Year : 1997 | Volume
: 45 | Issue : 4 | Page : 227--231
Possible role of lens collagen in cataractogenesis
G Mishra, GB Das, HN Behera
P.G. Department of Zoology, Berhampur University, India
P.G. Department of Zoology, Berhampur University
The solubility characteristics of collagen in human cataractous eye lenses were studied in relation to age. Post-operative cataractous lenses were collected form eye camps in Phulbani district of Orissa. These were preserved in 70 % alcohol and immediately transferred to the laboratory. Various fractions in the collagen obtained from lens capsule of each lens sample were estimated. The salt-soluble and the acid-soluble collagens showed positive correlation with age. The insoluble and total capsular collagen increased with increasing age. The solubility percentage of collagen in salt and acid solutions decreased significantly with increasing age. The possible role of lens collagen in the development of cataract with increasing age is discussed in the background of cross-link theory of aging.
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Mishra G, Das G B, Behera H N. Possible role of lens collagen in cataractogenesis.Indian J Ophthalmol 1997;45:227-231
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Mishra G, Das G B, Behera H N. Possible role of lens collagen in cataractogenesis. Indian J Ophthalmol [serial online] 1997 [cited 2023 Jun 5 ];45:227-231
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Defined on the basis of the pathomorphological process involved, cataract is an opacification or loss of transparency in the crystalline lens of the eye. Throughout the world atleast 50 million people have cataract-related decrease of vision and 17 millions are severely disabled by cataract. In India cataract accounts for a large majority of blindness. Cataract can be developmental in origin, or secondary to trauma, systemic diseases, drugs, and age related factors. Senile or age related cataract is responsible for more than 80 % of all cataracts.
Age related cataract is the main cause of blindness and visual impairment throughout the world. Cataract being an age-related disorder, its occurrence increases as longevity increases. In the West, the prevalence of cataract in people over 50 years is 15 %, while in developing countries it is above 40 %. Various risk factors have been identified in the pathogenesis of senile cataracts. Apart from aging, genetic factors, nutrition, diabetes mellitus, trace metals, ultra-violet radiation, and smoking have been implicated as significant risk factors in the causation of cataract.
The close correlation between the aged individuals and the occurrence of cataract clearly suggests that cataract is a disease of old age. Senile changes in lens proteins could go parallel with changes in other proteins, at least with respect to structural changes with age. The pathogenetic interpretations of cataracts must, therefore, take into account the great variety of etiological causes capable of modifying the protein component of the lens. It is an established fact that one of the major connective tissue proteins of body, collagen, undergoes extensive cross-linking leading to a decrease in solubility and increased stiffness, thereby blocking the passage of essential nutrients to the underlying tissues and cells as has been envisaged in the "crosslink theory" of aging. It is not sepculative to say that one of the major capsular protein is collagen. While a large majority of workers have reported a variety of factors involved in the development of cataract,[1,9,10] nothing has been reported so far on the role of capsular collagen in the process. Also, whether the lenticular collagen undergoes natural cross-linking during the aging process is not known. Therefore, it becomes an investigative issue to find out structural changes, if any, in the lenticular collagen with concomitant decrease in solubility characteristics during aging which might have played some role in depriving the lens of its essential nutrients, thereby creating deficiencies to initiate the development of opacity.
In view of the above, the present work was undertaken to assess the solubility characteristics of lens collagen in the capsule of cataractous lenses obtained from aged individuals.
Materials and Methods
Collection of cataractous lens
Cataractous lenses were collected from different eye camps organised by eye specialists of Phulbani district in Orissa State. These lenses were obtained from human individuals of ages 43-75 years belonging to both sexes. These were preserved in 70% alcohol and were brought to Berhampur University campus immediately after collection. Age of the individuals from whom the samples were collected was noted.
The capsular layer was separated from the cortex with a pair of fine forceps and was washed in distilled water. After blotting in filter paper, the capsular layer of each sample was weighed separately. The entire capsular tissue obtained from each lens was taken for extraction of different collagen fractions. The capsule sample was sliced and was suspended in a centrifuge tube in 3 ml of 0.14 M sodium chloride solution and kept at 8� C in a refrigerator for 24 hours. The salt-soluble and salt-insoluble fractions were separated by centrifugation at 5,000 rpm in a REMI centrifuge for 15 minutes. The salt-soluble fraction was taken in a glass sealing tube to which equal amount of concentrated (12 N) hydrochloric acid (HCl) was added. Residue was suspended in 3 ml of 0.45 M acetic acid and the samples were kept at 8� C in a refrigerator for 24 hours. Acid-soluble and insoluble fractions were transferred to sealing tubes containing 3 ml of concentrated HCl and 6 N HCl, respectively. The tubes were sealed by blowing flames through a nozzle. Salt-soluble, acid-soluble and insoluble fractions were hydrolysed at 110� C for 16 hours. The hydrolysed samples were transferred to 50 ml measuring cylinders and then neutralised in 10 N sodium hydroxide using one drop of methyl red as indicator. The final volume of neutralised samples was made upto 15 ml (for soluble part) and 50 ml (for insoluble part) with distilled water. The hydroxyproline content of the neutralised samples were estimated following the colorimetric method of Neuman and Logan as modified by Leach. Values for hydroxyproline were converted to collagen contents by multiplying with the factor 7.46 as suggested by Jackson and Cleary. The different collagen fractions were expressed as mg collagen per gm of tissue wet-weight. From the above values the total collagen content and solubility percent were calculated. Software assisted statistical data analysis with respect to each parameter against age was done using Pearson's correlation coefficient.
