Induction of buphthalmos in chicks fed an excess of glycine*

Ralph J. Helmsen, Douglas E. Gaasterland, and Max Rubin

A diet enriched in glycine leads to buphthalmia and growth suppression in.chicks. This was shown in newly hatched chicks when fed a nutritionally adequate purified diet supplemented with 8 per cent glycine, 5 per cent gelatin, and 10 mg. per cent nicotinic acid for a period of seven weeks; however, when the concentration of gelatin in the diet was increased to 8 per cent while the level of glycine was lowered to 6 per cent, the buphthalmic effect was retained while the suppression in body weight toas eliminated. Glycine-fed chicks, at seven weeks of age, appear to be essentially free of glaucomatous symptoms, i.e., increased intra- ocular pressure and decreased facility of aqueous humor outflow and the enlarged eye could almost wholly be accounted for by an increase in the size of the vitreous. The change in vitreous volume noted in such chicks was due primarily to an overgrowth of connective tissue rather than an imbalance in osmolarity of ocular fluids. The comparison of the glycine-induced state with other known buphthalmic conditions in experimental animals are discussed.

Key words: nutritionally-induced buphthalmos, glycine toxicity, globe enlargement, intraocular pressure, coefficient of outflow, vitreous, chickens.

I n 1948, Groschke, Anderson, and Briggs1 in developing chicks fed a purified diet observed a peculiar enlargement of the supplemented with high levels of glycine eyeballs (buphthalmos, macrophthalmos) in the presence of gelatin and nicotinic acid. Naber and co-workers2'3 stated subse- From the Laboratory of Vision Research and quently that intramuscular injections of Clinical Branch, National Eye Institute, National folic acid into vitamin B12 nourished chicks Institutes of Health, United States Department relieved, within a few hours, enlarged eye- of Health, Education, and Welfare, Bethesda, Md. 20014, and the Department of Poultry balls, nervous, and muscular symptoms Science, University of Maryland, College Park, associated with the buphthalmia caused by Md. 20742. glycine toxicity. Machlin and co-workers4 Manuscript submitted for publication Jan. 19, found that when vitamin Bl2 or folic acid 1973; manuscript accepted for publication was fed to vitamin B,..-depleted birds the March 2, 1973. prostration and leg weakness caused by Reprint requests: Dr. Ralph J. Helmsen, Bldg. 6, high-glycine diets was relieved but neither Rm. 224, National Institutes of Health, Be- thesda, Md. 20014. vitamin had any marked effect on the eye syndrome. "Scientific Article No. A1856. Contribution No. 4766 of the Maryland Agricultural Experiment Since the aforementioned authors pre- Station, Department of Poultry Science. sented no explanation for the physiologic 348

Downloaded from iovs.arvojournals.org on 10/02/2021 Volume 12 Induction of buphthalmos in chicks 349 Number 5

