The oxidative stress in the cataract formation

The lens of the eye is an avascular tissue surrounded by fluids such as the aqueous humor and vitreous body, with one side facing toward the outside of the body. We investigated peroxidative reactions occurring in cataractous lenses, examining changes within the lens tissues as well as in the surrounding environment. 1. Peroxidative reactions in lenses. 1) Aging and peroxidative reactions. The activity of superoxide dismutase (SOD) began to decrease in the lenses of rats at six months of age. Moreover, the level of lipid peroxide increased significantly in the lenses of rats at 24 months of age. Lipoproteins became increasingly oxidized with age. The levels of Na+, K+, and Ca++, ions that are important to the maintenance of membrane function, also varied significantly with age. In the lenses of six-month-old Senescence Accelerated Mice (SAM), there was a marked decrease in the ability of scavenge active oxygen and a marked increase in the amount of lipid peroxide. In human lenses, the level of autofluorescence increased as the lens fiber structure changed with age. 2) Generation of free radicals inside the lens. We verified that HO. and ascorbic acid radicals were being generated inside cataractous lenses using electron spin resonance (ESR). 3) Changes in oxidation-related substances in cataractous lenses. Senile cataractous lenses and diabetic cataractous lenses were classified as four types, cortical, nuclear, posterior subcapsular, and mature. In cataractous lenses from all types of diabetic patients, the levels of glucose, glycated protein, and lipid peroxide were higher than in senile cataractous lenses. Among the four types of cataracts, the accumulation of peroxides was the greatest in the nuclear type both diabetic and senile cataractous lenses. 4) Transitional metals. Iron ions and copper ions existed in lens tissue. In particular, the subepithelial region of the lens stained strongly for copper ions. The increased level of copper ions in cataractous lenses is likely to be related to the increased peroxidation in this tissue. 5) Changes in membrane. Lowered levels of phospholipids and a higher degree of saturation of fatty acids were observed in senile cataractous lenses as compared with normal lenses. The increased saturation of fatty acids indicated that there was a damage to the membrane structure due to peroxidative reactions. The receptors for low density lipoprotein (LDL) were shown to exist on the epithelium of normal lenses. Acetyl-LDL, a denatured lipoprotein was incorporated into senile cataractous lenses but not into normal lenses, suggesting that the barrier function of the membrane deteriorates in cataractous lenses. Moreover, in diabetic cataractous lenses, the levels of very low density lipoprotein (VLDL) and LDL significantly increased. 2. Change in the environment surrounding the lens and peroxidative reactions. 1) Changes in the levels of oxidation-related substances in blood, aqueous humor, and vitreous body from diabetic patients: all had decreased levels of reduced glutathione and superoxide scavenging activity, and increased levels of lipid peroxide and glycated protein. This may have been due to a reduction in the anti-oxidative potential in the environment surrounding the lens due to the enhanced glycation. Changes in the level of oxidation related substances in the vitreous body in particular, will likely have a significant impact on the lens. 2) Changes in lenses as the surrounding environment deteriorates. Human lenses were cultured for three weeks under conditions similar to those found in vivo utilizing the culture system that we had originally designed and constructed. When protective activity against peroxidation was reduced, the amount of lipid peroxide increased significantly. In the presence of high levels of glucose, the levels of lipid peroxide increased and the amount and activity of SOD decreased. 3. Effects of changes in the external environment on peroxidative reactions.     

Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate

The levels of alanine, aspartate and glutamine transaminase increase considerably in some diseases. We measured the activity of these enzymes and of the transaminase of 3-hydroxykynurenine, an aminoacid, which acts as a UV lens filter. Alanine and glutamine transaminases (carboxypeptidase) were not detected in normal and cataractous human lenses, and aspartate transaminase was found only in the cortex of normal lenses. 3-hydroxykynurenine transaminase was not found in lenses from persons below thirty years of age, but was found in lenses at about fifty years of age, and in cataractous lenses. Transamination of 3-hydroxykynurenine leads to the formation of xanthurenic acid and its derivatives. These substances appear to be responsible for the increase of lens fluorescence during cataract development.     

