phase of the development of cataract when opacification has developed. It would seem likely, that they are not causal in nature.
In the later stages of the development of cataract, proteolysis occurs whereby the soluble proteins are broken down by proteolytic enzymes. It has been found that the activity of the peptidases increases markedly in the cataractous lens. At this stage,therefore, there is an increase of amino acids in the lens and the aqueous humor. The proteinases become much more active in an acidic reaction which tends to develop in cataract so that in these circumstances all the proteins may be thrown out of action, the soluble proteins being hydrolysed to amino acids and the others being coagulated. Since the amount of albuminoid is small in young lens fibers, in these, practically all the proteins are thus broken up. Since complete hydrolysis increases the osmotic 43
pressure some 400 times, the initial effect is an enormous imbibition of water with a consequent swelling which may be followed as diffusion proceeds by an almost complete disappearance of the lenticular substance. In older fibers the presence of the insoluble protein makes disappearance impossible, although the lens as a whole shrinks owing to the loss of beta crystallin and albumin.
Peptides : The most important of these substances in the metabolism of the lens is glutathione. It tends to decrease, however, with age and diminishes markedly in all the forms of cataract, senile or experimental. This reduction is possibly due to a failure in synthesis since the ATP and enzymes necessary for this process have been found to be lacking in certain types of cataract. Ascorbic acid, which may participate with glutathione in the oxidation - reduction reactions and is also found in very high concentration in the cortex shows a comparable diminution with age or with the development of cataract.
Conflicting reports are available regarding the nucleotides (ATP, NADT NADH, NADP'^NADPH) in cataract.
The total lipids increase with age and in cataract; this principally affects the free lipids, especially cholesterol, and the triglycerides and protein bound lipid decreases. In the later stages of opacification the free lipids may appear in visible crystallin deposits.
The inorganic materials also suffer changes. All observers agree that the cataractous lens is rich in calcium. The content of potassium decreases and the sodium increases in the cataractous lens. Correspondingly the chlorides become increased as also do the inorganic phosphates. It has been suggested that this shift in the content of cations, due to free permeability of the cellular membranes, in place of their control by the mechanism 44
of active transport and cellular activity, may lead to an increase in osmotic pressure as necessitated by Donnan equilibrium and this participates in the intumescence of the lens.
Another important lenticular constituent whose concentration falls with age and development of cataract, is ascorbic acid. The significance of this change is unclear but it appears to be correlated with the diminishing metabolic activity in the ageing lens.
The other prominent constituent of the lens whose level change with age and cataract development is inositol. Here however, the change is in the direction of an increase with age and cataract development. This substance is thought to be metabolically inactive in the lens and it appears that its accumulation may result from failure to penetrate the lens membranes which develop decreased permeability upon ageing.
Ageing and cataract development also increases the concentrations of the two sorbitol pathway metabolites; i.e. sorbitol and fructose. A decrease in permeability with age is the most important factor governing this accumulation. The enzymes and co-enzymes associated with metabolic activity show a considerable diminution in the cataractous lens, with increase in activities of hydrolytic enzymes.
Transport in the cataractous lens : The entire metabolic activity of the lens depends on biological interchange through the capsule. Abnormalities in the permeability of this membrane have, from time to time, been considered as being etiologically concerned with the formation of cataract. It is postulated that lenticular opacities were due to an increased permeability of the capsule in old age, a view supported by observations that soluble 45
substances diffused out of the cataractous lens. But this hypothesis was contradicted by the findings of Friedenwald and Rytel (1955), who has suggested that the permeability of the capsule decreases in age and in early cataract, presumably by the structural constriction of its pores, although it probably increases sufficiently to allow the passage of proteins when the cataract becomes mature. There is now ample experimental evidence that the capsule acts as an inert membrane, permeable to small and impermeable to large molecules. It may be that a slight decrease in permeability may be a 'minor factor in the development of senile sclerosis and cataract by decreasing metabolic exchange.