mechanism which induced the ocular syn- upon dialysis were made in the appropriate cal- drome, a decision was made to reinvesti- culations. gate the phenomenon. Recently, Rubin and Soluble proteins were determined on the liquified fraction by the method of Lowry and Helmseiv' reported on the differentiation of co-workers.0 Hydroxyproline was assayed on both buphthalmos and growth syndromes in the neutralized hydrolysate of the residual and chicks fed high levels of crystalline glycine liquified fractions by the method of Stegemann7 as modified by Bergman and Loxley.s The proce- in a purified diet adequately supplemented 9 with all of the known nutrients required dure of Bitter and Muir was applied to liquified vitreous for the determination of hexuronic acid. for growth. Intraocular pressure and coefficient of outflow. Intraocular pressure and the coefficient of outflow Materials and methods were measured in 18 chicks. Both eyes of each Day-old chicks of mixed sexes were distributed bird were examined under an operating microscope in groups of twelve, by weight, to electrically before the parameters of intraocular pressure were heated metal cages and were given feed and measured in one eye. water ad libitum. In the first experiment, Barred The animals were anesthetized with sodium Plymouth Rock x White Leghorn chicks and the pentabarbital (40 mg. per kilogram of body New Hampshire breed used by Groschke, Ander- weight) administered intraperitoneally. One drop son, and Briggs1 were found to be equally sensi- of 2 per cent lidocaine hydrochloride was ad- tive to the ocular syndrome; the crossbreds were ministered topically to the eye for corneal anes- substituted for the latter in subsequent studies. thesia. The animal was positioned on its side, The improved basal diet A employed in these with the head supported in a head-holder and the experiments represents a modification of the basal eye being tested on the uppermost side. diet 113GN originally proposed by Groschke, An- The anterior chamber was cannulated from the derson, and Briggs.1 The following additions were anterior border of the cornea, next to the limbus, made to 100 Gm. of diet 113 GN: (1) Amino with a sharp 27-gauge needle held in a needle acids: L-arginine 0.7 Gm., L-tryptophan 60 mg.; holder. The lids were retracted during cannulation (2) Vitamins: vitamin B]2 2 /xg, folic acid 0.4 with sutures. Saline-filled polyethylene tubing mg., vitamin E 4.5 mg., and dry vitamin A and (PE 20) was attached to the needle and to a D3 (650 ng per 100 Gm. each) were added to three-way distribution valve. After penetrating replace the cod liver oil; (3) Minerals: zinc through the cornea, the tip of the needle was carbonate 15 mg., sodium selinite 200 ng, and advanced across the anterior chamber and through sodium molybdate 830 fig; (4) Antioxidant: the pupil until it rested in the posterior chamber ethoxyquin 10 mg. immediately behind the pupillary border opposite Chickens were weighed after seven weeks and the side of corneal penetration. The three-way killed by decapitation. One eye was selected in distribution valve connected the eye directly to a an alternate manner from each bird within the pressure transducer (Model 267 BC, Hewlett- group, immediately enucleated, and cleaned of Packard Corp., Waltham, Mass.) for the measure- adhering muscle and connective tissue. The intact ment of intraocular pressure. Care was taken to eye was weighed and frozen on Dry Ice. Following insure that no air bubbles were in the recording partial thawing, the cornea was excised with a system. Ten to 15 minutes were allowed to elapse pair of iris scissors and the weight of the ex- after cannulation to insure a stable base-line pres- tracted lens, devoid of its capsule, was determined sure. rapidly. Perfusion was done using the constant-pressure Vitreous was removed through the lens opening method of Grant10' ai with the modification that in the frozen state and freed of as much adhering the reservoir of sterile 0.9 per cent saline was retinal tissue and pigment as quickly as possible. elevated to 40.8 cm. above the level of the eye After weighing the connective tissue it was (corresponding to a reservoir pressure level of 30 homogenized immediately in a small Duall hand mm. Hg). The saline solution flowed to the eye homogenizer and stored at -10° C. through a previously calibrated polyethylene cap- Prior to chemical analysis, thawed vitreous was illary tube differential flowmeter. Calibration of transferred to 3 ml. test tubes and centrifuged at the flowmeter was made twice daily, and was 30,000" x g for 30 minutes in a J-21 Beckman found not to vary significantly during the course centrifuge at 4° C. The resulting supernatant of any one experimental day. During the course fluid was removed from the residual fraction and of the experiment, the facility of saline solution dialyzed overnight against isotonic saline buffered flow (the inverse of the resistance) through the in 0.005M phosphate, pH 7.0, and used as such in capillary tube varied from 0.22 to 0.29 fi\ per the designated determination. Compensation for minute per millimeter of Hg. Once started, perfu- changes in the volume of the supernatant fluid sion of the eye was maintained until the intraocu-

Downloaded from iovs.arvojournals.org on 10/02/2021 350 Helmsen, Gaasterland, and Rubin Investigative Ophthalmology Maij 1973

Table I. EfiFect of glycine on body and ocular tissue

Exp. Wt. (Gm.) Body Eye Vitreous Lens Basal A0 532135 (12)§ 1.93710.066 (12) 1.15410.044 (12) 58.1 + 1.5(12)H A 8% Clycine 410±20 (12) 2.222 + 0.065 (12) 1.43310.046 (12) 57.0+1.9 (11) P <0.01 < 0.01 < 0.001 NS Modified basal A°f 669 + 34 (11) 2.23610.078 (11) 1.399 + 0.051 (11) 62.811.8 (11) B 8% Clycine 543 + 27(9) 2.663 + 0.068(9) 1.778 + 0.070 (9) 60.511.5(9) P <0.05 < 0.001 < 0.001 NS C Modified basal A\ 681 + 15 (23) 1.95610.030 (23) 1.16510.023 (23) 55.810.8 (23) 6% Glycine 637117 (24) 2.369 + 0.057 (24) 1.46710.051 (24) 54.311.2 (23) P NS < 0.001 < 0.001 NS "Control diet also contains supplements of 5 per cent gelatin and 10 mg. per cent nicotinic acid. fSee text for diet change. } Supplements same as above, except contains 8 per cent gelatin instead of 5 per cent. ^Arithmetic mean ± S.E.M. Number in parentheses indicates number of animals in each group. IJExpressed in milligrams.

