Effects of near UV light photoproducts of epinephrine on aqueous humor proteins

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1 Effects of near UV light photoproducts of epinephrine on aqueous humor proteins Seymour Zigman At a level of 0.01 per cent in isolated, pooled calf aqueous humor, epinephrine bitartrate is converted into a series of pigmented. photoproducts by exposure to near UV light for only a few hours. The brown photoproducts, but not adrenochrome, bind to and alter the electrophoretic mobility of aqueous-humor proteins. The rapidity and mild conditions required for the above-described effects to occur, and other considerations of possible near UV photoproduct cellular toxicity, emphasize a need for physical or chemical measures to prevent near UV light exposure of the aqueous humor in patients treated with epinephrine, especially in the aphakic eye. Key words: epinephrine, near UV light, aqueous humor, protein electrophoretic mobility, ocular toxicity, photoproducts..last reports have indicated that tryptophan is photo-oxidized by exposure to near UV light into a series of brown-pigmented products whose chemical and physical properties were quite different from those of tryptophan. 1-2 Other results indicated that some of these photoproducts had a high affinity for polypeptides and proteins, especially for free amino groups. 2-3 Lens and aqueous humor proteins containing bound tryptophan photoproducts also ex- From the University of Rochester School of Medicine and Dentistry, 260 Crittenden Blvd., Rochester, N. Y This work was supported by a Research Grant from the United States Public Health Service (National Eye Institute EY 00459) and The Rochester Eye and Human Parts Bank, Inc. Manuscript submitted for publication Aug. 3, 1973; manuscript accepted for publication Sept. 12, hibited changes in their chemical and physical properties. 3 ' 4 While tryptophan is normally present in the aqueous humor and lens at only trace levels, epinephrine is used therapeutically in many cases at high levels (2 per cent topically) so that the aqueous humor concentration becomes appreciable. The ease of epinephrine oxidation into pigmented products at physiologic conditions has been known for many years, 5 and some of the products have been characterized. 6 When irradiated with low dosages of near UV light for only several hours in dilute solutions at the ph of aqueous humor (ph 7.4), epinephrine bitartrate has been found in our laboratory to be very rapidly converted to pink and then brown products. The brown products have affinity for aqueous humor proteins. This report shows that the proteins normally present in aqueous humor are altered in their electro-

128 Zigman Investigative Ophthalmology February 1974 AQUEOUS HUMOR ALONE AQUEOUS HUMOR PLUS EPINEPHRINE ALBUMIN 1 Fig. 1. Densitometric tracings of polyacrylamide gels of the proteins of calf aqueous humor exposed to near UV light for 5 hours with and without 0.

2 128 Zigman Investigative Ophthalmology February 1974 AQUEOUS HUMOR ALONE AQUEOUS HUMOR PLUS EPINEPHRINE ALBUMIN 1 Fig. 1. Densitometric tracings of polyacrylamide gels of the proteins of calf aqueous humor exposed to near UV light for 5 hours with and without 0.0 L per cent epinephrine bitartrate added and electrophoresed for 1 hour. See methods and materials for experimental details. phoretic mobilities by exposure to near UV light in the presence of epinephrine. Methods and materials Pools of fresh calf eye aqueous humor were obtained at a nearby abbatoir and were immediately placed on ice. The samples were withdrawn within one-half hour of the death of the animals and were processed within one hour of death. After nitration through Whatman No. 1 filter paper, 5 ml. portions were placed into the following experimental conditions: temperature, 22 C; samples with and without epinephrine bitartrate (Sigma Chemical Co., St. Louis, Mo.) at 0.01 per cent; dark controls; and samples exposed for five hours to near UV light (Ultraviolet Products, Inc.; PCQ 008L) at 3,000 /AV per square centimeter at 365 nm. The lamp has a ± 50 nm. spread, with much lower intensities at other wavelengths. At the completion of the incubations, aqueous humor samples were lyophilized to dryhess, redissolved in V& their original volume using Trisglycine, ph 8.4, buffer and 20 /*1 samples were subjected to polyacrylamide gel electrophoresis. 4 ' 7 Electrophoresis was carried out for 45 minutes or 60 minutes at room temperature, and gels were stained with Amido Schwarz for 2 hours. After destaining by soaking in many changes of 7 per cent acetic acid over 3 days, gels were scanned at 600 nm. in a Gilford recording spectrophotometer with a linear transport device. Distances from origin to peak of each band were determined by making use of the chart-paper markings. Results Fig. 1 illustrates densitometric tracings of typical stained polyacrylamide gels after electrophoresis of aqueous humor samples near UV irradiated with and without 0.01 per cent epinephrine bitartrate for 5 hours. The mobilities of the albumin and alphaglobulins are increased appreciably. Table I summarizes the results of a series of aqueous humor experiments so as to illustrate the reproducibility of the method and the large increase in the mobilities of albumin and alpha-globulin resulting from 5 hours of their near UV exposure in 0.01 per cent epinephrine. Results of experiments for 5 hours and 22 hours of incubation in near UV light are compared in Fig. 2. At 5 hours, mobility increases in the epinephrine containing near UV-irradiated proteins are clearly seen. Alpha-globulin bands are absent, having moved into the albumin region. Beta-globulin appears to be refractive to the treatment. Even greater increases in electronegativity are observed by 22 hours; but at this time the effects of near UV light on aqueous humor protein mobilities are just as great, even in the absence of epinephrine (see Fig. 4). When the epinephrine concentration of incubated aqueous humor was increased from per cent to 0.1 per cent and the same experimental procedures were repeated, the data in Fig. 3 were obtained. Protein showed little mobility dependence on epinephrine concentration in this range. It appears that even per cent epinephrine is capable of eliciting an appreciable increase in negative charge in aqueoushumor proteins in the presence of near UV light. Ascorbic acid was formerly found to inhibit the photo-oxidation of tryptophan, but the level of reducing agents (including ascorbic acid and glutathione) normally present in aqueous humor are not sufficient to prevent near UV oxidation of