Human age-related cataract and lens epithelial cell death

PURPOSE: To evaluate the importance of lens epithelial cell death in age-related cataract. To determine whether the large percentage of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive lens epithelial cells previously reported in human capsulotomy specimens results from apoptosis or necrosis. METHODS: Capsulotomy specimens from patients who had undergone cataract surgery and epithelia from cataractous lenses of eye bank eyes were compared with epithelia from noncataractous lenses of eye bank eyes. DNA fragmentation was assayed using the TUNEL method. Cell membrane integrity was tested using a fluorescent stain for DNA, BOBO-3, that is excluded from living cells. Cell proliferation was assayed by labeling with 5-bromo-2'-deoxyuridine (BrdU). The number of cells in different regions of the lens epithelium was measured by digital imaging and computerized counting of nuclei after staining with methyl green. RESULTS: TUNEL-positive cells were sometimes detected adjacent to denuded regions of capsulotomy specimens, especially when epithelia were not fixed immediately after surgery. TUNEL-stained cells usually stained with BOBO-3, indicating loss of plasma membrane integrity. No BrdU-labeled cells were detected in capsulotomy specimens. Cell density in cataractous lens epithelia was similar to that in normal lens epithelia. In cataractous lenses from eye bank eyes, cell density in the region of the epithelium overlying the cataract was higher than cell density in the region of the epithelium overlying the transparent part of the lens. No correlation was found between cell density and cataract severity or between cell density and age. CONCLUSIONS: TUNEL staining of lens epithelial cells in capsulotomy specimens most likely results from necrotic cell death caused by damage during or soon after cataract surgery. Loss of cells from the lens epithelium, by apoptosis or other mechanisms of cell death, does not seem to play a major role in age-related cataract formation.     

Histological comparison of morphea and lichen sclerosus et atrophicus

To clarify the role of the lens capsule in cataract formation, changes in the protein conformational structure of immature cataractous lens capsules from patients with systemic hypertension or glaucoma have been investigated, as compared to normal lens capsules. The protein secondary structure and composition of these capsular samples were determined using Fourier transform infrared microspectroscopy with second-derivative, deconvolution and curve-fitting methods. We found that the composition of both random coil and beta-type (beta-sheet and beta-turn) structures in the immature cataractous human lens capsules was increasingly induced by systemic hypertension or glaucoma, but alpha-helix content clearly decreased, leading to the alteration of protein conformational structures in lens capsules. A possible pathway of cataract formation exacerbated by systemic hypertension or glaucoma is discussed. According to the results, we propose that systemic hypertension or glaucoma induce changes in the protein conformational structures of the lens capsule, then cause alteration of membrane transport and permeability for ions, and finally increase intraocular pressure, resulting in the exacerbation of cataract formation. The effect on the conformational structure of cataractous human lens capsules is more pronounced for systemic hypertension than for glaucoma. The present study implies that systemic hypertension or glaucoma can exacerbate cataract formation in senile patients by modifying the protein secondary structures in the lens capsule.     

Oxygen free radical on interleukin-1 activity of hemorrhage/resuscitation rat

OBJECTIVE: To investigate the effects of oxygen free radical on the enhancement of IL-1 activity in vivo and in vitro on hemorrhagic and resuscitated rat. METHODS: 30% of rats total blood volume was withdraw by carotid artery catheter and resuscitated with 2 times of lost blood volume 1 h later. RESULTS: Plasm IL-1 activity and MDA content increased and SOD activity decreased significantly 1-4 hours after resuscitation. There was a marked correlation between IL-1 activity and MDA content as well as SOD activity. Treatment with SOD as resuscitation prevented the postresuscitation increase in plasma IL-1 activity significantly. Hemorrhage and resuscitation also caused significant decrease of SOD activity and elevation of MDA in peritoneal macrophage 2 hours after resuscitation. After preincubation with SOD for 1 hour, the macrophage presented a much lowered capacity to release IL-1. CONCLUSION: Oxygen free radical may be one of the most important factors that contribute to elevation of IL-1 level after hemorrhage and resucitation.     