lar pressure reached a new, sustained, elevated added at the 8 per cent level to this dietary level. The rate of flow of saline solution into the regimen, a growth suppression was still eye at the higher, steady-state pressure level was produced in the crossbred bird at seven determined by multiplying the pressure differential of the flowmeter (reservoir pressure minus intra- weeks of age which was accompanied by ocular pressure) by the facility of the flowmeter. an increase in eye size and vitreous weight. The outflow facility, "C", (microliters per minute Elevation of the level of gelatin from 5 per millimeter of Hg) of the eye was calculated by to 8 per cent in the diet and reduction of dividing the rate of flow of saline solution into glycine from 8 to 6 per cent (Experiment the eye by the induced change of intraocular pressure, from the base-line steady-state level to C, Table I) resulted in the elimination of the elevated, perfusion steady-state level (C = the growth suppression in the crossbreds Flow/A P)- The steady-state intraocular pressure with retention of the ocular effect. during perfusion was about 20 mm. Hg in all To elucidate whether glycine feeding in- experiments. duced glaucomatous symptoms in develop- Results ing chicks, 18 birds were selected from The feeding of 8 per cent glycine to various experimental and control diets for newly hatched chicks raised for a period of measurement of intraocular pressure and seven weeks on a basal diet supplemented coefficient of outflow. Examination with with 5 per cent gelatin and 10 mg. per cent the operating microscope before pressure nicotinic acid produced an enlargement of measurements were taken, disclosed that globe (buphthalmia) and a suppression in the anterior chamber was formed with the body growth (Experiment A, Table I). iris set well back from the cornea in all of This increase in eye size was almost totally the birds. due to an expansion in the size of the Successful measurements of intraocular vitreous and was observed to occur in both pressure were obtained in 15 eyes from 15 breeds of birds studied. Histologic sections chicks. Technically satisfactory evaluation prepared from these eyes and stained with of the coefficient of outflow was obtained hematoxylin-eosin revealed no apparent in 13 eyes of the 15 animals. ocular pathology. The results are summarized in Table II. Addition of a variety of supplements The mean base-line intraocular pressure in (see Methods) to the basal diet of Grosch- the eyes of the control chicks was 10.8 mm. ke, Anderson, and Briggs1 to provide a Hg and it was 10.2 mm. Hg in the glycine- ration judged adequate in all known nu- treated birds. The mean coefficient of out- trients effected a significant increase in flow in the eyes of control birds was 0.38 body weight in control birds (Experiment /J per minute per millimeter of Hg and in B, Table I). However, when glycine was the eyes of the glycine-treated animals was

Downloaded from iovs.arvojournals.org on 10/02/2021 Volume 12 Induction of buphthalmos in chicks 351 Number 5

Table II. EfiFect of glycine on aqueous humor dynamics in 7-week-old chicks

Animal* IOPt No. Dietf Wt. (Gm.) base-line "C"t A. Control group : 504 Basal A 376 11 0.35 641 Basal A + 5% gel 521 9 0.23 583 Basal A + 8% gel 451 13 0.96 655 Modified basal A + 5% gel 631 13 0.20 661 Modified basal A + 5% gel 647 8 0.15 Mean 10.8 0.38 S.D. 2.3 0.33 B. Treated group: 513 Basal A + 8% glycine 183 10 0.20 516 Basal A + 8% glycine 198 9 — 518 Basal A + 8% glycine 211 7 0.55 522 Basal A + 8% glycine 194 11 — 662 Basal A + 8% glycine + 5% gel 391 10 0.13 679 Basal A + 8% glycine + 5% gel 611 14 0.23 458 Basal A + 8% glycine 289 8 0.45 460 Basal A + 8% glycine 279 9 0.20 462 Basal A + 8% glycine 259 14 0.23 468 Basal A + 8% glycine 293 10 0.38 Mean 10.2 0.30 S.D. 2.3 0.15 "Animals numbers 504 through 679 are Barred Plymouth Rock x White Leghorn chicks; the remainder are New Hampshire chicks. +All diets cited are supplemented with 10 mg. per cent nicotinic acid. t Intraocular pressure (IOP) base-line expressed as millimeters of Hg; mean coefficient of outflow ("C") expressed as microliters per minute per millimeter of Hg.