3 Volume 13 Number 2 Effects of near UV light photoproducts 129 AQUEOUS HUMOR ALONE AQUEOUS HUMOR PLUS 0.01% EPINEPHRINE HR. 5HR. UV R ' U-CN D A R K 5HR.22HR. UV UV D A R K 5HR.22HR. UV UV 1.0- GAMMA BETA ALPHA ALBUMIN Fig. 2. Changes in electrophoretic mobilities of calf aqueous humor protein resulting from 5 and 22 hours of exposure to near UV light with and without 0.01 per cent epinephrine bitartrate. Electrophoresis was for 1 hour as detailed in methods and materials. Standard errors indicated by vertical line at top of each bar of the graph. aromatic compounds within it. Fig. 4 shows that when ascorbic acid is added to calf aqueous humor at several concentrations, the lower ( per cent) and intermediate (0.005 per cent) levels did not protect its proteins from mobility alterations due to exposure to near UV light with epinephrine added. A five-fold excess, however, prevented the increased protein negativity from occurring. Some insight into the near UV photochemical alterations of epinephrine and the manner in which proteins are altered by exposure to them can be obtained from the following data. The time course of the accumulation of epinephrine bitartrate photoproducts is illustrated in Fig. 5. Conditions are as above, except that 0.05 M phosphate buffer, ph 7.4, was used. Pink adrenochrome forms first. This is then converted into brown products. While the color intensity plateaus at about 5 hours, the high 360 nm. excited-440 nm. emitting fluorescence of the products decreases rapidly after 4 hours. The brown material has higher molecular weight and color intensity than adrenochrome, which has a O AQUEOUS HUMOR ALONE 2. AQUEOUS HUMOR PLUS 0.001% EPINEPHRINE 3. AQUEOUS HUMOR PLUS 0.01% EPINEPHRINE 4. AQUEOUS HUMOR PLUS 01 % E PI NEPHRINE GAMMA BETA ALPHA ALBUMIN Fig. 3. Changes in the electrophoretic mobilities of calf aqueous humor proteins resulting from 5 hours of exposure to near UV light with 0, 0.001, 0.01, or 0.1 per cent epinephrine bitartrate added. Electrophoresis was for 1 hour as detailed in methods and materials. Results are the averages of at least two independent experiments with very close agreement between duplicates. much greater 360 nm. excited-440 nm. emitting fluorescence, as shown in Fig. 6. The brown photoproducts of epinephrine have high affinity for aqueous humor and other proteins, while adrenochrome

