Human Senile Cataractous Lenses Research Articles

Elevated Concentrations of Kynurenic Acid, a Tryptophan Derivative, in Dense Nuclear Cataracts

Purpose: Kynurenines and their glycoside derivatives in the ocular lens absorb ultraviolet radiation and thus possibly help protect the retina from ultraviolet light. The current study analysed kynurenine aminotransferase I (KAT I) activity and kynurenic acid (KYNA) concentrations in human senile cataractous lenses and in experimentally induced cataracts in diabetic rats treated with streptozotocin (STZ). Methods: KYNA levels and KAT I activity were investigated with HPLC and detected fluorimetrically in the nuclei of 91 human cataractous lenses collected during planned extracapsular extraction. The lenses were classified on the Lens Opacity Classification System III scale and compared with clear lenses regarding KYNA concentrations. Cataractous lenses from STZ-treated rats were compared with control lenses. Results: KYNA concentration was 0.95 ± 0.22 in human NC0 (nuclear color) control lenses, 0.8 ± 0.72 in NC1, 1.18 ± 0.88 in NC2, 1.31 ± 0.70 in NC3, 1.78 ± 0.92 in NC4, 8.80 ± 8.28 (p < 0.05 vs. NC0) in NC5, and 14.0 ± 11.1 (p < 0.05 vs. NC0) in NC6. A correlation was found between KYNA concentrations and the grade of cataract (r = 0.047, p < 0.001). KAT I activity in human cataracts was 0.44 ± 0.16 pmol/mg protein− 1 hr− 1. Elevated KYNA concentrations in rat cataractous lenses were also observed (p < 0.05). Conclusions: KYNA levels are elevated in senile nuclear human cataracts and in cataractous lenses of rats with experimentally induced diabetes.

Antioxidant markers in human senile and diabetic cataractous lenses.

To determine the role of free radicals and antioxidant enzymes in patients suffering from cataract due to aging and diabetes. Experimental study. Department of Biochemistry, University of Karachi from December 2004 to May 2005. The study was conducted on 80 cataract patients. Lens and plasma samples from senile and diabetic subjects suffering from cataract were analyzed and activity of Cu, Zn-superoxide dismutase (Cu, Zn-SOD) was measured using xanthine oxidase system. Catalase (CAT) activity was estimated. Levels of malondialdehyde (MDA) was measured by thiobarbituric acid reaction. Student's t- test was employed for analysis of results. Significant difference (p<0.001) was found in activity of Cu, Zn-SOD in cataract lenses from diabetic patients as compared to senile subjects, CAT activities was also lower (p<0.001) in lenses from diabetic cataract patients when compared to lenses from senile cataractous subjects. Lens MDA levels were significantly higher (p<0.001) in cataract lenses from diabetic patients than in senile subjects. Plasma MDA levels were lowest (p<0.001) in controls as compared to senile and diabetic cataractous patients. Results of present study suggest that increased production of high levels of free oxygen species is linked to glucose oxidation and non-enzymatic glycation of proteins. Furthermore, elevated glucose levels might be involved in the insufficiency of antioxidant enzymatic activity and in the progression of lipid peroxidation.

Alteration in the ubiquitin structure and function in the human lens: a possible mechanism of senile cataractogenesis

High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73–76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins.

Advanced glycation end products in human senile and diabetic cataractous lenses.

The authors prepared water-soluble (WSF), urea-soluble (USF), alkali-soluble (ASF), sonicated (SF), sonicated insoluble (SIF) and membrane (MF) fractions of lens proteins from human senile and diabetic cataractous lenses and age-matched clear lenses. Levels of advanced glycation end products (AGEs) including carboxymethyl lysine (CML), a glycoxidation product, were determined by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA). Distribution of AGEs in the various protein fractions was ascertained by SDS-PAGE and Western blotting. An overall increase in the levels of AGEs in diabetic cataractous lenses as compared to senile cataractous lenses and clear lenses has been observed. ASF and SF, both of which originated from the urea-insoluble fraction, showed the highest levels of AGEs. However, no clear-cut differences in CML levels were seen among clear lenses and senile and diabetic cataractous lenses. AGEs were found to be distributed mostly in the high molecular aggregates in all the fractions. These data suggest that AGEs contribute to protein aggregation and subsequent insolubilization.