Superoxide dismutase and malonyl dialdehyde in human pulp tissue

A total of 21 pulps were collected from 12 inflamed and 9 normal cases. SOD activity and MDA content were identified in the normal and inflamed pulpal tissues. In the inflamed pulpal tissues, SOD activity and MDA content were significantly increased than those in the normal tissues. The results demonstrated that the inflammation of pulpal tissues resulted in the increasing of the reactivity of superoxide radical and lipid peroxide (LPO). The results also indicated that human dental pulp possessed an endogenous defense mechanism to protect the tissue components from the toxic effects of the reactive oxygen intermediates.     

Confocal microscopy of human lens membranes in aged normal and nuclear cataracts

PURPOSE: To visualize the structure and determine the continuity of lipid membranes in lens fiber cells (LFCs) from human aged normal and cataractous lenses. METHODS: Thick sections from human nuclear cataracts and aged normal lenses were stained with the lipophilic probe DiI, and then analyzed by confocal microscopy. Staining patterns of membranes were observed in individual optical sections or three-dimensional projections of z-series taken in longitudinal section and cross-section of LFCs from different regions within the lens nucleus. RESULTS: DiI bound to and delineated the plasma membrane of LFCs from all regions of the lens nucleus. Three-dimensional projections of z-series from aged normal and cataractous lenses suggested that some of the stained lipid membranes were not continuous with LFC plasma membrane of cataractous lenses. CONCLUSIONS: The results obtained using these methods demonstrated that lipid membranes, discontinuous with the plasma membrane of LFCs, were indicative of a novel process occurring predominately in cataractous human lenses.     

Distribution and type of morphological damage in human nuclear age-related cataracts

The distribution and type of fiber cell damage was evaluated in human age-related nuclear cataracts and in aged normal (non-cataractous) lenses. Ten age-related nuclear cataracts (53 to 89 years old) and four normal lenses (59 to 67 years old) were examined by electron microscopy of fixed Vibratome sections. Images from the adult, juvenile, fetal and embryonic nuclear regions were compared. Each cataractous lens contained a central region of increased light scattering which involved the embryonic and fetal regions with progressively less involvement in the juvenile and adult nuclear regions. Some damaged fiber cells were observed in all specimens, although damage was minor and infrequent in the normal lenses. Degeneration of single or groups of fiber cells was noted in all the adult nuclei of the cataractous lenses, becoming less frequent in the juvenile nuclei. The types of damage included localized voids, multilamellar membrane aggregates, globular bodies, enlarged cells and regions of highly convoluted membranes. The fetal and embryonic nuclei of the cataractous lenses exhibited rare and minor morphological defects, and were virtually identical to the equivalent regions of the normal aged lenses. Examination of cell interfaces in opaque regions of cataractous lenses revealed that the oldest fiber cells sustained apparent membrane loss. Extracellular spaces in the embryonic, fetal and juvenile regions of the cataractous lenses often contained dense deposits, presumably cytoplasmic material lost from adjacent fibers. The results indicate that the region of greatest nuclear opacity, located in the lens center, does not contain any significant cellular damage. This suggests that older fiber cells respond differently to pathological and senescent changes than younger cells made after fetal development. The observed loss of membranes and cytoplasmic material from the oldest fiber cells may be a contributory mechanism in the formation of age-related human nuclear cataracts.     

Cerebellar dysgenesis in infants and children: an experience of 22 cases

BACKGROUND: Scanty information is available on the changes in conformational structure and composition of human lens capsule in cases of hereditary congenital cataract. The purpose of this study was to use Fourier-transformed infrared (FT-IR) spectroscopy to determine the secondary structure and composition of hereditary cataractous human lens capsule, as compared with normal human lens capsule. METHODS: FT-IR spectroscopy with the Fourier self-deconvolution and curve-fitting program was performed, and second-derivative analysis was used to verify the peak positions and assignments of the IR spectra. RESULTS: The curve-fit FT-IR spectra revealed that the content of hydroxylysine and arginine were clearly higher in the lens capsule of the hereditary congenital patient, but the content of aspartic acid significantly lower, than in normal human lens capsules. The secondary conformational changes in alpha-helix, triple helix and random coil structures were important findings in the lens capsule of a hereditary cataractous patient. CONCLUSION: Possible alterations in secondary structures and compositions of lens capsule are observed in the hereditary congenital cataractous patient by using FT-IR spectroscopy with curve-fitting and second-derivative analysis.     

