Identification of unknown quaternary ammonium compounds in corneal epithelium and aqueous humor.

Bovine corneal epithelium and bovine aqueous humor are investigated for their content of quaternary ammonium compounds. In total, four compounds are found. Three of these are identified. For the fourth compound, a proposal for its structure is made on the basis of tandem mass spectrometry fragmentation spectra. The compounds investigated have m/z values of 146, 160, and 174. The compounds with m/z 146 are confirmed as acetylcholine (in corneal epithelium) and (3-carboxypropyl)-trimethylammonium (in both corneal epithelium and aqueous humor). The compound with m/z 174 is identified as butyrylcholine (in corneal epithelium). The compound with m/z 160 is probably acetyl-g-homocholine (in both corneal epithelium and aqueous humor). For both butyrylcholine and acetyl-g-homocholine, it is the first time the presence of these compounds in corneal epithelium or aqueous humor (or both) is described. Both acetylcholine and butyrylcholine are unstable compounds, which are probably susceptible to enzymatic degradation by acetylcholine-esterase and butytrylcholine- esterase, respectively.

Sample preparation and determination of acetylcholine in corneal epithelium cells using liquid chromatography-tandem mass spectrometry.

A sample preparation method with subsequent liquid chromatography (LC)-mass spectrometry (MS)-MS analysis for acetylcholine in corneal epithelium is developed. The sample preparation is developed with a focus on compatibility with the LC-MS-MS system and the stability of acetylcholine because acetylcholine esterase is present in the tissue. It appears that both acetylcholine as well as the internal standard (IS) used (acetyl-beta-methylcholine) have fragments at m/z values in the tandem MS spectrum, which correspond with the m/z values of fragments of endogenous substances. Acetylcholine and (3-carboxypropyl)triethylammonium both have 146-->87 and 146-->60 transitions. Acetyl-beta-methylcholine and an unknown compound both have 160-->101 and 160-->60 transitions. This makes it necessary to use a chromatographic step, which has a baseline separation between these endogenous compounds, acetylcholine, and the IS. The analytical procedure has linearity from 1 ng/mL (30 pg/mg corneal epithelium tissue) to at least 250 ng/mL (7.55 ng/mg corneal epithelium tissue). The limits of detection and quantitation are 15 and 45 pg on column, respectively. Relative standard deviation and bias values are within the range of acceptance for all concentration levels.

UV absorption by uric acid in diurnal bird aqueous humor.

PURPOSE: To analyze the components responsible for the UV absorbance in diurnal bird aqueous humor. METHODS: The absorbance studies were carried out using a Hitachi spectrophotometer (U 2000). Uric acid was determined by high-performance liquid chromatography (LC-10 system; Shimadzu, Kyoto, Japan). Chicken and turkey eyes were examined. RESULTS: The UV absorbance in chicken aqueous was largely accounted for by the presence of protein, tryptophan, tyrosine, ascorbic acid, and uric acid. Ascorbic acid was low (23 micromol/l). Uric acid was, on the other hand, remarkably high (151 micromol/l) compared with that in mammals (cattle, 16 micromol/l). Principally the same results were obtained in chicken and turkey. CONCLUSIONS: Uric acid is a significant UV-absorbing substance in the aqueous humor of diurnal birds with its peak absorbance at 292 nm. The hypothesis that the aqueous humor acts as a UV filter seems to be valid also for the avian eye. However, in these eyes uric acid fulfills the role that ascorbic acid does in mammals.

Distribution of ascorbate in the anterior bovine eye.