As<^, M »? f. ' V%. T-^**- -V- ^ \<^ (. \ A:\\ • nz.y ! . . ' , ^•^.'i ; -'> 1 ' <^J••^\-^ -
y' -V ; k '^tf-'V*' ^ J. \-. ^ ...^^ 46
CLASSIFICATION OF CATARACT
Previously because of non-availability of normal human lenses, it was not possible to compare biochemical changes in cataract with those of the normal lenses. Therefore, a number of methods of classifying cataracts were adopted and biochemical changes were compared with each group. However, now a days, normal lenses could be obtained from 'Eye banks' and the results could be compared.
There are several ways by which cataractous lenses can be classified. Morphologically cataractous lenses can be classified into nuclear cataracts and cortical cataracts. The nuclear cataract in which the nucleus of the lens becomes yellow or brown, is probably an exaggeration of the normally occurring senile changes. The cortical cataracts, on the other hand, represent opacities developing in more recently • laid-down lenticular fibres, and thus reflect and response to some abnormality in metabolism.
Nuclear Cataracts : These cataracts were completely different, the early stages being essentially a lenticular sclerosis accompanied by yellowing, and a diffuse scattering of light, with no large foci of scatter. In the advanced stages, usually associated with yellow, brown pigmentation, the electron-microscopical picture was normal, so far as extracellular spaces were concerned, but in the dense whitish nuclear cataracts large aggregates of intracellular matrix of diameter 50 to lOOnm became obvious. Such particles would be capable of scattering light and might account for the nuclear opacity (Benedek,1971).
Cortical cataracts : The gross morphology of these cortical cataracts consists in the appearance of aberrant forms of epithelial cells, which become large and round and are called 'balloon' cells. 47
Lens fibres may become fused into discrete masses,called morgagnian globules. If this process goes far enough, the whole cortex becomes a milky liquid mass; and material may escape from the lens into the aqueous humor and vitreous body.
In the human lens the cortical cataracts characteristically begin as localized points of opacity in surrounding transparent material in the subcapsular or supranuclear regions. In the electron microscope, Philipson (1973), found essentially the same changes, consisting of an initial loss or damage to cell membranes leading to cellular dissolution with formation of large intercellular spaces or vacuoles.
As a result of slit-lamp examination human lenses have been classified as follows : 1. Clear lens. 2. Nuclear cataract - the cortex remains clear and the nuclear opacity appears more grey or mainly yellow brown. ' 3. Deep cortical cataract (typical senile cataract, supranuclear cataract) wedge shaped or spoke like opacities, lamellar dissociation and water clefts. These opacities are covered by clear superficial layers of fibres.
4. Subcapsular cataract - most of the cortex remains clear. Initially, only a thin posterior subcapsular opaque area is visible (weak subcapsular cataract) but as the cataract progresses additional opacities develop under the anterior capsule; often a grey nuclear opacity is also visible (intense subcapsular cataract).
5. Totally opaque lens. 48
Co-operative Cataract Research Group (Chylack,1984) -has classified cataract lenses as follows : 1. Immature : An opacity which does not totally obscure all normal anatomical regions of the lens. 2. Mature : A totally opaque lens in which no recognizable normal anatomical zone remains, but in which there is no appreciable antero-posterior swelling. 3. Hypermature : A totally opaque lens, that has undergone marked swelling in the antero-posterior dimension.
Pirie (1968) examined some 328 human cataractous lenses and classified them in accordance with their appearance as follows : 1. Uniform pale yellow. 2. Pale cortex with pale brown coloured nucleus. 3. Pale cortex with dark brown coloured nucleus.