All the collagen fractions showed significant positive correlation with age indicating their increased levels with age: salt-soluble (r=0.30, p<0.02; [Figure:1], acid-soluble (r=0.23, p<0.1; [Figure:2], insoluble (r=0.35, p<0.001; [Figure:3] and total (r=0.61, p<0.001; [Figure:4].
On the other hand, the solubility percent (soluble fraction as percentage of total collagen) in salt (r= -0.521, p<0.001; [Figure:5] and dilute acid (r= -0.686, p<0.001; [Figure:6] media showed significant negative correlation with age indicating decreased levels of solubility in these solutions.
One of the well known parameters to assess aging in vertebrates is the change associated with characteristics of the connective tissue protein, collagen. Basic structural unit of collagen is a tropocollagen which consists of three polypeptide chains (alpha-chains) unusually rich in glycine, proline, hydroxyproline, and hydroxylysine. A set of intra-molecular cross-links joins the three alpha-chains together in the collagen molecule and the chains themselves are established in the fibres nu inter-molecular cross-links, derived from specific lysine and hydroxylysine residues through oxidative deamination and subsequent condensations. With advancing age, the number of intra and inter-molecular cross-links increase in the collagen molecule. This eventually leads to a derangement in physiological functions of various tissues. Aging of collagen might affect the aging of the organism as a whole. Such changes in the characteristics of collagen during aging provides excellent support to the "cross-link" theory of aging. The development of such cross-links decreases the solubility of collagen in dilute salt and acid solutions and makes the tissue less susceptible to digestion by bacterial collagenous.
Cataract primarily being a disease of old age, the present study was undertaken to assess the solubility characteristics of lens collagen in order to appreciate its role in the process of cataractogenesis. Collagen is distributed in almost all forms of occular tissues except the crystalline lens, with the highest content in the lens capsule. The biochemical detection of type-IV collagen in mammalian lens capsule has been widely reported., Bernard suggested that type-IV collagen supplies strength and flexibility to this basement membrane. However, their X-ray diffraction studies did not show chicken-wire meshwork of fibres, so characteristic of type-IV collagen, but demonstrated 10 nm banded microfibrils which might have been the result of the association of collagen types I, III, and IV. An age related increase in thickness of lens capsule has been observed. Such type of accretion of collagen is also apparent in primary open angle glaucoma, attributed chiefly to enhanced synthesis and decreased degradation of collagen.
The results of the present study show increased salt-soluble collagen in the lens capsule from individuals of both sexes. This fraction represents the newly synthesized collagen, not covalently linked into fibres by labile aldimine type bonds. An increase in the content of such newly synthesized collagen possibly shows an increased synthesis. Acetic acid extracts a form of collagen cross-linkages. The fact that acid-soluble collagen from lens capsule showed positive correlation with increasing age possibly reflects a higher degree of cross-linked collagen.
Insoluble collagen from lens capsule increased significantly with increasing age, an observation very often marked in aging collagen and is taken as a very good indicator of increased number cross-linkages. Moreover, there are indications that lens proteins form disulphide bonds with other proteins leading to the formation of insoluble protein complexes., This view is further supported by a marked increase in cystine in senile cataracts at the expense of cysteine. Thus, it appears that opacification of lens is preceeded by oxidation of -SH groups in the proteins, amounting to a qualitative alteration. The total collagen content of a tissue reflects a balance between its synthesis and degradation. An age-related increase in the total collagen in lens capsule from individuals indicates an enhanced synthesis and/or decreased degradation, followed by its incorporation into fibres, a fact supported by higher level of salt-soluble, acid-soluble and insoluble collagens. This view is further corroborated by the observation that the lens capsule increases in thickness with age and this accretion process appears to be due completely to uniform assembly of three types of collagen (I, III, and IV), a process possibly intrinsic to aging and that these collagens may not be present in the human capsule in the early decades.
An increase in the number of cross-linkages of collagen leads to alterations in the physiological activities of the cells, eventually leading to aging of the whole organism. The molecular stability of collagen increases with concomitant decrease in its solubility during aging.,,, The results of the present study that there is an overall decrease in the solubility percentage of collagen in both salt and acid media with increasing to age are consistent with the above view. It appears that the various forms of collagen being synthesized and incorporated into the lens capsule develop cross-links among themselves and/or with other proteins leading to increased stability of this macromolecule. It is very likely that such stabilized form of collagens might be responsible for reduced transport of essential nutrients and other substances into the lens leading to the development of opacities during cataractogenesis.
We acknowledge the Pool Scheme of the Government of India for financial support during the present work. We thank the Council of Scientific and Industrial Research, New Delhi for providing Pool Officership (Senior Research Associate) to Dr. Gitanjali Mishra; Berhampur University for providing laboratory facilities; Dr. P.K. Das, District Headquarters Hospital, Phulbani for providing cataractous lenses; and R.K. Padhi, Maintenance Engineer, Computer Centre, Berhampur University for biostatistical assistance.
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