Metabolic production of a blue-green fluorophor in lenses of dark-adapted mice and its increase with age.

A blue-green fluorophor (496 nm emission/406.7 nm excitation) occurs in the mouse lens; its increase with age is more pronounced in the nucleus than in the cortex. The level of fluorophor and its rate of production are the same for animals reared in the dark as for animals reared in the light. Thus, the fluorophor is not generated by a photochemical reaction but is a purely metabolic product.

Fluorophors and chromophors from rat lens crystallins in UV with hydroxykynurenine.

Isolated alpha-, beta-, and gamma-crystallins from young rat lenses were incubated in solution for 16 hr with 3-hydroxykynurenine under ultraviolet (366 nm) light. Controls included: incubation without light, without kynurenine, and with 2-mercaptoethanol. These procedures generated several chromophors (with absorption maxima or shoulders at 340, 370, and 470 nm) and fluorophors (with excitation/emission at 407/515, 458/550, 515/555, 647/664, and 647/740 nm). The formation of these pigments was inhibited by 2-mercaptoethanol. The findings are discussed in relation to the chromophors and fluorophors found in aged and brunescent human lenses.

Fluorescence/Raman intensity ratio for monitoring the pathologic state of human lens.

The authors have put quantitation of human lens fluorescence on a rational basis by using the accompanying Raman signal from lens protein as a normalization factor. The intensity ratio, Fluorescence/Raman (F/R), may be used to compare lenses of different ages when the exciting wavelength is long enough to give a measurable Raman signal. In younger lenses excited at 457.9 or 514.5 nm, the F/R shows a log increase with age. Older lenses, above 60 years of age, excited at 647.1 nm give a steeply rising sigmoid curve. In developing this procedure, the authors found that for each lens there is a characteristic wavelength that is called the critical wavelength (lambda critical). At wavelengths longer than lambda critical the Raman signal appears in the absence of a broad fluorescence peak; at shorter wavelengths the fluorescence intensity increases enough to overwhelm the Raman signal. For normal lenses, clear and not heavily pigmented, the lambda critical is age dependent, giving a curve that is a flattened sigmoid approximating a straight line.

Tryptophan Raman/457.9-nm-excited fluorescence of intact guinea pig lenses in aging and ultraviolet light.

Normal age-matched guinea pig lenses were compared with those exposed to (1) long-term ultraviolet (UV) light (9 months, 353-nm peak) in vivo, and (2) short-term UV light (3.5 hr, 325 nm) in vitro from a helium-cadmium laser. Tryptophan Raman and 457.9-nm-excited fluorescence profiles along the visual axis (VA) were obtained by taking 21 (for tryptophan) or 11 (for fluorescence) successive spectra for each intact lens using the Raman optical dissection technique. To indicate the extent of UV exposure, fluorescence spectra were obtained along the VA (excitation/emission = 457.9/497 nm); these spectra indicated that the major alteration by UV was in the nucleus with the least in the posterior cortex. Normal aging lenses had no apparent change in the tryptophan profile between 3 days and 12 months. The UV-irradiated lenses also showed no appreciable difference from the normal aging patterns. These results indicate that there is no detectable tryptophan photolysis in the intact guinea pig lens by longwave UV light.

A Raman study of disulfide and sulfhydryl in the Emory mouse cataract.

Emory mice (EM) are genetically predisposed to late-onset cataract formation. Our early work has shown UV-exposure slightly enhanced the expected 2 SH----SS conversion of normal mouse lenses only in the cortical regions. There was essentially no difference in the disulfide profiles of the nuclear region between UV-exposed and control lenses. Since the first noticeable change in the Emory mouse is a hazy nucleus when a lens is examined in vitro, we wondered if cataractogenesis in this model is different from the UV-produced cataract. This question was answered by comparing the visual axis profiles for SH and SS in early EM cataracts and in clear lenses from age-matched controls. The sulfhydryl profiles show that the SH level of 8.5-month-old EM lenses is essentially the same as that of the controls. Likewise, the disulfide profiles show no significant difference. The results clearly demonstrate that EM lenses do not undergo accelerated disulfide production. Therefore for the EM lens, the early stage of cataract formation must involve factors other than just accelerated oxidation of protein SH or glutathione SH. Invest Ophthalmol Vis Sci 29:823-826, 1988

Red fluorescence in older and brunescent human lenses.