Clinical observation on relationship of antioxidation criteria and immune function of erythrocyte in patients with asthenia syndrome

Results of observation on 31 patients of Qi-Yin Deficiency Syndrome showed that as compared with normal person, the superoxide dismutase (SOD) activity of erythrocyte in patients was lowered, the content of serum lipid peroxide (LPO) increased, the rosette rate of red cell C3b receptor (RC3 bRR) lowered but that of red cell immune complex was elevated significantly (P < 0.01). There were a positive correlation between erythrocyte SOD activity and RC3bRR (r = 0.381, P < 0.05), and a negative correlation between serum LPO level and RC3bRR (r = -0.395, P < 0.05).     

Differential cataractogenic potency of TGF-beta1, -beta2, and -beta3 and their expression in the postnatal rat eye

PURPOSE: Transforming growth factor-beta has been shown to induce cataractous changes in rat lenses. This study assesses the relative cataractogenic potential of TGF-beta1, TGF-beta2, and TGF-beta3 and their expression patterns in the rat eye. METHODS: Lens epithelial explants and whole lenses from weanling rats were cultured with TGF-beta1, TGF-beta2, or TGF-beta3 at concentrations ranging from 0.025 ng/ml to 4 ng/ml for 3 to 5 days. Cataractous changes were monitored daily by phase contrast microscopy and by immunofluorescent detection of cataract markers alpha-smooth muscle actin and type I collagen. Expression of TGF-beta was studied by immunofluorescence and in situ hybridization on eye sections from neonatal and weanling rats. RESULTS: All three isoforms induced morphologic changes in lens epithelial explants and cultured lenses that are typically associated with human subcapsular cataract. Transforming growth factor-beta2 and TGF-beta3 were approximately 10 times more potent than TGF-beta1. All three isoforms were expressed in the eye in spatially distinct but overlapping patterns. Transforming growth factor-beta1 and TGF-beta2 and their mRNA were detected in most ocular tissues, including the lens. Although TGF-beta3 was immunolocalized in lens epithelium and fibers and in other ocular tissues, its mRNA was detected only in the retina and choroid. CONCLUSIONS: All three isoforms of TGF-beta are potentially available to lens cells and have the potential to induce cataractous changes. The results suggest that TGF-beta activity is normally tightly regulated in the eye. Activation of TGF-beta in the lens environment, such as may occur during injury, in wound healing, or in pathologic conditions may contribute to cataractogenesis in vivo.     

Quantitation of vitamin E and a carotenoid pigment in cataractous human lenses, and the effect of a dietary supplement

The quantitation of tocopherols and carotenoids in lipid extracts of cataractous human lenses was performed in parallel with those of matched samples of plasma, which was also analysed at the same time. Alpha-tocopherol in cataractous lenses from elderly human subjects was present at 4.4 mumoles/kg wet weight, much less than the mean of 33 mumoles/l in plasma from these subjects. The mean ratio of alpha- and gamma-tocopherols was 3.5 in the lenses, and 11.3 in plasma. Lens extracts contained no detectable alpha- or beta-carotene, lycopene, or beta-cryptoxanthin. However, all the lens extracts contained a pigment with the retention time and spectrum of lutein and zeaxanthin. Using the molar extinction coefficient of lutein this was present at ca. 0.03 microM, compared with 0.2 microM in plasma. Seven patients with bilateral cataracts had one of their cataractous lenses removed and analysed, and were then given either an oral placebo, or an oral supplement of ascorbate, alpha-tocopherol and beta-carotene. Three months later, the second cataractous lens, and a blood sample, were analysed. Three of the seven had received the active supplement, as confirmed by substantially raised blood levels of alpha-tocopherol and beta-carotene, and raised aqueous humour levels of vitamin C. However, lens tocopherol levels remained unchanged, and no beta-carotene could be detected in the lenses after supplementation. This preliminary evidence needs to be confirmed in larger studies.     