0.30 /A per minute per millimeter of Hg. liquified vitreous derived from glycine-fed The difference between these values is not male chicks. A value of 14 /*g per milliliter significant (p < 0.40 for difference of the was found in the ocular fluid sample ob- intraocular pressure and p > 0.50 for tained from eight birds as compared to 4 difference of the coefficient of outflow). p.g per milliliter from a similarly pre- To see what effect excess glycine in the pared sample from 11 control chicks. diet had on the chemical composition of the vitreous, various tissue parameters were Discussion examined in the eyes of male chicks as Chicks fed a high glycine diet for a illustrated in Table III. Along with a sig- period of seven weeks are characterized by nificant increase in fluid content, it should increases in eye size and weight of vitreous be noted that the concentration of hydroxy- over that of control chicks. The present proline remains the same as in the control study reveals that the depth of the an- chicks. However, since the total amount of terior chamber appears normal; the intra- vitreous has increased in the glycine-treated ocular pressure and coefficient of outflow, birds, the total amount of hydroxyproline determined with the animals under penta- is also elevated. Thus, it appears that an barbital anesthesia, were the same in con- increase in vitreous is paralleled by an in- trol and experimental animals. The ocular crease in collagen. Total soluble proteins, enlargement observed in the treated chicks on the other hand, remain constant in the does not appear to be due to an elevation experimental birds and therefore show a of intraocular pressure. small but significant decrease in concentra- On the other hand, several observers tion in the vitreous. have noted that newly hatched chicks de- In addition, a sizeable rise in the con- velop ocular hypertension with a concomi- centration of hexuronic acid, a constituent tant enlargement of the eyeballs when ex- of hyaluronic acid, was observed in pooled posed to continuous light.1210 In addition,

Downloaded from iovs.arvojournals.org on 10/02/2021 352 Helmsen, Gaasterland, and Rubin Investigative Ophthalmology May 1973

Table III. Alterations in vitreous composition with glycine feeding

Glycine Male (7 Weeks) No glycine (6%) P Eye weight (Gm.)° 2.020 ± 0.047 (11)1[ 2.504 ± 0.062 (14) < 0.001 Vitreous weight (Gm.)f 1.197 ± 0.040 (11) 1.590 ± 0.051 (14) < 0.001 Sol PR (mg./Gm. vitreous )\ 2.00 ± 0.23 (11) 1.48 ± 0.12 (14) < 0.05 HyPro (/ig/Gm. vitreous )\ 7.5 ± 2.0 (11) 6.8 ± 1.3 (14) NS Hex Acid (^/ml.)§ 4.0 14.0 °Control diet also contains supplements of 8 per cent gelatin and 10 mg. per cent nicotinic acid. fLiquified vitreous in milliliters. No glycine group 1.0 ± 0.0(11); 6 per cent glycine group 1.3 ± 0.0(14); p < 0.001. (Expressed per grams of wet tissue. {Expressed per milliliter liquified vitreous; represents pooled samples from 8 and 11 birds of no glycine and 6 per cent glycine groups, respectively. ^Arithmetic mean ± S.E.M. Number in parentheses indicates number of animals in each group.

a shallow anterior chamber and a narrow- thalmos in the chick is unclear at the pres- served in these eyes. Thus, it is evident that ent time. It is interesting to note, however, ing of the chamber angle has been ob- that the amino acid has been found to be the glycine-induced buphthalmos differs stimulatory for uridine 5'-diphosphoglucose from the light-induced buphthalmos. (UDPG) dehydrogenase (UDPG:nicotin- The span of time during which the in- amide adenine dinucleotide oxidoreductase, duction of buphthalmos takes place, in the E.G. 1.1.1.22) in extracts isolated from calf developing chick, corresponds normally to a vitreous hyalocytes.18 This enzyme cata- period of rapid growth in the size of the lyzes the conversion of UDPG to uridine eyeball with a marked increase in the 5'-diphosphoglucuronic acid, the first step amount and percentage of the liquid phase in hyaluronic acid biosynthesis. Glycine has in the vitreous. It can be determined from also been suggested as a possible inhibitory the values calculated by Balazs and co- neurotransmitter in certain specified areas workers17 that there is a gradual decline in of the nervous system19 and more recently the hexuronic acid and hydroxyproline con- in the retina.20 centration per gram of gel and liquid The buphthalmos induced by glycine vitreous following hatching. However, the feeding thus provides a third animal model total amounts of each of the above con- for experimental study of this clinical con- stituents in the tissue remain almost con- dition along with the photo-induced en- stant during this phase when expressed per largement in fowl1210 and the genetic whole vitreous. buphthalmos observed in rabbits.21-22 The The increase in total hydroxyproline and role of light in controlling the severity of soluble hexuronic acid in the vitreous of the eye enlargement is presently under chicks fed an excess of glycine suggest that investigation. the eye abnormality observed in these birds is due primarily to an overgrowth of The authors wish to acknowledge the excellent technical assistance of Mr. Luther Dowell. the connective tissue and not merely to an optic fluid imbalance. Although no similar REFERENCES studies have been performed on the 1. Groschke, A. C., Anderson, J. O., and Briggs, vitreous of chicks exposed to continuous G. M.: Peculiar enlargement of eyeballs in 12 light, Jensen and Matson did not detect chicks caused by feeding a high level of a difference in the diy weight of fluid glycine, Proc. Soc. Exp. Biol. Med. 69: 488, withdrawn from the posterior chamber of 1948. the eye of experimental birds or their con- 2. Naber, E. C., Snell, E. E., and Cravens, W. W.: The effect of folic acid on glycine toxicity trol birds. in the chick, Arch. Biochem. Biophys. 37:158, How glycine effects the onset of buph- 1952.