Brunescent lenses and normal human lenses more than 70 years old exhibit red fluorescence due to a fluorophor with emission maximum at 672 nm under excitation by the 647.1 nm line of krypton ion laser. The properties and mode of occurrence of this fluorophor suggest that its formation is highly pertinent to senile nuclear pathology.

Proteases in the Emory mouse cataract.

Exopeptidases identified as dipeptidyl peptidase III and leucine aminopeptidase, and an endopeptidase, prolyl endopeptidase, were found in the Emory Mouse cataract and the Cataract Resistant mouse lens extracts. The specific activity measured on Arg-Arg-2-NNap for DPP III and the hydrolysis of Boc-Arg-Pro-2-NNap for prolyl endopeptidase were higher in the Emory Mouse cataractous lens extract. A relatively high rate of hydrolysis of the beta-naphthylamide of leucine aminopeptidase was present in both mouse categories; however, the Cataract Resistant mouse lens had approximately double the protease activity of the Emory Mouse cataract.

Alterations in fiber cell membranes of Emory mouse cataract: a morphologic study.

Morphologic alterations in cortical fiber cell membranes of the developing Emory mouse cataract were studied with scanning, transmission and freeze-fracture electron microscopy. Scanning electron microscopy revealed the extensive formation of prominent ridges on the surfaces of normal-appearing fibers, greatly enlarged degenerating fibers and globular structures in the relatively superficial cortical regions of the cataractous lenses where such a surface pattern was not found in the normal controls. Transmission electron microscopy showed undulating 13 nm pentalamellar structures, which were thinner than 17 nm heptalamellar (or pentalamellar) structures of gap junctions, were distributed within the cell membranes having ridge patterns. Some globular structures were encircled by repeated undulating 13 nm pentalamellar structures and multilamallar membranes. Freeze-fracture studies demonstrated that 13 nm pentalamellar structures consisted of square crystalline arrays of 6 nm intramembrane particles whereas 17 nm heptalamellar profiles showed randomly-packed 9 nm intramembrane particles of typical lens fiber gap junctions. It is suggested that the extensive formation of ridges in the relatively superficial cortical regions of the Emory mouse lenses may be associated with a degenerative process of lens fiber cell membranes during cataractogenesis.

The Emory mouse cataract: accumulation of free leucine and incorporation into protein by cataractous and clear lenses.

Leucine metabolism has been studied in the Emory mouse lens to determine if there is a correlation between the rates of leucine accumulation and incorporation and the stage of cataractogenesis. Developing cataracts were graded in vitro according to severity and then incubated in tracer leucine. There was no significant difference between grades with respect to leucine incorporation or accumulation of free leucine, although there was a gradual decrease in CL/CM as the cataracts increased in severity. The results showed a high degree of variability, especially for incorporation, leading to the conclusion that the morphological appearance of these cataracts is not predictive of their metabolic behavior as revealed by leucine accumulation or incorporation.

The Emory mouse cataract: an animal model for human senile cataract.

The Emory mouse cataract is a late-appearing lens opacity which may serve as an animal model for some human senile cataracts. It is inherited as an autosomal dominant trait and has a typical course of development. Lens opacities may become readily apparent as early as 6-8 months in mice having a familial history of early cataracto-genesis. Many gross morphologic and microscopic features resemble findings in human senile cataract. As an animal model it has many desirable characteristics. Its slow development permits studies of the lens at the pre-cataractous stage and makes it a good assay system for drugs or other factors affecting cataractogenesis. In this paper are given some morphologic and histologic aspects of the developing cataract.

Glutathione metabolism in lenses of Emory and cataract-resistant mice: activity of five enzymes.

The activities of five enzymes of glutathione metabolism were determined in lenses from cataract-resistant and cataract-prone (Emory) mouse variants at three different ages (5 weeks, 10 weeks and 6 months). The enzymes included those required for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione-S-transferase, glutathione peroxidase and glutathione reductase. The differences in the activities of the five enzymes in the two mouse variants were not remarkable at any of the three ages. Activity of each enzyme was noted to be in excess of the preceding one in this integrated metabolic pathway, with the exception of glutathione reductase. gamma-Glutamylcysteine synthetase appears to be the pacesetting enzyme of this metabolic scheme in the mouse lens. The activity of each enzyme was compared with that earlier reported for human, rabbit and dog lenses.