Identification and synthetic pathway of sialyl-Lewis x-containing neolacto-series gangliosides in lens tissues. 2. Enzymatic synthesis of sialyl-Lewis x gangliosides in monkey and rat lenses

In Japanese monkey lenses, 3H-labeled fucose and N-acetylneuraminic acid were enzymatically transferred to neolactotetraosylceramide (nLc4) and III 3FucnLc4, respectively, suggesting the presence of a synthetic pathway of IV 3NeuACIII 3FucnLc4 via III 3FucnLc4 in monkey lenses. Six rat strains, Wistar, Sprague-Dawley and pigmented strains, contained sialyl-Lewis x gangliosides in non-cataractous lenses in a strain-specific manner. Glycosyltransferase assay revealed that the transfer of 3H-labeled fucose to nLc4 occurred in all the strains, but that the transfer of 3H-labeled N-acetylneuraminic acid to III 3FucnLc4 was strain-specific. These results suggested that sialyl-Lewis x gangliosides were generally synthesized from neolactotetraosylceramide via Lewis x glycolipid (III 3FucnLc4) in lens tissues, differing from other tissues. Combining our results, we propose two synthetic pathways of sialyl-Le x-containing neolacto-series gangliosides and A-pathway ganglio-series gangliosides in human senile cataractous lens: one to sialyl-Lewis x gangliosides from nLc4 via Lewis x glycolipid, and the other to GD1a from GM3, via GM2 and GM1.

Biochemical evidence for adhesion-promoting role of major intrinsic protein isolated from both normal and cataractous human lenses

In this study, we tested the adhesion-promoting role of major intrinsic protein from both normal human (cadaver) and senile cataractous lenses. Junctional membrane solubilized proteins and pure major intrinsic protein obtained from both type of lenses were reconstituted in neutral phosphatidylcholine liposomes. The interaction of these liposomes with phosphatidylserine vesicles was studied by resonance energy transfer. Our results show that normal human lens junction solubilized proteins and pure major intrinsic protein isolated from them promote adhesion. No quenching effect was observed when major intrinsic protein was omitted in the vesicle reconstitution, no other intrinsic protein of normal human junctional membrane provoked the adhesive effect. In contrast, major intrinsic protein isolated from human senile cataractous lens fails to induce adhesion. The proteolytic cleavages that in vitro originate major intrinsic protein 22,000 Da did not blunt its adhesive capability, suggesting that the proteolytic modifications that major intrinsic protein undergoes in senile cataract were not related with the incompetence of cataractous lens junctions to induce adhesion. Cataractous lens junctional membranes showed protein aggregates. These membranes were treated with sodium hydroxide and reconstituted into liposomes. The sodium hydroxide treatment removed the protein aggregates and restored the adhesive capability. Furthermore, the supernatant obtained after the sodium hydroxide treatment of cataractous junctional membranes, inhibited the adhesive effect of vesicles reconstituted with bovine solubilized proteins. These experiments prove that the failure to induce adhesion of human senile cataractous lens junction proteins is due to the interaction with protein aggregates, which can be removed by sodium hydroxide.

Identification and synthetic pathway of sialyl-Lewis x-containing neolacto-series gangliosides in lens tissues. l. Characterization of gangliosides in human senile cataractous lens

Human lens accumulates gangliosides in association with aging and senile cataract progression. In this study we purified and characterized five major gangliosides in human cataractous lenses. Structural analyses and immunological studies revealed the presence of ganglio-series gangliosides, GM3, GM2, GM1 and GDla, and a sialyl-Lewis x-containing neolacto-series ganglioside, NeuAc α 2-3Ga1 β1-4(Fuc α l-3)GlcNAc β 1-3Ga1β1-4Glc β1-l ceramide (IV 3NeuAcIII 3FucnLc4). Slow-moving gangliosides, although minor components, were also found to have sialyl-Lewis-related structures, based on anti-Lewis' antiserum binding to their asialo forms. However, sialyl-paragloboside, a possible precursor of the sialyl-Lewis x ganglioside, was not identified.