Physiological investigations by image analysis

Cataract formation in diabetic lenses has been attributed to polyol-osmotic pressure-generated influx of water. The ensuing swelling in the form of pocket and lake accumulations cause light scattering. The authors tested whether clear lenses of diabetic patients show different hydration properties than age matched normal lenses. Normal and diabetic human lenses were investigated for their nonfreezable water content by differential scanning calorimetry. The total water content of the lens sections were studied by thermogravimetric analysis. Non-cataractous diabetic lenses in all three regions showed a higher total water content than normal lenses. The nonfreezable water content, seems to increase with age in diabetic lenses and decrease with age in normal human lenses. Thus, hydration changes in human diabetic lenses precede cataract formation. While syneresis, the release of bound water into the bulk, is part of the normal aging process, it appears to occur in the younger diabetics only. In older diabetics syneresis is halted or even reversed. This may be due to the glycation of lens proteins in diabetic patients which tends to immobilize water and therefore, reverse the syneresis due to aging.     

Disulfide bond formation of cysteine-37 and cysteine-66 of beta B2 crystallin during cataractogenesis of the human lens

Beta B2 crystallin has been prepared from total protein of normal (transparent) and cataractous human lenses. After digestion with endoprotease lys-C, the crude digests were resolved on a C18 reverse phase column. The lys-C digests of beta B2 crystallin from cataractous lenses consistently showed the presence of a major peptide that was not present in the lys-C digests from normal lenses. Treatment of this peptide with dithiothreitol resulted in the production of two peptides, corresponding to beta B2 crystallin sequences 18-41 and 48-67, containing cysteine-37 and cysteine-66, respectively. The results demonstrated that intramolecular disulfide bonding of these two residues had occurred in vivo. Based upon previous knowledge of the three dimensional structure of beta B2 crystallin, formation of this disulfide bond suggests that significant denaturation of the beta B2 crystallin molecule has occurred during the process of human lens opacification.     

Protein associated with human lens 'native' membrane during aging and cataract formation

Plasma membrane contains extrinsic as well as intrinsic proteins. Changes in the extrinsic proteins of lens membrane during human aging and cataract formation have not been investigated in detail. Unlike previous studies which examined lens membrane after being stripped of extrinsic proteins by treatment with chaotropic agents, we have isolated whole or 'native' lens membrane on a sucrose gradient by ultracentrifugation of the total water-insoluble protein. Essentially all of the water-insoluble protein from young to aged to cataractous human lens appeared membrane associated. In young lens (20-37 years old), most of the membrane banded at the 25/45% sucrose interface fraction. This fraction contained relatively little urea-soluble protein and likely represents fiber-cell plasma membrane with its physiologically associated extrinsic and intrinsic proteins. With aging (62-80 years old), about one-third of the membrane, as judged by the distribution of cholesterol, banded at a much higher density (50/58% sucrose fraction). The higher density was due to a great increase in the membrane's relative protein content (protein/cholesterol). Although this extra protein was composed of both urea-insoluble and -soluble fractions, the urea-soluble protein predominated in all lenses. Cataractous lens differed from aged-clear lens in that much more of the total membrane (70-75%) had shifted to the high density and participated in this massive binding of cytosolic proteins. Although alpha-crystallin was the principal extrinsic-membrane protein in young lens, high molecular weight aggregate of modified (acidic) crystallins accounted for the increased extrinsic protein in aging. The extrinsic proteins bound to both clear-aged and cataractous lens membrane were aggregated. In conclusion, examination of human lens native membrane fractions revealed that the association of crystallins with membrane in both aging and cataracts was much greater than previously recognized and most of this increased protein was non-covalently bound to the membrane. Much more of the lens total membrane from cataractous than clear-aged lens was involved in this massive protein association and the protein bound to cataract membrane appeared more highly aggregated.     

