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before and after the addition of beta-glucuronidase.4 ported by the Juvenile Diabetes Foundation and by the University In circumstances where the actual plasma free fluo- of London, Central Research Fund. The HPLC was purchased with an MRC grant to Dr. Michael J. Neal. Submitted for publi- rescein has not been measured, the term "plasma- cation: April 12, 1984. Reprint requests: Dr. P. S. Chahal, Depart- free fluorescence" should be quoted. It is then better ment of Medicine, Hammersmith Hospital, Du Cane Road, London to measure overall fluorescence (fluorescein and the W12 OHS, England. glucuronide metabolite) in protein-free plasma ultra- filtrate and the fluorescence appearing in the ocular References compartments using the same excitor and emission filters. 1. Araie M, Sawa M, Nagataki S, and Mishima S: Aqueous Fluorescein glucuronide is a potential source of humor dynamics in man as studied by oral fluorescein. Jpn J Ophthalmol 24:346, 1980. variability in studies of blood-ocular dynamics using 2. Zeimer RC, Blair NP, and Cunha-Vaz JG: Pharmacokinetic fluorescein. Its exact role has yet to be established. interpretation of vitreous fluorophotometry. Invest Ophthalmol VisSci 24:1374, 1983. Key words: Blood-ocular barriers, diabetes, plasma ultrafil- 3. Chen SC, Nakamura H, and Tamura Z: Studies on metabolite trate, fluorescein glucuronide, fluorescence, protein-binding of fluorescein in rabbit and human urine. Chem Pharmacol Acknowledgments. Technical assistance was given by Dr. Bull 28:1403, 1980. J. Cunningham, Ian Joy, and Margaret Foster. 4. Chen SC, Nakamura H, and Tamura Z: Determination of fluorescein and fluorescein monoglucuronide excreted in urine. From the Department of Medicine, Hammersmith Hospital,* Chem Pharmacol Bull 28:2812, 1980. and the Department of Pharmacology, St. Thomas's Hospital 5. Lund-Andersen H, Krogsaa B, and Jensen PK: Fluorescein in Medical School,f University of London, London, England. Sup- human plasma in vivo. Acta Ophthalmol 60:709, 1982.

Diosynrhesis of Neopterin, Sepioprerin, ond Diopterin in Rar ond Humon Oculor Tissues

Gadiparrhi N. Rao and Edward Corlier

Neopterin, , and synthesis by lens, animals have been published,8'9 and a pathway shown retina, and ciliary body-iris of rat and human indicates in Figure 1 has been proposed.8 The aim of the formation from their precursor, GTP. The pteridine present investigation, indeed, is to know whether biosynthesis was higher in the retina (neopterin 422 ± 27, lenticular tissues possess a biosynthetic pathway to 260 ± 24; sepiapterin 135 ± 12, 118 ± 14; biopterin 76 synthesize . ±10, 68 ± 8 nanomoles/g soluble protein/hr, in rat and human, respectively) than in the ciliary body-iris and lens. Materials and Methods. Neopterin, sepiapterin, The light-sensitive pteridines may protect eye tissues against and biopterin were purchased from Dr. Schrick's the effects of sunlight in addition to their role in the laboratory, Switzerland. NADPH, Tris and Dowex- hydroxylation of aromatic amino acids. Invest Ophthalmol 50H+ were obtained from Sigma Chemical Company Vis Sci 26:768-770, 1985 (St. Louis, MO). (U-I4C)-GTP was procured from Amersham (Arlington Heights, IL). All other reagents Yellow and red eye pigments of Drosophila mela- used were of analytic grade. Albino rats weighing nogaster were believed to be due to the presence of 200-300 g body weight were used. Human eyes were pteridines, sepiapterin, and drosopterin, respectively.1 supplied by the Connecticut Eye Bank & Visual Studies on fluorescent compounds in the mammalian Research Foundation, Inc. (New Britain, CT). Ciliary ocular lens have suggested the occurrence of glucoside body-iris, lens, and retina were dissected out carefully of 3-hydroxykynurenine,2 /3-carbolines,3 and pteridine- from the eyeballs. Tissues were homogenized in ho- like substances.4'5 Protective role of pteridines against mogenizing medium consisting of 10 mM Tris-HCl light-induced effects has been proposed in lens and buffer-40 mM KC1 (pH 8.0) using Potter-Elvehjem retina,4 however, evidence on the presence of these homogenizer. The homogenates were centrifuged at compounds in ocular tissues is lacking. Since the 17,000 X g for 1 hr in Sorvall R. C. 2B centrifuge at discovery of , a reduced form of 0°C, and the supernatants were used for pteridine , as a natural cofactor for aromatic biosynthesis. All studies utilizing experimental animals amino acid hydroxylases,67 a number of reports on conformed with the ARVO Resolution on the Use of the biosynthesis of pteridines in the tissues of higher Animals in Research.