No Immunoglobulins in Cataractous Lenses

All 47 human senile cataractous lenses studied by a direct immunohistochemical method for the presence of immunoglobulins yielded negative results. Consequently, cataract cannot be an autoimmune disease as has been suggested by some authors.

Characterization of neutral glycosphingolipids in human cataractous lens

Neutral glycosphingolipids were purified from human senile cataractous lenses by a combination of solvent extraction, Folch's partition, acetylation, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major glycosphingolipids (A-F) from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis, secondary ion-mass spectrometry, glycosidase digestion, and chromium trioxide oxidation. Their structures suggested that they were closely related in their metabolism: their sugar chains were in sequence and their ceramide moieties were similarly composed, namely C16:0 and C24:1 constituted most of the fatty acids, and long-chain base components were mostly C18-dihydrosphingosine with a small portion of C18-sphingosine. The sugar chains implied two pathways branching from lactosylceramide: one to globotriaosylceramide and the other to lactotriaosylceramide, which leads to the production of Le(x) glycolipid via neolacto type 2 core chain.

Intercellular junctions in thin sections of human senile cataracts

The ultrastructural analysis of normal and cataractous lenses is often hindered by incomplete fixation especially of the centers of the lenses. To overcome this limitation, we have achieved good preservation by chemically fixing 50-200 um thick vibratome sections1 of six human senile cataractous lens nuclei obtained after extracapsular surgery. The remaining nuclear core of each lens was deep yellow to amber and contained central opacities. Fresh vibratome sections (15-30 per lens) were examined in the light microscope (FIG. 1) or were prepared for EM. Fixed and embedded whole vibratome sections were usually bisected and examined along a diameter by trimming successive plateaus (FIG. 2 inset) which gave us access to most regions within the lenses.Whole sections in the light microscope (FIG. 1) consistently showed rings of varying density. Undetectable in uncut lenses, the rings may represent slight discontinuities in composition, refractive index or adherence of layers of fiber cells. These rings roughly correspond to the location of cracks in sections which appear after EM processing (FIG.2 &amp; 3). Fiber cells within these lenses generally displayed complex and irregular shapes, but the cells were well preserved with distinct cell boundaries. We emphasize here the common features of the nuclear cataracts which all contained severe enough opacities to warrant surgical removal but in which the cells appeared to remain intact. Adjacent cells interdigitate with irregular protrusions (FIG. 4). Differential cytoplasmic density between neighboring cells is suggestive of variations in refractive index which might cause excessive light scattering. In this same region in FIG. 4 we also note that dense staining material is present in the extracellular space appearing between the undulating membranes of adjacent cells (FIG. 4, inset). We know of no other report of such deposits in normal or abnormal lenses. The thickness of these deposits may also be sufficient to scatter light. A prominent feature of the lens preparations regardless of location is the presence of undulating membranes which are part of tongue-and-groove junctions (FIG. 5). It is noteworthy that the convex side of the curves is consistently well-preserved and is probably the crystalline array of protein noted in other studies, including in human autopsy lenses. It is not known whether cell communication occurs in the nucleus via gap junctions, but structures with the distinctive morphology of gap junctions are present even within senile cataracts (FIG. 5). FIG. 5 also shows a complex clustering of undulating junctions. The multilamellar membrane cluster in FIG.6, which is similar to structures reported in Emory mouse cataracts, is not commonly seen within the mass of lens tissue but is seen at cut surfaces and within cracks where cells are pulled apart.

