Sidney Lerman

Biophysical aspects of corneal and lenticular transparency

The chemical composition and morphology of the cornea and lens can provide significant information regarding what wavelengths of nonionizing radiation these two tissue should absorb and transmit. Such data, including a variety of parameters determined by biophysical techniques, can provide us with information regarding the molecular basis for corneal and lenticular transparency and the subtle changes occurring with aging and ambient radiation exposure during our lifetime. The biophysical approach (fluorescence and NMR spectroscopy) has already provided new clinical tools for studying and delineating the initial events responsible for eventual opacification in these two tissues, months to years before they become manifest by current conventional clinical methods of examination.

Investigations on lens transparency and its disturbances by microdensitometric analyses of Scheimpflug photographs

During the aging process, the lens is subjected to changes of its pertinent properties which condition the transmission of light of different wavelengths. Between the second and the third decade, for instance, wavelengths below 400 nm are almost completely absorbed. The increase in lens thickness, effected by the appositional growth during life, might be considered responsible for this phenomenon, if the newly developing lens fibers in the outer periphery would synthesize proteins which, with respect to light transmission, differ from those formed during the embryonic phase. For such a phenomenon, however, no indication was found either by clinical observations or biochemical research. -Microdensitometric analysis of Scheimpflug-photographs of the anterior eye segment allows measurements of lens transparency in the single lens which directly depend on the respective wavelengths used in the photographic procedure. Investigations performed with this method on a normal age-grouped population, show that the changes in light transmission are most evident in two of the lens segments. The lens nucleus shows a continuous increase in light scatter during aging. Also the anterior cortex - particularly in the deeper layers - shows changes in light transmission. With advancing age (beginning between 30 and 40 years of age), increased lens fluorescence is found in the region of the deeper anterior cortex, which can be excited by UV wavelengths of 330-380 nm. This phenomenon is not found in the lens nucleus. The localization of the changes within the lens clearly shows that they are due to age-related modifications of the protein properties earlier designated as posttranslational or postsynthetic molecular modifications.(ABSTRACT TRUNCATED AT 250 WORDS)

Lens Fluorescence in Aging and Cataract Formation

The phenomenon of lens fluorescence in aging and cataract formation is reviewed and two specific fluorogens are demonstrated in the normal aging lens. Evidence is presented regarding the role of UV radiation (above 290 nm) in the generation of at least one fluorogen by means of a free radical induced photodegradation reaction involving protein bound tryptophan in the lens (particularly in the nucleus). The fluorogens can serve as aging parameters and are associated with the development of two other aging parameters; the increase in insoluble protein and decline in SH with age. A mechanism is proposed to account for lenticular aging and nuclear (brown) cataract formation as related to the effects of prolonged exposure of the lens to UV radiation above 290 nm.

Fluorescence spectra of tryptophan residues in human and bovine lens proteins

Abstract The water soluble proteins from human and bovine lenses have been studied using fluorescence spectroscopy. Excitation of aqueous solutions of α, β and γ-crystallins at 290 nm produced fluorescence emission due solely to tryptophan residues. No emission attributable to tyrosine or phenylalanine residues could be detected. The fluorescence emission maxima for all of the human and bovine crystallins in aqueous solution were in the range 332±2 nm, and the bandwidths were in the range 52±2 nm. These results indicate that the tryptophan residues in these proteins exist mainly in hydrophobic environments in aqueous solution. All of the human and bovine crystallin fractions could be denatured by addition of 8 m -urea, 5 m -guanidine hydrochloride, or 1% sodium dodecyl sulfate (SDS). The former two reagents shifted the fluorescence maxima into the range 349±2 nm, indicating that the majority of tryptophan residues are exposed to water in these solutions. Addition of SDS produced only a small spectral shift to 335±1 nm, but produced a marked spectral broadening to 59±1 nm, again indicating significant protein denaturation. The fluorescence data for lens crystallins have been compared with corresponding data for α-chymotrypsin in an effort to classify the lens crystallin data into discrete spectral classes based on specific fluorescence properties of three types of tryptophan residues.

Evidence for a free radical mechanism in aging and u.v.-irradiated ocular lenses

Abstract ESR spectra are reported for whole rat lenses and for the central 3-mm cores from human lenses exposed to u.v. radiation in the ESR cavity at 77°K. The observed signals are attributed to a free radical which is produced via photoionization of tryptophan residues in lens protein. The intensity of the ESR signal was found to be greater in young than in old human lenses and greater in normal lenses than in nuclear (brown) cataracts of the same age. Incubation with penicillamine in the dark reduced the observed ESR signal intensity in rat and human lenses. It is concluded that penicillamine functions as a radical trap within the lens. The ESR experiments reported here, together with evidence from fluorescence spectroscopy, suggest that u.v.-induced damage to the ocular lens could proceed via a free-radical mechanism.