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The method for pteridine biosynthesis followed here is essentially that of Yoshioka et al9 with minor modifications. For neopterin and biopterin biosyn- thesis, reaction mixture (total 1.0 ml) containing 100 GTP PPP-O-H2C /umol Tris-HCl buffer (pH 7.2), 5 /umol MgCl2 • 6H2O, 14 NADPH, 0.2 Mmol (U- C)-GTP (SA 4.51 OH OH ), and 0.3 ml of the supernatant was incu- bated for 3 hr at 37°C in the dark. The reaction was -COOH

terminated by the addition of 150 /x\ of 20% (W/V) OH OH

TCA and centrifuged at 9,000 Xg for 30 min to HN C-CH2- O-PPP remove protein. To 300 MI of the clear supernatant, I H H2 neopterin-PPP 50 ^1 of 30 JUM each of neopterin and biopterin were H H added as internal carriers, and the entire mixture was n, Mg2+ 3 Pi treated with equal volume of iodine solution for 1 hr 0 0 0 (2 g KI and 1 g iodine in 100 ml of double-distilled ^N^_ " n HN^ water). Excessive iodine was destroyed by the addition C CH3 Compound 'x' of 1% (W/V) ascorbic acid. The mixture (pH 1.0) H2N^N'I was then passed through 0.6 X 20 mm of Dowex-50 W(H+, 12X, 200-400 mesh) and washed in the in , NADPH column with double-distilled water. The water wash 0 0 H was discarded. Pteridines were eluted with 3.0 ml of Sepiapterin 1.0 M NH4OH, and the eluate was evaporated to 0H dryness under nitrogen. The residue was dissolved in known amount of water. Ten microliters were then spotted on Whatman No. 1 paper and developed in Inr, NADPH O H H the following solvent systems: (1) 3% NH4C1; (2) 1-

propanol: 1% NH4OH (2:1); (3) l-propanol:ethyl OH OH biopterin acetate:water (7:1:2), and (4) l-butanol:acetic acid: water (4:1:2). After identification of the spots under Fig. 1. A proposed pathway of dihydrobiopterin biosynthesis. I: UV light, they were cut and placed in scintillant vials GTP cyclohydrolase; II: sepiapterin-synthesizing enzyme 1; III: containing 10 ml of scintillant fluid, aqualyte. The sepiapterin-synthesizing enzyme 2; IV: sepiapterin reductase. radioactivity was measured in Nuclear-Chicago Mark II liquid scintillation system. For the estimation of synthesizing activity was found high in retina as sepiapterin formed, the reaction mixture as described compared with ciliary body-iris and lens (Table 1). above was incubated for 3 hr in the dark at 37°C, The pteridine-synthesizing activity in rat and human and the reaction was terminated by the addition of 4 lenses was only 14% and 21%, respectively, of that of volumes of ice-cold ethanol. The protein was removed retina. The ciliary body-iris and retina of rat possess by centrifugation, and the supernatant was evaporated to dryness. The residue was dissolved in known the high activity of neopterin, sepiapterin, and bio- amount of distilled water. Ten microliters of this was synthesis as compared with the ciliary body- spotted on Whatman No. 1 paper and developed in Table 1. Neopterin-, sepiapterin-, and biopterin- different solvent systems as mentioned above with synthesizing activities in ocular tissues carrier sepiapterin. The rest of the procedure is the (nanomoles/g soluble protein/hr) same as above. Neopterin Sepiapterin Biopterin Protein content was determined according to Lowry et al10 with the use of bovine serum albumin as Ciliary body-iris standards. Rat 21A ± 16 (6) 91 ± 15 (6) 53 ± 6(6) Human 182 ± 31 (5) 34 ± 3 (5) 38 ± 4(5) Results and Discussion. From our results it is clear Lens that the ciliary body-iris, lens, and retina possess the Rat 20 ± 5 (6) 24 ± 4 (6) 30 ± 6(6) Human 45 ± 12 (5) 22 ± 3 (5) 27 ± 4(5) biosynthetic pathway to synthesize pteridines. The Retina pteridine-synthesizing activity in the ocular tissues Rat 422 ± 27 (6) 135 ± 12(6) 76 ± 10(6) was found linear with respect to the duration of the Human 260 ± 24 (5) 118 ± 14(5) 68 ± 8(5)

incubation time (1-4 hr) and to the protein concen- Parentheses indicate number of determinations. tration (0.5-5.0 mg) in the assay system. The pteridine- Values are mean ±:SE.