In the present study, the Pirie's classification was used to analyse the lenses biochemically. In senile cataracts, change in colour to yellow, then to pale brown, then to dark brown and hardening of the lens nucleus parallels decreased transparency. Many theories on the pigment formation have been proposed. The majority of these theories revolve around the following assumptions :
1. Oxidation or photo-oxidation of tryptophan and/or tyrosine in lens proteins (Pirie,1971 and Zigman,1973a). 2. Formation of sugar amino acid bonds with subsequently 'browning' of lens proteins (Scharff and Montgomery, 1972 and Stevens et al,1978). 3. Formation of lipid peroxidation products (Bhuyan et al,1979 and Cotlier et al,1978). 49
Oxidation or photo-oxidation of tryptophan or tyrosine can be induced by ultraviolet light or enzymes. Exposure of intact lenses or lens proteins to the photo-oxidation products results in yellowing or browning of the protein. Among repeatedly confirmed findings in the nuclear yellow or brown cataracts are the progressive decreases in reactive SH groups, and the presence of a fluorescent material in the insoluble lens proteins. Further characterization of human lens fluorogens has been accomplished by isolation of fluorescent polypeptides. These fluorescent polypeptides are rich in water soluble amino acids- mainly aspartic acid and glutamic acid. This yellow fluorescent polypeptide increases in concentration with progression of lens density. It also . differed in amino acid composition from other soluble or insoluble proteins or peptides from the human lens. Previously, van Heyningen (1973) found Kynurenine derivatives in the human lens, but their concentrations were not augmented in cataracts. Dillon and co-workers (1976) have described beta carbolines as major fluorogens in human cataracts.
Because of the progressive increases in tryptophan and the kynurenine complex, a derangement of tryptophan metabolism probably occurs in the most advanced senile cataract. Pirie (1971) however, claims light could photo-oxidize lens tryptophan in situ with formation of formyl kynurenine. 50
TYPES OF CATARACT
1. Senile cataract 2. Diabetic cataract 3. Galactosemic cataract 4. Xylose cataract 5. Toxic cataract 6. Radiation cataract 7. Nutritional cataract 8. Traumatic cataract 9. Cold cataract 10. Genetic cataract.
The term cataract is used loosely to mean the occurrence of an optical discontinuity in the lens of such magnitude as to cause a noticeable dispersion of light. In a more restricted sense, it is an accumulation of irreversibly coagulated lenticular protein. The alterations in protein during cataract formation may present so many variations in morphology, and be the result of so many kinds of physical and biochemical disturbances that the term cataract is more properly used in the plural. Cataracts, then, are symptomatic, and are the end-products of a diversity of insults to the lens. If the etiology is known, the entity may be distinguished by a specific name, e.g. radiation cataract. Many agents, including trauma, chemicals, radiation, electricity, viruses and vitamin or amino acid deficiencies, diabetes can induce cataracts in humans or in experimental animals. Cataracts may be associated with skin, central nervous system, and skeletal diseases, chromosomal abnormalities, and other ocular disease or malformation. Other cataracts of unknown etiology are senile cataracts. The great majority of human cataracts are senile cataracts. 51
Senile Cataract
The human lens normally undergoes changes with age : it slowly increases in size as new lens fibers develop throughout life; older lens fibers in the depths of the lens become dehydrated, compacted, and "sclerosed", a yellow brown pigment accumulates. The increase in the optical density of the nucleus tends to increase the refractive power of the lens so - that less hyperoptic spectacle correction may be needed in old age. The yellow brown pigment may become so dense as to constitute nuclear sclerosis and later brunescent cataract. Cortical cataract, however, is the development of vacuoles and water clefts in the lens cortex that tend to increase in extent and in the advanced state give the lens a pear, like appearance. Approximately 60% of human beings have some alterations in lens transparency after 60 years of age. Progression of lens changes differ among individuals and lens opacities can cause visual deficits in a shorter or longer period of time.
Diabetic Cataracts
Increased levels of glucose in the aqueous and lens are found in patients with diabetes mellitus. In general, the glucose concentration in the aqueous parallels the concentrations in the plasma. From the aqueous, glucose diffuses rapidly into the lens. The lens metabolizes glucose through four main pathways. 1. Anaerobic glycolysis 2. The Hexose Monophosphate shunt 3. Citric Acid Cycle 4. Sorbitol Pathway.