PURPOSE: To analyze the ascorbate distribution in the anterior eye wall to better understand the functional significance of this compound in the eye. METHOD: Ascorbic acid was determined by high-performance liquid chromatography using an LC-10 system (Shimadzu, Kyoto, Japan). Bovine eye samples were used. RESULTS: The highest ascorbate concentration was observed in the corneal epithelium, with significantly higher values in the central (1.56 mg/g) than in the peripheral (1.39 mg/g) area. The ascorbate content was similar in the corneal stroma (0.22 mg/g), the Descemet's membrane (DM)/endothelium (0.22 mg/g), and the aqueous humor (0.21 mg/ml). By comparison, the sclera (0.15 mg/g) and the conjunctiva (0.11 mg/g) showed lower values, as did the lacrimal gland (0.09 mg/g) and the serum (0.0008 mg/ml). CONCLUSIONS: (1) Peak ascorbate concentration was observed in the central corneal epithelium covering the pupillary area. This is compatible with the idea that the ascorbate may act as an UV filter shielding internal eye structures from radiation damage. (2) The ascorbate concentration in the corneal stroma and DM/endothelium was as high as in the aqueous humor, and it is suggested that the aqueous humor plays a key role in the distribution of ascorbate to the anterior eye wall.

Epidemiology of the pseudo-exfoliation syndrome.

1) Pseudo-exfoliation syndrome (PE syndrome) is a condition with worldwide distribution. 2) Marked geographical variations have been demonstrated. 3) Environmental factors may be of etiological significance for the development of PE syndrome. 4) The PE syndrome's ability to promote glaucoma may vary from one area to another. 5) There seems to be a trend towards lower debut age of PE syndrome at lower latitudes. 6) More prevalence studies from different parts of the world are needed. A standardized set-up of such works would facilitate comparison of the results, and so some guidelines have been proposed.

Toxicological evaluation of cyclosporine eyedrops.

PURPOSE: The short-term toxicological effects of two cyclosporine A eyedrop formulations are compared. METHODS: Formula A was based on Sandimmune (Novartis, Switzerland) infusion concentrate with a final ethanol concentration of 1% (w/w), and formula B on Sandimmune oral solution. Both formulations were diluted in sterile peanut oil (10 mg/ml). The left eyes of 12 rabbits were treated with the cyclosporine eyedrops over a two-week period. The right eyes served as controls. Slit-lamp and scanning electron microscopic examinations were performed, and the total protein concentration in aqueous humor was measured in treated and control eyes. RESULTS AND CONCLUSION: Both Sandimmune oral solution and infusion concentrate-derived eyedrops were found to be non-toxic to rabbit eyes and there were no significant differences between the two formulations. More definite conclusions as to the safety of these cyclosporine formulations cannot be made without long-term trials.

Ascorbate in the corneal epithelium of diurnal and nocturnal species.

PURPOSE: To compare the amount of ascorbic acid in the corneal epithelium of various species to unveil possible differences between diurnal and nocturnal mammals. METHODS: Ascorbic acid was determined by high-performance liquid chromatography, using an LC-10 system (Shimadzu, Kyoto, Japan). RESULTS: Diurnal animals show a higher ascorbate concentration in the corneal epithelium than nocturnal animals. Ascorbate concentration is higher in the corneal epithelium than in the matching aqueous humor in diurnal and nocturnal species. The highest ascorbate concentration is found in the corneal epithelium of the reindeer. CONCLUSIONS: Ascorbate level in the corneal epithelium seems to vary in accordance with ambient radiation exposure of the respective species, just as in the aqueous humor. Both phenomena are regarded as environmental adaptations, and the ascorbic acid is suggested as protecting against photokeratitis and as acting as an ultraviolet filter for internal eye structures.

Corneal epithelium and UV-protection of the eye.

PURPOSE: To study UV-absorption and UV-induced fluorescence in the bovine corneal epithelium. METHODS: Spectrophotometry and spectrofluorimetry. RESULTS: The corneal epithelium absorbs UV-B radiation mainly owing to its content of protein, RNA, and ascorbate. Some of the absorbed energy is transformed to the less biotoxic UV-A radiation by fluorescence. RNA and ascorbate reduce tissue fluorescence. CONCLUSIONS: The corneal epithelium acts as a UV-filter, protecting internal eye structures through three different mechanisms: (1) Absorption of UV-B roughly below 310 nm wavelength. (2) Fluorescence-mediated ray transformation to longer wavelengths. (3) Fluorescence reduction. The extremely high ascorbate concentration in the corneal epithelium has a key role in two of these processes.