The Emory mouse cataract: changes in the beta and gamma-crystallins during aging and cataractogenesis as revealed by isoelectric focusing of the native soluble proteins.

Isoelectric focusing on ultra-thin polyacrylamide gels was employed to study alterations in the distribution patterns of soluble beta and gamma-crystallins during the development of cataract in the Emory (EM) mouse. These alterations were compared with corresponding changes occurring in clear control lenses of the same age from the cataract-resistant (CR) strain. At two months of age both strains gave similar patterns. At four and fourteen months the EM lens showed depressed beta peaks compared to the CR lens. At fourteen months the EM lens had reduced gamma 2 and gamma 5 compared with the CR lens. At twenty-four months the EM lens had all gamma's markedly reduced, especially gamma 5, with the complete disappearance of gamma 4 which was however still present in the CR lens at twenty-six months. At twenty-four months the EM lens had a relatively large amount of beta 5 but no beta 5a or beta 5b; the latter two were present in the CR lens surrounding beta 5 which was less prominent than in the EM lens. The decrease in gamma's in the twenty-four months EM lens was approximately equalled by an apparent increase in beta's. Of the above changes only the decrease in beta's at four months can be considered precataractous and perhaps a primary change in cataractogenesis. The later changes in both beta and gamma may be secondary although they are certainly at least associated with cataractogenesis. The invariable decrease in gamma with age is noticeably accelerated in cataractogenesis.

Laser raman spectroscopy of the lens in situ, measured in an anesthetized rabbit.

We have obtained the first Raman spectrum from the lens of a live animal. A laser beam (514.5 nm; 15 mW) was directed into the eye of an anesthetized rabbit at 60 degrees from the visual axis and Raman emission was collected at 90 degrees from the incident beam. The power density at the retina was estimated at 0.5 W/cm2. The entire scattering column in the lens can be imaged on the entrance slit of a spectrometer with so little distortion that Raman "optical dissection" analysis (Askren, Yu and Kuck (1979) Exp. Eye Res. 29, 647) can be performed on the in situ lens. The advantages of multichannel detectors over photomultiplier tubes with respect to in situ measurements are discussed.

Oxidative stress on lens and cataract formation: role of light and oxygen.

The mechanism of oxidative damage to the lens through intraocular photochemical generation of superoxide and its derivatization to other oxidants such as singlet oxygen, hydroxyl radical and hydrogen peroxide has been studied. Rat lenses when organ cultured aerobically in TC 199 containing additional amounts of riboflavin were damaged as demonstrated by an inhibition of the uptake of Rb 86 against a concentration gradient. The pump was not affected by light if the culture was conducted in the basal TC 199. However, light was observed to induce significant peroxidative degradation of the tissue lipids even in the basal medium, the degradation being indicated by the formation of malonaldehyde. Both the inhibition of the pump as well as the peroxidative degradation of the tissue lipids, were attenuated considerably by scavengers of superoxide and hydrogen peroxide. In addition, the lipid degradation was prevented by vitamins C and E. The results suggest that the photodynamic injury to the lens cation pump as well as to membrane lipids is incumbent upon an initial generation of superoxide and its derivatization to other oxidants. Thus, the ocular lens is susceptible to oxidative insult and physiological damage through photocatalytic generation of various oxygen radicals. Large concentrations of ascorbic acid in the aqueous humor seems to be able to provide significant protection against such an insult. Thus, this may be one of the functions of high concentration of ascorbic acid in the aqueous humor. The implication of oxidative stress has also been examined in the genesis of cataracts in vivo. Treatment with vitamin E of the Emory mouse led to a decrease in the rate of cataract progression suggesting that at least in some instances an oxidative stress could participate in the formation of cataracts. Oxygen radicals may inflict damage at multifarious biochemical sites. Human lens lipids were also shown to have an absorption maxima at 239 nm indicating their susceptibility to oxidative degradation. In addition the lipid extract has fluorescence similar to that of lipofuscins. The levels of MDA were higher in the brunescent cataracts as compared to that in the nonbrunescent cataracts. The implications of oxidative stress towards the genesis of cataracts in humans is being explored further.

Increased lipid peroxidation and altered membrane functions in Emory mouse cataract.