4 130 Zigman Investigative Ophthalmology February AQUEOUS HUMOR ALONE 2. AQUEOUS HUMOR PLUS 0.01% EPINEPHRINE 3. AQUEOUS HUMOR PLUS 0.01 % EPINEPHRINE PLUS % ASCORBIC 4. AQUEOUS HUMOR PLUS 0.01% EPINEPHRINE PLUS 0009% ASCORBIC 5. AQUEOUS HUMOR PLUS 0.01% EPINEPHRINE PLUS 0.025% ASCORBIC I.0-- I GAMMA BETA A B S E N T l-r- ALPHA ALBUMIN Fig. 4. Influence of ascorbic acid on the changes in electrophoretic mobilities of calf aqueous humor proteins resulting from 5 hours of exposure to near UV light with and without 0.01 per cent epinephrine bitartrate added. Electrophoresis was for 1 hour as detailed in methods and materials. The averages of at least two independent experiments are represented by each bar of the graph. There was very close agreement between duplicates. does not. Brown pigment remains with the proteins when aqueous humors are pressure dialyzed after near UV irradiation for 5 hours with 0.01 per cent epinephrine added. Pink color passes through the dialysis membrane and is therefore not protein bound. To further illustrate this finding, the results in Fig. 7 are included. Polyarginine (0.1 per cent) (Sigma Chemical Co., St. Louis, Mo.) in distilled water adjusted to ph 7.4 with 0.1M NaOH, was near UV irradiated with 0.01 per cent epinephrine bitartrate, and pressure dialysis was carried out at 0, 1, 5, and 9 hours. While the 480 nm.-absorbing adrenochrome is dialyzable, the brown photoproducts are bound to the macromolecules whose absorption spectra develop an absorption shoulder at 280 nm. The same result occurs when the macromolecules are precipitated with ethanol or phosphate buffer. Thus, affinity of the photoproducts for amino groups is found. Discussion Before discussing the data, it is interesting to put the in vitro conditions used in the experiments into the perspective of in vivo circumstances. The intensity of near UV lamps used herein was 3,000 /xw per square centimeter at 365 nm., while that of sunlight at ground level is approximately 5,000 [xw per square centimeter (on a clear, sunny 80 F. day in July, and a clear, sunny, 20 F. day in January in the northeastern United States at noon). A battery of two cool white fluorescent lamps (40 W.) emit only 50 JU.W per square centimeter, while daylight fluorescent lamps at 30 W. emit about 150 pw per square centimeter at 365 nm. Note, however, that the visible wavelengths in the sunlight and fluorescent lamps also would contribute strongly to epinephrine oxidation. The extent to which light at this wavelength penetrates the cornea has been estimated as approximately 75 per cent in the calf and 50 per cent in the human. Epinephrine-supplemented calf aqueous humor was also found to form fluorescent photoproducts when calf corneas were placed between the light source and the aqueous humor. The findings show that when aqueous

5 Volume 13 Number 2 Effects of near UV light photoproducts Fig. 5. Changes in the color density at 440 nm. and in the 360 ran. excited/440 emitting fluorescence of solutions of epinephrine bitartrate (0.01 per cent) exposed to near UV light at ph 7.4 (0.05 M phosphate buffer) with increasing time of exposure. Details of irradiation in methods and materials. A Cary 14 spectrophotometer and a Turner 111 fluorimeter were used for measurements. humors containing levels of epinephrine of 0.01 per cent to per cent are exposed to 3,000 ixw per square centimeter of near UV light for several hours, the formation of a number of pigmented photooxidation products of epinephrine is rapidly induced. Some of these products bind to, and thereby alter, the electrophoretic mobilities of aqueous humor proteins, especially albumin and the alpha-globulins. The brown photoproducts, but not the initially formed pink adrenochrome, appear to bind to amino groups of these proteins, and being negatively charged themselves, cause an increase in protein electronegativity. The mechanism of formation of near UV photoproducts of epinephrine in the aqueous humor and the resulting protein mobility alterations appear to parallel those occurring when aqueous humors containing much higher levels of tryptophan are thus treated, 4 but they occur about 5 to 10 times more rapidly. While an attempt has been made to sort out which epinephrine photoproducts bind to aqueous humor proteins, no definitive identification of the active material can yet be proposed. A detailed report on the oxidation products of epinephrine by Lund 5 reveals the multitude of substances implicated in the further oxidation of adrenochrome to brown products. The present report indicates that these products are of higher molecular weight than adrenochrome. Photo-oxidized products of tryptophan that bind to proteins have also been shown to have higher molecular weight than tryptophan or its initial oxidation product, N-formylkynurenine. 2

6 132 Zigman Investigative Ophthalmology February O Fig. 6. Elution profile from Sephadex G 10 column of the products of the near UV irradiation for 5 hours of 0.01 per cent solutions of epinephrine bitartrate. Eluent was 0.01 M (NH 4 ) 2 CO:i, ph 8.8.; 2 ml. were collected per tube. Column size was 1.8 by 30 cm. Instrumentation as in Fig. 5 legend. A Gilson Medical Electronics fraction collector was used to separate eluted proteins. Table I. Electrophoretic mobilities of calf aqueous humor proteins on polyacrylamide gels (7.5 per cent) Protein Conditions Gamma Beta Centimeters Alpha moved in 45 minutes Albumin Aqueous dark for 5 hours Average Aqueous plus UV for 5 hours ± ± ± ± Average Aqueous dark plus epinephrine for 5 hours Average Aqueous UV plus epinephrine for 5 hours Average 0.39 ± ± ± ± ± ± ± ± 0.02 L O ST 3.33 ± ± ±