Basically the initial rate of glucose metabolism in the lens is determined by hexokinase, a regulatory enzyme. In diabetes excessive glucose in the lens (over 200mg%) saturates hexokinase. 52
Glucose then piles up and is converted partly into sorbitol and fructose (Kinoshita et al,1963).
NADPH ^4ADP
Glucose ^=»^ '^ > Sorbitol Aldose reductase
NAD Polyol dehydrogenase
ATP iNADH<- Hexokinase
^ G6PD Glucose-6-Phosphate > HMP Shunt
Pyruvic acid LDH
Lactic Acid
Aldose reductase catalyzes the glucose to sorbitol reaction. High levels of sorbitol and fructose were found in diabetic animal lenses by Kuck (1961) and in human diabetic lenses by Pirie and van Heyningen (1964). The excess of sorbitol is also found in rabbit lenses cultured in vitro in high glucose media (Kinoshita et al, 1963).
The effects of excessive sorbitol and fructose in the diabetic lens results in increased hydration and sodium ions gain with subsequent loss of K ions, amino acids and inositol (Kinoshita, 1965b). The swelling of the lens has led to postulate that 'high levels of sorbitol in the diabetic lens draw water, thus rupturing 53 lens fibers and causing vacuolation in lens cortex. The rate of cataract progression depends on the glucose levels in serum; the higher the glycemia, the faster the cataracts become mature (Patterson, 1952). In humans, diabetic cataracts are rarely seen in juveniles but may appear suddenly in adults with uncontrolled diabetes mellitus. Other mechanisms beside accumulation of sugar alcohols are also involved in cataract formation in diabetes. The known damage to basement membrane in these patients may be the cause of the decreased fragility of their lens capsule (Caird et al, 1968). Furthermore, excessive red blood cell glycosylation is found in uncontrolled diabetics, and it may have an effect on the lens as well. Stevens and coworkers (1978) have found evidence of increased protein glycosylation by animal lenses incubated in high glucose media. The formation of sugar amino acid (lysine) bonds may result in protein aggregation and decreased transparency.
Galactosemic Cataract Inability to metabolize galactose occurs in the galactosemias, which may be caused by inherited defects in any of the 3 enzymes marked (1), (2) and (3) as shown below :
NADPH NAD?
Galactose -> Galactitol Aldose reductase
ATP Galacto (1) ( kinase ADP Galactose-1-P UDP-Glucose
Galactose-1-P Uridyl (2) Transferase UDP Glucose Glucose fr UDP Galactose -^ 1-P Uridine Diphosphogalactose (3) 54
Excessive galactose in the serum and the lens results in a shift toward the formation of galactitol. The accumulation of galactitol has deleterious effects on the lens and leads to cataract formation. Galactosemic cataract also results due to the excessive amounts of sugar alcohols CGalactitol) drawing water into the lens. Water gains by the lens fibers rupture their nmembranes , with resultant loss of K , amino acids, and inositol.
Xylose Cataract Xylose is the third member of the series of cataractogenic sugars (the others are glucose and galactose). Like other sugar cataracts, Xylitol and Xylulose accumulate in Xylose cataract, but levels are never as high as those of sorbitol and fructose in diabetes.
Toxic Cataract Toxic cataract may be defined as lens opacification due to the effects of chemicals upon the lens. These include a number of drugs and poisons' which cause cataract. Naphthalene induced experimental cataracts are studied in some more detail. According to van Heyningen and Pirie (1967) Naphthalene is converted into Naphthalene diol. This can be converted in the eye to 1-2 dihydroxynaphthalene which is probably responsible for the blue fluorescence in the eye of the naphthalene-fed rabbit. This is oxidised to beta nophthaquinone (Rees and Pirie, 1967). It reacts with ascorbic acid to form H^O, which is toxic. One of the early changes is a marked swelling of the lens. An unusual result is thickening of the epithelium to several layers early in the process. Much later (nearly 30 days) when the cataract is mature, the entire capsule is lined with epithelium, the cortex is liquefied although the nucleus remains intact. In the postmature 55
stage the epithelium is lost and the fibers fall to pieces within the thickened capsule.