Lipid peroxidation has been shown to be involved in the pathogenesis of some types of cataract. The possibility of such a mechanism was investigated in Emory mouse cataract. Malondialdehyde, a breakdown product of lipid peroxides, increased 4-fold in advanced cataract. Studies on cation transport revealed that in early cataract there was no alteration in permeability and active transport of cations. However, these functions were significantly altered in advanced cataract as evidenced by about 300% increase in cellular influx of 22Na+ (140 mM) and 50% fall in cellular uptake of 86Rb+ (5 mM). At this stage of cataract, the ouabain- inhibitable component of uptake of Rb+ was drastically decreased, whereas the ouabain-resistant component was unchanged. The mannitol-space increased markedly with progression of cataract. Altered transport of cations in cataract was indicative of damaged membranes which may be due to peroxidation of unsaturated fatty acids in the lipid bilayers concomitant with oxidation of sulfhydryl groups of proteins of the plasma membrane. Superoxide dismutase, catalase and glutathione peroxidase, the defensive enzymes against reactive species of oxygen, were decreased 54%, 57% and 62% respectively in cataract, exposing the lens to oxidants such as 02(-), H202, 0H. and 1 delta 02, which can initiate lipid peroxidation and/or oxidation of protein.

Sodium-potassium-dependent-ATPase activity in Emory mouse lens.

Previous morphological and biochemical studies indicate that a late appearing hereditary Emory mouse cataract may be a good model for certain human senile cataracts. The development of lenticular opacity in the Emory mouse is a slow process which provides an opportunity to conduct analysis of the progression of alterations that lead to cataract development. Biochemical investigations have not yet demonstrated any specific correlation between alterations in the lens and the extent of opacity. We have conducted studies to determine the role of Na+K+-ATPase in the development of cataract in the Emory mouse. In this report we present results obtained on the site and level of activity of Na+K+-ATPase in six- and twelve-month-old Emory mouse lenses in which visible cataractous changes are beginning to appear. CFW mice (the parent strain) were used for controls in this study. Ultrastructural cytochemistry for the localization of Na+-K+-ATPase exhibited the enzyme reaction product for this enzyme to be present mainly between the lateral epithelial cell membranes and between the apical epithelial cell membranes and superficial cortical fiber membranes. In cortical fibers the reaction product was localized between fiber membranes. Although there was very little or no significant differences in the extent of reaction product in epithelial cells, the reaction product in the cortical fibers of six-month-old Emory mouse was less extensively distributed as compared to lenses from control CFW mice of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of long-wave ultraviolet light on the lens. IV. Leucine metabolism in normal human lenses in vitro.

A series of lens pairs (each pair from the same donor) from human eyebank eyes were incubated in a medium containing tracer leucine. One lens of each pair was exposed to long-wave ultraviolet light. The other was shielded. The irradiated lenses accumulated free leucine to a level only 53% of that in the shielded lenses. Irradiation depressed leucine incorporation to 29% of the control level. These results show that UV may adversely affect important parameters of human lens protein metabolism in vitro over a period of 10-30 h.

Leucine metabolism by same-donor pairs of human lenses from eye bank eyes.

29 pairs of same-donor human lenses were incubated 6 h in radioactive leucine. The accumulation (CL/CM) and incorporation (cpm/mg protein) were measured. Donor ages were 4-86 years old. The total storage time was recorded. Same-donor (mated) pairs gave highly concordant results for the two members of a pair but there was extreme variability between different pairs, even when the age was comparable. Storage time did not appear to be a significant factor during the first 24 h but beyond that time continued storage led to marked deterioration of lens metabolism. Because of high variability, no significant dependence of metabolism on age was apparent. A consideration of these results along with comparable investigations from the literature leads to the conclusion that differences among individuals are the chief cause of the wide variation among human lenses although there is some evidence that storage conditions may produce a significant effect. It was not possible to derive a normal value for the parameters measured in this investigation.

Effect of long-wave ultraviolet light on the lens. I. Model systems for detecting and measuring effect on the lens in vitro.

Rat mouse, and chick lenses incubated with 3-aminotriazole under long-wave ultraviolet (UV) show reduced accumulation and incorporation of leucine and a loss of glutathione. The effect on leucine incorporation is strikingly enhanced when capsule-epithelium pools are incubated. The procedure may identify photosensitizers or metabolic inhibitors which are cataractogenic when acting in conjunction with UV.