Downloaded from iovs.arvojournals.org on 10/02/2021 Volume 12 Induction of buphthalmos in chicks 353 Number 5

3. Naber, E. C, Cravens, W. W., Baumann, C. J.: Effects of exposure to continuous light on A., et al.: The relation of dietary supplements the eye of the growing chick, Proc. Soc. Exp. and tissue metabolites to glycine toxicity in Biol. Med. 106: 871, 1961. the chick, J. Nutr. 60: 75, 1956. 14. Frankelson, E. N., Lauber, J. K., and Boyd, 4. Machlin, L. J., Lankenau, A. H., Denton, C. T. A. S.: The role of angle closure in light- A., et al.: Effect of vitamin B« and folic induced avian glaucoma, Can. J. Ophthalmol. acid on growth and uricemia of chickens fed 4: 59, 1969. high levels of glycine, J. Nutr. 46: 389, 1952. 15. Smith, M. E., Becker, B., and Podos, S.: 5. Rubin, M., and Helmsen, R. J.: Differentiation Light-induced angle-closure glaucoma in the of growth and buphthalmos syndromes in domestic fowl, INVEST. OPHTHALMOL. 8: 213, chicks fed high levels of crystalline glycine, 1969. Poultry Sci. 51: 1855, 1972. (Abst.) 16. Lauber, J. K., Boyd, J. E., and Boyd, T. A. 6. Lowry, O. H., Rosebrough, N. J., Farr, A. L., S.: Aqueous humor inflow in normal and et al.: Protein measurement with the phenol glaucomatous avian eyes, Exp. Eye Res. 13: reagent, J. Biol. Chem. 193: 265, 1951. 77, 1972. 7. Stegemann, II.: Mikrobestimmung von hy- 17. Balazs, E., Toth, L. Z. T., Jutheden, G. M., droxyprolin mit chloramin-T und p-dimethyl- et al.: Cytological and biochemical studies on aminobenzaldehyd, Z. Physiol. Chem. 311: the developing chicken vitreous, Exp. Eye 41, 1958. Res. 4: 237, 1965. 8. Bergman, I., and Loxley, R.: Two improved 18. Jacobson, B.: Synthesis and degradation of and simplified methods for the spectrophoto- UDP-glucuronic acid in the hyalocytes of metric determination of hydroxyproline, Anal. calf vitreous, Exp. Eye Res. 6: 332, 1967. Chem. 35:1961, 1963. 19. Aprison, M. H., and Werman, R.: The distri- 9. Bitter, T., and Muir, H. M.: A modified uronic bution of glycine in cat spinal cord and roots, acid carbazole reaction, Anal. Biochem. 4: Life Sci. 4: 2075, 1965. 330, 1962. 20. Bruun, A., and Ehinger, B.: Uptake of the 10. Crant, W. M., and Trotter, R. R.: Tono- putative neurotransmitter, glycine, into the graphic measurements in enucleated eyes, rabbit retina, INVEST. OPHTHALMOL. 11: 191, Arch. Ophthalmol. 53: 191, 1955. 1972. 11. Grant, W. M.: Experimental aqueous per- 21. McMaster, P. R. B.: Decreased aqueous out- fusion in enucleated human eyes, Arch. flow in rabbits with hereditary buphthalmia, Ophthalmol. 69: 783, 1963. Arch. Ophthalmol. 64: 388, 1960. 12. Jensen, L. S., and Matson, W. E.: Enlarge- 22. McMaster, P. R. B., and Macri, F. J.: The ment of avian eye by subjecting chicks to rate of aqueous humor formation in buphthal- continuous incandescent illumination, Science mic rabbit eyes, INVEST. OPHTHALMOL. 6: 84, 125: 741, 1957. 1967. 13. Lauber, J. K., Shutze, J. V., and McGinnis,

Downloaded from iovs.arvojournals.org on 10/02/2021