Human and experimental lens repair and calcification

Lens repair and calcification have been studied in an experimental rabbit model of anterior segment necrosis. Findings were compared with those in a human senile cataractous lens with subcapsular calcification. Rabbit lenses subjected to anterior segment ischemia underwent a repair process similar to that observed in perforating lens injuries. Cellular response included the formation of fibroblast-like cells that covered epithelial defects of the anterior pole. These observations suggest that the lens epithelium can transform into fibroblast-like cells. The calcification process was a non-cell-induced, and the observed mineral was probably nucleating on organic molecules. Elemental analysis demonstrated that crystals contained calcium and phosphorus with a ratio of 2:1. The mineral was probably hydroxyapatite. Since morphological findings in rabbit lenses closely resemble those of the studied cataractous human lens, the rabbit model appears to simulate one type of lens calcification in senile cataract.

Membrane permeability characteristics of perfused human senile cataractous lenses

Human cataractous lenses were removed by the cryoprobe technique and were maintained for up to 24 hr in a solution of similar ionic composition to human aqueous humour. The bimodal distribution of internal sodium concentrations was similar to that previously reported for unincubated human lenses. Lenses with lower total and free sodium contents had relatively higher membrane potentials and they lost 86Rb at a slower rate than lenses with high internal sodium. The 86Rb efflux in these lenses was stimulated four-fold by removing external calcium. The efflux was reduced by inereasing external calcium, but was increased during a small (60 mosmol) hyperosmotic shock. A similar hyperosmotic shock also surprisingly increased 86Rb efflux. Lenses with increasing internal sodium (and calcium) levels showed an inereasing rate of loss of 86Rb and the stimulation by calcium removal was progressively diminished. The efflux from lenses with disturbed ion levels was also relatively to changes in external osmolarity and to increasing external potassium. Lenses with raised sodium concentrations also had an increased inulin space. Frog, rat and rabbit lenses were also exposed to the same range of stimuli and only frog lenses responded to the low calcium solution with more than a four-fold increase in efflux rate. Although only a two- to four-fold increase in efflux rate was obtained from rabbit lenses exposed to Ca-free conditions, this was the only type of animal lens so far tested that, like the human lens, responded to both hyperosmotic and isosmotic shocks with an increase in efflux rate. All three species of mammalian lenses responded with an increase in efflux rate when exposed to a hyperosmotic test solution while in the frog, the efflux rate from the lens decreased. The glucose efflux from human cataractous lenses was inhibited by cytochalasin B in a similar manner to the efflux from rat and frog lenses. It was concluded, therefore, that the cryoprobed human lens can be kept for a limited period in a relative simple artificial aqueous humour solution. The potassium permeability characteristics of low sodium cataracts remained relatively intact and showed a unique response (relative to lenses from other animals) when exposed to various stimuli that are known to be potentially cataractogenic.

Alpha neoprotein molecules in normal lenses from animals of different ages and in cataractous lenses

Alpha neoprotein molecules are formed by an association of A and B subunits resulting in a different quaternary structure than in alpha crystallin. The content of alpha neoprotein vs. alpha crystallin was estimated separately on the crystallin, cryoprotein and albuminoid fractions in lenses of animals of different species and ages, in lenses with experimentally induced cataracts and in human cataractous lenses. Alpha crystallin was determined on immunoadsorbents with bound antibodies restricted to quaternary determinants of this protein. Alpha neoprotein molecules were determined in the filtrate containing lens proteins not bound on the above immunoadsorbent. The filtrate was applied on an anti-A chain immunoadsorbent, and after desorption the A chain-containing molecules were applied on an anti-B chain immunoadsorbent. The amount of lens proteins bound to the latter immunoadsorbent gave a measure of molecules formed by an association of A with B chains other than in alpha crystallin, that is of alpha neoproteins. No alpha neoprotein was present in lenses from 2-week-old rats or in the 3- to 6-month-old calf lens cortex. The 2-year-old bovine lens nucleus did contain alpha neoproteins, but only in the albuminoid fraction. The lenses of adult rats (i.e. 3- to 4-month-old) contained alpha neoprotein molecules mainly in the albuminoid fraction: small amounts were also found in the cryoprotein fraction. All three fractions of lens proteins from 1-year-old rats contained alpha neoprotein molecules. No significant differences in the content of alpha neoprotein molecules were found in lenses with experimentally induced X-ray and galactose cataracts and in normal controls. This finding does not exclude possible structural differences between normal and cataractous alpha neoproteins. In human senile cataractous lenses, the content of alpha neoprotein molecules in all fractions analyzed equaled or exceeded the content of alpha crystallin present. The present findings demonstrate an age-dependent formation in the mammalian lens of alpha crystallin neoproteins lacking native quaternary determinants. The data obtained point to the possibility that the structural lability of alpha crystallin may be a contributing factor in cataractogenesis.