Another suggested mechanism for naphthalene cataract was that based on the finding that one of the elimination products is urinary alpha-naphthyl mercapturic acid. The detoxification of large amounts of naphthalene would theoretically utilize enough cystine to cause a deficiency of this compound.
In addition to naphthalene, many other drugs often used therapeutically, at one time, such as dinitrophenol, myeleran and dimethyl sulphoxide, would cause cataracts but not necessarily in man. For example, dimethyl sulphoxide has not reportedly caused any eye damage in man, although it has, in experimental animals, such as the dog (Rubin and Bernett, 1967).
Radiation Cataract
Ionizing radiation (X-rays, gamma rays, neutrons, beta rays) induced lens vacuoles and lens opacities. The lens is affected only by radiation of wavelengths that are absorbed. Wavelengths between 293 and 400 are transmitted by the cornea and effectively absorbed by the lens, so this is the range of ultraviolet light, potentially most damaging to the lens. Above 1100-1400 m^, the infrared may be injurious, since in this range, the rays are transmitted through the cornea and absorbed by the lens. Radiation affects the permeability of the lens. Lenses exposed to radiation are more leaky to K , inositol, and glutathione. These effects may occur at the level of the membranes of the lens fibers. Radiation affects the synthesis of lens proteins. Decreased incorporation of amino acids into proteins with decreased levels of soluble lens protein is produced (Dische et al,1959). 56
Nutritional Cataract In experimental animals, cataracts could be produced by feeding diet deficient of essential amino acids and certain vitamins. The essential amino acids are necessary for the elaboration of lenticular proteins and vitamins are necessary for the continuance of the metabolism.
The occurrence of cataract due to lack of essential amino acids is more firmly established. Most of the experimental work has been done on rats. In this connection the most important amino acid is tryptophan. The mechanism for tryptophan deficiency cataractogenesis is unknown, but the available evidence suggests that there is a failure in the supply of nicotinic acid which is produced from tryptophan. Less dramatic results have been reported following the lack of other indispensable amino acids with the exception of arginine, phenylalanine, histidine and methionine. It would seem that the occurrence of cataract is due to the arrest of the synthesis of soluble proteins and is associated with a general arrest of growth, cutaneous lesions and vascularization of the cornea.
A deficiency of vitamins, particularly those of the B2 (Riboflavin) has been claimed to result in the development of cataract.
Traumatic Cataract Trauma to the eye, or more specifically to the lens, can cause cataract, but the mechanism is obscure. With a rupture of the capsule, different conditions arise owing to the entrance of the aqueous, the loss of essential diffusible substances, and rapid proteolysis with the development of a localized or a complete opacity. 57
Cold Cataract If the isolated lens of a young animal is frozen, it turns completely opaque and on thawing clears up from the periphery. The reaction is due to the precipitation of gamma crystallin when cooling reaches a temperature below 10°C, a reaction is reversed on warming above this temperature. The cold precipitation possibly occurs as this protein possesses a large number of exposed hydrophobic groups. If the fraction of gamma crystallin is greater than 0.3%, other proteins increase the solubility of the total protein by hydrophobic bonding with the gamma crystallin. Therefore, young animals are more susceptible to cold cataract since at an early age the gamma crystallin constitutes the major part of the proteins of the lens.
Genetic Cataract Lowe's syndrome is a recessive sex-linked trait characterized biochemically by hyperamino aciduria and abnormal ammonia metabolism. Among other symptoms it affects the eyes by causing cataract or glaucoma. Its effect on the lens appears to be mediated by reducing the availability of amino acids for synthesis of lenticular proteins.
Galactosemia in humans is the result of an inborn error of metabolism. Galactosemic cataracts result from an excess of galactose in the serum and in aqueous humor.
The high incidence of diabetes among the population places it among the more important inherited defects leading to cataract.