Glutathione levels of the human crystalline lens in aging and its antioxidant effect against the oxidation of lens proteins

This paper reports the role of glutathione (GSH) in the crystalline lens as an antioxidant against the oxidation of lens protein. GSH levels in normal lenses decreased gradually with increasing age, from approximately 5 mumol per g lens (wet weight) to 3 mumol per g lens (wet weight). On the other hand, levels of oxidized GSH in the lenses increased until the age of 40. After that, it remained almost constant at the level of approximately 0.9 mumol per g lens. Protein-bound GSH levels in both soluble and insoluble lens proteins dropped noticeably in the 50-year and older age groups, although there were significant differences in levels between both fractions. A decrease of tryptophan and tyrosine residues in lens proteins was proportional to a decrease in GSH levels in the lens as a result of aging. Those residue levels in the cataractous lenses were approximately half those in the normal lens proteins, and GSH levels in such lenses were almost one-tenth that in the normal lens. This study revealed that GSH may play an important role in preventing the oxidation of lens proteins from various oxidants. Furthermore, it is conceivable that these normal changes in GSH levels in the lenses increase the vulnerability of the lens to senile cataractogenesis.     

Morphologic changes in the lens epithelium in patients with age-induced cataract, radiation and steroid cataract and cataract following eye contusion

The lens epithelium of the human eye plays a crucial role in the pathogenesis of primary and secondary cataract. Sixty hematoxylin-eosinstained lens epithelia were examined using a light microscope. Cell parameters were compared to one another in the various age groups of patients, between patients with secondary cataract (radiation cataract, steroid cataract and traumatic cataract) and patients with senile cataract, and between male and female patients. The t-test was used for statistical comparison. The median cell density was 3116.5 cells/mm2. The median nucleus-plasma ratio was 1:2.74. Patients with secondary cataract had a larger medium cell and nucleus area as well as a higher nucleus-plasma ratio and a lower cell density than patients with senile cataract. A part from degenerative and proliferative cell changes we found various cell types. The age of patients and the cause of cataract correlate with morphometric changes of lens epithelium cells. However, there was no correlation between specific cytologic changes and causes of cataract.     

Erythrocyte superoxide dismutase activity and plasma malondialdehyde levels in children with Henoch Sch?nlein purpura

Reactive oxygen molecules (ROM) have been suggested to contribute to many pathological conditions including vasculitides and renal diseases. In the present study we measured the activity of superoxide dismutase (SOD) as an antioxidant enzyme in red blood cells and the level of malondialdehyde (MDA), which is a product and an indicator of lipid peroxidation, in the plasma of 16 children (7M, 9F) with Henoch Sch?nlein purpura (HSP) at the onset of the disease (SOD 1 and MDA 1) and at the remission period (SOD 2 and MDA 2). The results were compared with the results of 17 healthy children studied as a control group. There was no significant difference for SOD activities between the patients in each period and the control group (p > 0.05). There was a statistically significant difference between MDA 1 and MDA 2 levels (p < 0.01), each of which were also significantly different from the MDA levels of control group (p < 0.001 and p < 0.01, respectively). The effect of ROMs on different clinical conditions of HSP was also examined and lipid peroxidation was found to be increased more in patients with renal involvement. It is concluded that oxidant stress especially lipid peroxidation plays an important role in the pathogenesis of HSP and in development of renal injury.     

Clinical aspects of Kvamatel (famotidine) administration

An experimental model was used to determine the changes in TNF, MDA and SOD in granulation tissues during natural wound healing after skin excision on rats. Our results indicated that the changes in TNF and SOD exhibited a curve of V. The levels of TNF and SOD were lower on day 7 than day 3. The levels of MDA rose gradually, especially on day 7. A positive correlation was shown between TNF and MDA (at days 3, 5, 7), also between TNF and SOD (at day 3, 7). When the concentration of TNF was lower than 90 Pg/mg protein, the process of wound healing was best, while wound healing was hindered when the levels of SOD were low. The results suggest that in the process of wound repair there are influential changes in the contents of TNF, MDA and SOD. Lower levels of TNF and higher levels of SOD are apparently beneficial to wound healing after trauma.