Pseudo-exfoliation and mortality.

The hypothesis that ocular pseudo-exfoliation syndrome is part of a generalized disorder has been tested by suggesting that subjects having this syndrome would have increased mortality. However, no association was found between presence of ocular pseudo-exfoliation syndrome and mortality.

In vitro evidence for UV-protection of the eye by the corneal epithelium mediated by the cytoplasmic protein, RNA, and ascorbate.

PURPOSE: (1) To evaluate the effect of ribonucleic acid (RNA) and ascorbate on UV-absorption and their impact on ultraviolet-induced (UV) fluorescence from various proteins, and (2) to compare RNA and DNA reduction of protein fluorescence. These informations will be useful for later work on the UV-filtering effect of the corneal epithelium. METHOD: Spectrophotometry and spectrofluorimetry. RESULTS: (1) RNA and ascorbate caused a significant UV-absorption and reduced the fluorescence from various water-soluble proteins, the degree of reduction varying independently from one protein to the other. (2) RNA and DNA showed protein fluorescence reduction of roughly the same order. CONCLUSION: The results are discussed both in the context of UV-protection of the cell nucleus in general, and the possible UV-filtering effect for the eye of bovine corneal epithelium.

The significance of ascorbate in the aqueous humour protection against UV-A and UV-B.

It has previously been suggested that the aqueous humour acts as a UV-filter protecting the structures behind. This hypothesis has here been further evaluated by use of spectrophotometry and spectrofluorimetry. Principally three different aspects of the protecting mechanism have been unveiled: absorption, fluorescence quenching and wavelength transformation. The extremely high ascorbate values obviously play a key role in all of them. Thus, aqueous ascorbate both increased absorption and suppressed fluorescence of radiation below roughly 310 nm wavelength. In addition, as a consequence of ascorbate quenching, fluorescence emission to the UV-A range (320-400 nm) is substantially reduced.

Epidemiology of glaucoma in northern Europe.

PURPOSE: To survey glaucoma epidemiology in Northern Europe. RESULTS OF LITERATURE REVIEW: Open-angle glaucoma is defined here as the simple and capsular types taken together. On this basis open-angle glaucoma makes up by far the largest group of glaucoma in Northern Europe. These are the target groups of this study, and pigmentary glaucoma is excluded because of the small numbers. CONCLUSION: Open-angle glaucoma is less frequent in Southern Sweden and Denmark than in Finland, Middle Sweden, Norway, and iceland. This may to some extent be explained by the suggested low prevalence of pseudoexfoliation in the southern areas, but other factors are presumed to be of importance as well.

Quenching of UV-induced fluorescence by ascorbic acid in the aqueous humour.

Spectrophotometry and spectrofluorimetry of bovine aqueous humour have been performed. The question of whether mechanisms for fluorescence quenching exist in the eye media was evaluated. It turned out that the marked fluorescence of tryptophane and protein was considerably quenched by the ascorbic acid, an observation not shown before. Thus, the aqueous humour minimizes UV-radiation to the lens in diurnal animals through three different mechanisms: 1) Absorption. 2) Fluorescence-quenching. 3) Fluorescence-mediated ray transformation of energy-rich short wavelengths to less potent longer wave-lengths. The impact of each of them is significantly influenced by the ascorbate concentration in the aqueous humour. The phenomenon of fluorescence-mediated ray transformation is, of course, per se independent of ascorbate, but the transformational input is significantly reduced due to ascorbate.

Increased IgA antibodies to gluten and gliadin in serum of persons with ocular pseudo-exfoliation.

Three of 12 persons with pseudo-exfoliation syndrome revealed increased IgA serum antibodies to gluten and gliadin vs none in the control group. The phenomenon is suggested to indicate barrier breakdown in the gut wall.