Distribution of taurine in the crystalline lens of vertebrate species and in cataractogenesis

Marked heterogeneity was observed in the distribution of taurine in different regions of the lens in various species. In general low taurine pools were observed in the nucleus of all species except frog and human. The distribution of taurine in human senile cataractous lenses at different stages of maturation showed decreased contents in all the regions except capsule epithelium as compared to the normal human lenses. This decrease is progressive upto the ‘mature’ stage of cataract. In rat lenses with galactose cataracts taurine contents decreased by about 83–94% of the normal values in the equatorial, anterior, posterior cortical and nuclear regions.

Cation pump activity and membrane permeability in human senile cataractous lenses

The majority of human senile cataractous lenses show a progressive alteration of electrolyte levels resulting in increased lens sodium and decreased potassium, and a concomitant increase in lens water. These changes are known to be associated with the cortical type of senile cataract. The aim of this study was to investigate the mechanisms responsible for the osmotic imbalance in cortical cataract. Determination of Na,K-ATPase activity gave a wide range of values that were not significantly different from normal controls, but rubidium influx showed a normal accumulation in cataractous lenses demonstrating a normal functioning of the cation pump. Both sodium influx and rubidium efflux are increased in the cataractous lenses and the increase is proportional to the absolute level of lens sodium. These results suggest that the biochemical change responsible for the osmotic imbalance in human senile cataract is not a defect in the cation pump mechanism but rather an increase in lens membrane permeability.

Human senile cataract. Light- and electron-microscopic studies of the morphology of the anterior lens structures, with special reference of anterior capsular/subcapsular opacity.

Light- and electron-microscopic studies of human senile cataractous lenses with and without biomicroscopically detectable anterior capsular/subcapsular opacity (ACSCO) revealed the main difference between the two types of cataract to be in the subepithelial cortex, where ACSCO lenses showed disintegrating cortex fibres, fibres of the deep cortical type and even in some cases collapsed fibres. These findings were considered to be associated with the decomposition and disappearance of the superficial cortex in ACSCO lenses. Numerous mitochondria in the epithelium of ACSCO lenses point towards a high oxidative metabolism, which may facilitate active transport across the epithelium. No difference in capsular surface morphology between cataractous lenses with and without ACSCO was found by scanning electron microscopy. Transmission electron microscopy showed in both categories of lenses granular inclusions in the capsules, most pronounced in totally opaque lenses. Large intercellular vacuoles were seen in the anterior part of the epithelium, both light- and electron-microscopically in both categories. Consequently, these ultrastructural changes do not seem to form part of the biomicroscopical picture of ACSCO. Based on our study, we prefer the term: ASCO (anterior subcapsular opacity) in place of ACSCO, the latter term having been used by us previously.

A Study of Human Senile Cataract: Growth and Differentiation of Lens Epithelial Cells in in vitro culture

Epithelial cells of human senile cataractous lenses from aged patients (over 60) were cultured <i>in vitro</i>. They began to grow from the 4th to 6th day of culture and formed typical epithelial cell sheets. The lentoid identified by immunochemical methods as a mass of lens fibers were found to develop at the terminal period of the primary culture. From the secondary passage of the culture, the growth potential of the cells decayed rapidly. It was also demonstrated clearly that the senile cataractous lenses still contained a small number of active epithelial cells exhibiting rather high growth and differentiation potentials, No signficant relationships between activities of epithelial cells and the condition of cataract have been observed so far.