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Table 2. Biopterin-synthesizing activity in rat lens tryptophan hydroxylase activities, ie, the initial en- and retina extracts in various incubation conditions zymes in the production of catecholamines and se- (nanomoles/g soluble protein/hr) rotonin, respectively. Lens is devoid of nerve supply and, as such, the existence of tyrosine and tryptophan Incubation medium Lens Retina hydroxylase activities in lens is doubtful. The other possibility is that the conversion of to Complete system 26.48 ± 4.16 (5) 84.62 ± 12.47 (4) tyrosine may be beneficial in the lens, and, hence, -NADPH 8.05 ± 2.24 (5) 22.14 ± 4.35 (4) the pteridines in lens may serve as cofactor for -MgCI2-6H2O 10.36 ±2.68 (5) 28.10 ± 3.80(4) -(U-MC)-GTP ND ND phenylalanine hydroxylase activity. The sepiapterin- Boiled homogenate ND ND and biopterin-synthesizing activities in rat retina were Parentheses indicate number of determinations. 30 and 120% higher than the values of rat brain Values are mean ± SE. reported by Yoshioka et al.9 The ability of ciliary ND: not detectable. body-iris to synthesize pteridines is the same as found in rat brain by Yoshioka et al.9 The higher activity iris and retina of human (Table 1). Control experi- of pteridine synthesis in retina as compared with that ments run without GTP or supernatant of the tissue of brain deserves further studies on these compounds homogenates failed to generate neopterin, sepiapterin, to understand their physiologic function in ocular and biopterin. Addition of boiled homogenate to the tissues. assay mixture did not produce pteridines. In the Key words: Neopterin, pteridine, sepiapterin, lens, retina, absence of either NADPH or magnesium chloride ciliary body-iris, rat, human from the incubation mixture, the biopterin synthesis was depleted by 60 to 70% in lens and retina of rat From the Eye Biochemistry Laboratories, Department of Oph- (Table 2). The remaining biopterin-synthesizing ac- thalmology, Cornell University Medical College, New York, New York. Submitted for publication: August 8, 1984. Reprint requests: tivity in the lens and retina of rat in absence of either Dr. Gadiparthi N. Rao, Department of Medical Microbiology and NADPH or magnesium chloride from the incubation Immunology, Albert B. Chandler Medical Center, University of mixture could be due to the tissue levels of the latter Kentucky, Lexington, KY 40536-0084. compounds. Further, in the absence of (U-14C)-GTP in the assay mixture, no radioactivity was observed References in neopterin, sepiapterin, or biopterin. From these 1. Taira T: The metabolism of sepiapterin in Drosophila mela- results it is evident that GTP is the precursor for nogaster; emphasizing its tetrahydroform. Jpn J Genet 36:244, pteridine biosynthesis and that the pathway is 1961. NADPH and magnesium ion-dependent. 2. Van Heyningen R: Fluorescent glucoside in the human lens. Nature 230:393, 1971. Sepiapterin is a naturally occurring yellow pteridine 3. Dillon J, Spector A, and Nakanishi K: Identification of j8- eye pigment in Drosophila melanogaster and was carbolines isolated from fluorescent human lens proteins. Nature isolated from the same by Forrest and Mitchell" for 259:422, 1976. the first time. These pteridines in insect eye were 4. Cremer-Bartels G: A light sensitive fluorescent substance in thought to provide light protective pigmentation.12 bovine and rabbit lenses. Exp Eye Res 1:443, 1962. 5. Cooper GF and Robson JG: The yellow colour of the lens of Cremer-Bartels isolated a light-sensitive fluorescent the grey squirrel. J Physiol 203:403, 1969. substance from the rabbit and bovine lenses and 6. Kaufman S: The structure of the phenylalanine hydroxylation thought that it may be pteridine.4 Cremer-Bartels cofactor. Proc Natl Acad Sci USA 50:1085, 1963. further suggested that these pteridine-like substances 7. Nagatsu T, Levitt M, and Udenfriend S: Tyrosine hydroxylase: may have a role in the protection of these tissues the initial step in norephinephrine biosynthesis. J Biol Chem 239:2910, 1964. from the light-induced effects. Though it is evident 8. Tanaka K, Akino M, Hagi Y, Doi M, and Shiota T: The from the present study that ocular tissues synthesize enzymatic synthesis of sepiapterin by chicken kidney prepara- pteridines, the role of these substances in the mam- tions. J Biol Chem 256:2963, 1981. malian eye tissues towards pigment formation is not 9. Yoshioka S, Masada M, Yoshida T, Inoue K, Mizokami T, yet known. It may be mentioned that neopterin, and Akino M: Synthesis of biopterin from dihydroneopterin triphosphate by rat tisues. Biochim Biophys Acta 756:279, sepiapterin, and biopterin are intermediates towards 1983. the synthesis of tetrahydrobiopterin, a natural cofactor 10. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ: Protein for aromatic amino acid hydroxylation reactions. The measurement with the Folin phenol reagent. J Biol Chem 193: cofactor activity of tetrahydrobiopterin, a reduced 265, 1951. form of dihydrobiopterin, in the hydroxylation of 11. Forest HS and Mitchell HK: Pteridines from Drosophila. II. 67 Structure of the yellow pigment. J Am Chem Soc 76:5656, aromatic amino acids is well-established. The syn- 1954. thesis of pteridines in retina and ciliary body-iris 12. Ziegler J and Harmsen R: The biology of pteridines in insects. may provide a pteridine cofactor for tyrosine and Adv Insect Physiol 6:139, 1969.

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