7 Volume 13 Number 2 Effects of near UV light photoproducts W A V E L E G T H (nm) Fig. 7. Binding to polyarginine of products of epinephrine bitartrate resulting from exposure to near UV light for 5 hours. Macromolecules separated from low molecular weight products by pressure dialysis through a UM 10 (Amicon, Inc.) membrane filter (10,000 molecular weight cut off). Spectra were measured in a Cary 14 spectrophotometer. Ascorbic acid at much higher than physiologic concentrations has been shown to inhibit epinephrine photo-oxidation, as was found for tryptophan photooxidation. For tryptophan, an equimolar level was sufficient to prevent photoproduct formation in near UV light. However, a much greater concentration of ascorbic acid than of epinephrine (5 times as great) was required to prevent the formation of its photoproducts and protein mobility changes. It is not yet possible to directly relate ocular toxicity to the changes in aqueous humor proteins resulting from near UVinduced epinephrine photoproducts. The intent of this communication is to show that these photoproducts can be formed and can influence the properties of ocular proteins. A short discussion of the possible hazards to the eye of near UV light-induced epinephrine photoproducts in the aqueous humor follows. Because of the rapid turnover of the aqueous humor in living eyes, it is unlikely that free photo-oxidized tryptophan could accumulate in sufficient amounts to alter its proteins. However, when epinephrine therapy is in effect, enough photooxidized epinephrine could form there quickly so as to combine with and alter proteins. The rapidity of formation of near UV-induced epinephrine photoproducts could result in high enough levels to promote changes in aqueous humor proteins. Other forms of ocular toxicity may occur as a result of epinephrine photoproduct formation in the aqueous humor. The near UV photoproducts of tryptophan have been found to have antimetabolic effects on a variety of cells In addition, brown epinephrine photoproducts have been found to penetrate into the calf lens in vitro and to bind to the crystallins, thereby increasing their electronegativity. A preliminary experiment has also shown that the incorporation of amino acids into calf

134 Zigman Investigative Ophthalmology February 1974 retina photoreceptor proteins was markedly inhibited by near UV photoproducts of tryptophan and epinephrine bitartrate.

8 134 Zigman Investigative Ophthalmology February 1974 retina photoreceptor proteins was markedly inhibited by near UV photoproducts of tryptophan and epinephrine bitartrate. The formation of epinephrine photoproducts in the aqueous humor may be a serious problem in aphakic eyes in which these substances would have easier access to other ocular tissues, especially the retina. An epinephrine maculopathy has already been reported by Kolker and Becker, 11 who found that 20 per cent of the aphakic eyes receiving epinephrine therapy in their study developed a reversible retinitis. It is interesting to speculate whether near UV light and epinephrine photoproducts play a role in this clinical situation, and if near UV light filters or ascorbic acid supplementation would prevent it. REFERENCES 1. Zigman, S., Schultz, J. B., Yulo, T., et al.: The binding of near UV photo-oxidized amino acids to proteins, Fed. Proc. 30: 1198, Zigman, S., Schultz, J. B., Yulo, T, et al.: The binding of photo-oxidized tryptophan to a lens gamma crystallin, Exp. Eye Res (in print). 3. Zigman, S., Schultz, J. B., Yulo, T., et al.: Effects of near UV irradiation on lens and aqueous humor proteins, Isr. J. Med. Sci. 8: 1590, Zigman, S., Griess, G., Yulo, T, et al.: Ocular protein alteration by near UV light, Exp. Eve Res. 15: 255, Lund, A.: Fluorometric determination of adrenaline in blood. II. The chemical constitution of adrenolutine (the fluorescent oxidation product of adrenaline), Acta Pharmacol. 5: 121, Udenfriend, S.: Fluorescence assay, In Biology and Medicine, New York, 1962, Academic Press, pp Ornstein, L., and Davis, B. J.: Disc Electrophoresis. Rochester, N. Y., 1960, Distillation Products Industries. 8. Zigman, S., and Yulo, T.: How near UV photoproducts of tryptophan inhibit dogfish lens protein synthesis, Biol. Bull. 142: Rustad, R. C, Antonellis, B. C, and Zigman, S.: A near ultraviolet photoproduct of tryptophan inhibits the mitosis and development of arbacia eggs, Biol. Bull. 172: Johnson, S., and Zigman, S.: Inhibition of dogfish retina protein synthesis by near ultraviolet light, Biol. Bull. 141: 391, Kolker, A. E., and Becker, B.: Epinephrine maculopathy, Arch. Ophthalmol. 79: 552, 1968.

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