Control of pyruvate kinase activity in the lens

Lens pyruvate kinase (PK) was found to be a variant of the M 2 PK isozyme. It was inhibited by ATP and a number of l-amino acids; Mg 2+ abolished the ATP effect, whereas l-serine counteracted the amino acid inhibition. Pre-incubation of lens PK with fructose-1,6-diphosphate promoted the enzyme affinity toward phosphoenolpyruvate. This property was preserved in human senile cataractous lenses. A decrease of PK specific activity was noted in the epithelium of some cataractous lenses, but the residual activity was at least 15 times that of phosphofructokinase. The activity of PK was apparently determined by the catalytic activity of phosphofructokinase, and also by the cellular concentrations of ATP and amino acids such as alanine.

Relative contents of sodium, potassium, water and dry matter in human senile cataractous lenses in relation to anterior capsular/subcapsular opacity.

In 49 human senile cataractous lenses the sodium and potassium concentrations of the lens water as well as the water and dry weight percentages were examined. It was found justifiable to classify the lenses into three categories on the basis of correlated biomicroscopic and biochemical findings: 1. Immature cataractous lenses without anterior capsular/subcapsular opacity (ac-sco) were characterized by low CNaL+, high CKL+ and low sums of CNaL++CKL+. 2. Immature cataractous lenses with ac-sco were characterized by intermediate value of CNaL+ and CKL+, as well as high sums of CNAL++CKL+. 3. Totally opaque lenses (these lenses had 80-100% of ac-sco) were characterized by high CNaL+, low CKL+, high sums of CNaL++CKL+, high water, and low dry weight percentages. It was found that in immature cataractous lenses increasing extension of ac-sco was correlated to increasing CNAL+ and increasing ratios of CNAL+/CNAA+ as well as to decreasing CKL+ and decreasing ratios of CKL+/CKA+. The sums of CNaL++CKA+ increased. There was a correlation of the extent of ac=sco to the water and dry weight percentages of the immature senile cataractous lenses with ac-sco, viz. a negative correlation for water and a positive one for the dry weight. However, these latter two correlations may be due to chance significances, the level of significance being only greater than P greater than 0.02 in both instances. Lenses which were estimated to have larger than or equal to 30% of ac-sco were found to be more opaque than lenses with less than or equal to 25% of ac-sco.

Concentrations of some ribonucleotides, L-lactate, and pyruvate in human senile cataractous lenses with special reference to anterior capsular/subcapsular opacity.

The concentrations of some ribonucleoside tri- and diphosphates, adenosine-5'-monophosphate, L-lactate and pyruvate were determined in human senile cataractous lenses removed during cataract operations. Pyruvate concentrations were found to be negligible (median = 56 mumol/kg lens wet weight) in 15 human senile cataractous lenses. On the basis of correlations between the biomicroscopic appearances of the senile cataractous lenses (N = 80) and the concentrations and ratios of the metabolites in question, the following classification was found to be justified: 1. Immature cataractous lenses without anterior capsular/subcapsular opacity: high levels of ribonucleoside triphosphates (RTP), high sums of RTP, ribonucleoside diphosphates (RDP), and adenosine 5'-monophosphate (AMP) as well as high levels of L-lactate and high ratios of L-lactate in the lens/L-lactate in the aqueous. 2. Immature cataractous lenses with anterior capsular/subcapsular opacity; intermediate levels of RTP, intermediate values for the sums of RTP, RDP, and AMP, high L-lactate levels, and intermediate values of the ratios of L-lactate in the lens/L-lactate in the aqueous. 3. Totally opaque lenses, which all had extensive anterior capsular/subcapsular opacity; low values for the concentrations of lens RTP, for the sums of RTP, RDP and AMP, and for lens L-lactate. Low ratios of L-lactate in the lens/L-lactate in the aqueous.