Further Studies on Allopurinol Therapy and Human Cataractogenesis

We examined 11 cataractous lenses (or aspirated lens matter from extracapsular extractions) from patients ranging in age from 55 to 84 years who used allopurinol on a long-term basis (more than two years). Phosphorescence analyses demonstrated the characteristic allopurinol triplet in these lenses. When we analyzed normal lenses from patients taking allopurinol in a similar manner we found no evidence of allopurinol photobinding. These data indicated that allopurinol has a cataractogenic action only in patients in whom the drug has become photobound within the lens. Long-term allopurinol therapy does not necessarily cause or enhance cataracts in all patients. There may be a relationship between ultraviolet radiation exposure and circulating allopurinol levels (and perhaps renal function) in the genesis of photosensitized allopurinol cataracts.

Ultraviolet-visible slit lamp densitography of the human eye

A new slit lamp densitographic apparatus (based on the Scheimpflug principle) capable of accurately and reproducibly recording visible changes in lens density as it ages was recently introduced. We have modified this apparatus to utilize u.v. radiation (300–400 nm) to measure and quantitate the age related fluorescence levels in the normal lens in vivo and correlate them with our in vitro data reported in previous communications. These studies demonstrate the feasibility of obtaining in vivo lens fluorescence data which are objective, reproducible and can be quantified. Several laboratories have demonstrated that u.v. radiation (longer than 300 nm) can play a role in the generation and enhancement of non-tryptophan fluorescence of lens proteins. Furthermore, we have previously reported that 8-methoxypsoralen, a known u.v. photosensitizer (used in treating psoriasis) can become photobound to nucleic acids and proteins within the lens resulting in its accumulation and the enhancement of lenticular fluoresence and phosphorescence. Thus, u.v. slit lamp densitography can be used to monitor objectively one parameter of lens aging (fluorescence) as well as photosensitized lens damage at a molecular level years before visible opacities become manifest by conventional slit lamp examination.

Ultraviolet action spectrum for fluorogen production in the ocular lens.

Abstract— …Previous work has demonstrated that fluorescent material (360nm excitation, 440nm emission), whose concentration normally increases with age in human lenses, can be generated artificially by exposing cultured human or animal lenses to UV radiation. In the present paper we report measurements of the rate of production of this fluorescent material in rat lenses in vitro as a function of UV irradiation wavelength. A plot of the observed rate of fluorogen production normalized to constant photon flux vs irradiation wavelength shows little action at 360 or 320nm, increases sharply at 300nm, remains relatively constant in the range 300–280nm, and then exhibits a further gradual rise from 270–250nm. The results on rat lenses are compared with results reported elsewhere for tryptophan in aqueous solution. The action spectrum for photochemical destruction of tryptophan in solution closely parallels that for fluorogen production in rat lenses. This result and other evidence suggest that photochemical destruction of tryptophan might be the initial event in UV‐induced fluorogen production in the ocular lens.

The rates of photodestruction of tryptophan residues in human and bovine ocular lens proteins

Abstract The relative rates of photodestruction of tryptophan residues in α-, β-, and γ-crystallin fractions from both human and bovine lenses have been measured. The destruction of tryptophan was monitored by observing loss of tryptophan fluorescence, changes in the UV absorption spectra, and changes in the near-UV CD spectra. Photolysis experiments using monochromatic 290 nm radiation were performed on air-equilibrated and vacuum-degassed 1·0 mg/ml protein solutions at 25°C. Both neutral aqueous buffer solutions and the denaturing solvents 8 m -urea and 5 m -guanidine hydrochloride were used in the photolysis studies. The observed rates of destruction of tryptophan residues in all three human crystallins were the same. Denaturing the human crystallin fractions with urea or guanidine hydrochloride did not significantly alter the photolysis rates. The same photolysis rates, and the same lack of effect of denaturation, were also observed for the bovine proteins. Vacuum degassing the lens protein solutions, prior to UV irradiation, resulted in reduction in the photolysis rates by a factor of about one-half.

Allopurinol Therapy and Cataractogenesis in Humans

Long-term ingestion of allopurinol, an antihyperuricemic agent used to treat gout, may be related to the development of lens opacities in relatively young patients (second to fifth decades of life). Cataracts obtained from three patients taking allopurinol were subjected to high-resolution phosphorescence spectroscopy. The characteristic allopurinol triplet was demonstrated in all three cataracts. Identical spectra were obtained for normal human lenses incubated in media containing 10(-3)M allopurinol and exposed to 1.2 mW/cm2 ultraviolet radiation for 16 hours; control lenses (irradiated without allopurinol) showed no allopurinol triplets. Similar data were obtained for lenses from rats given one dose of allopurinol and exposed to ultraviolet radiation overnight. These data provide evidence that allopurinol can be photobound in rat and human lenses and suggest its cataractogenic potential.