- Email: [email protected]
Hypoxanthine and xanthine levels in human aqueous humor from cataractous eyes
Life Sciences, Vol. 59, Nos 25f26, pp. PL 387-390.19% CopyrightQ IS96 Elticr Science Inc. Printed in the USA. All rights nwrwd m4-32m~% SlS.00 + .M)
PI1 SOOZ4-3205(96)00579-6
ELSEVIER
Ph%RMACOLOGY LElTER.3 Accelemted Communication
HYPOXANTHINE
LEVELS IN HUMAN AQUEOUS FROM CATARACTOUS EYES
AND XANTHINE
G.Spoto, L.Mastropasqua,
F.Gizzi, A.Arduini, P.Del Gallo, M.Ciancaglini, A.Giacomello.
Institutes of Biochemical
Science , Ophthalmology, Medical Physiophathology, “G. D’Annunzio” 66013 Chieti - Italy
HUMOR
P.E.Gallenga,
University
(Submitted April 30, 1996, accepted May 14, 1996, received in final form October
8, 1996)
Abstract: Purine nucleotide degradation products have been determined by HPLC in aqueous humor obtained during cataract surgery and from plasma of 22 patients (12 women). Uric acid, cytosine, guanosine monophosphate, uracyl, guanine, adenosine, adenosine monophosphate, thyminc. adenine, inosine, cyclic guanosine monophosphate, hypoxanthine and xanthine were evaluated. Uric acid and the last two were the only compounds detectable in measurable amounts in aqueous humor and in plasma of all patients. Aqueous humor xanthine levels were not significantly different from plasma; aqueous humor hypoxanthine concentrations were lower than those of xanthine and then plasma oxypurine levels. In 8 patients, treated with allopurinol, oxypurinol concentrations in aqueous humor and in plasma were comparable suggesting that oxypurines are transported through the bloodaqueous humor barrier. Key Wwd.s: hypoxanthine,
xanthine, cataracts,
aqueous humor
Introduction Few studies on purine metabolism in the human eye have been carried out. Elevated levels of hypoxanthine in vitreous humor from victims of infant sudden death syndrome have been reportled (1) and high uric acid concentrations in human aqueous humor of some glaucomatous eyes have been described (2). However data concerning the levels of nucleobases and nucleosides in human aqueaus humor are lacking. Purpose of the present paper was to determine the main purine compounds present in human aqueous humor from cataractous eyes and to compare their concentrations with those of plasma; the distribution of oxypurinol across the blood-aqueous barrier, after allopurinol administration, has been also studied.
Patients: The study population consisted of 22 subjects (12 women). admitted at the Division of Ophthalmology of the University of Chieti for cataract extraction. Eight (8) patientsl(4 males) were subjected to oral allopurinol(300 mg/day) for 3 days before ocular surgery. Local ethical committee approval was obtained and patients gave their informed consent to participate in the study. Venous blood and aqueous humor were obtained at the time of surgery. Specimens were centrifuged within 15 min after collection, and plasma was used for compound determinations. Materials: Purine and pyrimidine compounds were purchased Mannheim or Sigma. All other reagents were of analytical grade from Merck.
from Boehringer-
Cor=pondence: Dr. Giuseppe Spoto, Institute of Biochemical Sciences, University “Gabhele D’ Annunnzio”, via dei Vestini 31- 66013 Chieti, Italy. Fax: +39 871 355356; E-mail: [email protected]
Vol. 59, No.s 25/26,1996
Pa&e in Human Aqueous Humor
PL-388
Extraction procedure: Aqueous humor and plasma samples were deproteinized by adding 8 M perchloric acid at l/20 of the sample volume. After mixing, the samples were kept in crushed ice for 20 min and then centrifuged (9000xg) for 5 minutes at room temperature. The excess pemhloric acid was cold precipitated as potassium perchlorate by adding a mixture containing 1 mol of potassium hydroxide and 4 M K2HP04 (this adjusted the pH range of the sample to 6.0-6.7). The neutralized extract was again centrifuged and filtered through a nylon-66 filter, 0.22 pm (Rainin corporation). The clear filtrate obtained was used directly for HPLC assay or stored at -80 “C. Chromatographic apparatus. HPLC system was from Beckman and consisted in a two 11OA pumps, a variable wavelength spectrophotometer Spectroflow 783 (Kratos Analytical) measuring at 254 nm and an autosampler Promis (Spark Holland). Chromatographic conditions. Purine and pyrimidine bases and nucleosides determinations were done as previously described (3). The column used was a 5ym Spherisorb S5 0DS2, (250 x 4 mm) with a pre-column. The mobile phase employed consisted in 47 mM KH2PO4 pH 4.5. The flow rate was 0.7 ml/mm; detection was performed at 254 nm. Peak identities were confirmed by coelution with standards and by disappearance of the peak after incubation with the appropriate enzymes (4). Quantitative comparisons were carried out using standard solutions of known concentrations. Statistical Analysis. Statistical analysis was conducted using version 5 of the Systat software, (Systat Inc, Evanston,
IL).
Results Uric acid, cytosine, guanosine monophosphate, uracyl, guanine, adenosine, adenosine monophosphate, thymine, adenine, inosine, cyclic guanosine monophosphate, hypoxanthine and xanthine were evaluated. Uric acid and the last two were the only compounds detectable in measurable amounts in aqueous humor and in plasma of all patients. In subjects treated with allopurinol, oxypurinol was also assayable, while allopurinol was found in very small amounts in both fluids. The means (SD) of the measured variables are presented in the Table. TABLE. Means (SD) of the measured variables in patients treated and untreated with allopurinol Patients untreated with allopurinol
Patients treated with allopurinol
Variable
(n=14)
(n=8)
Age (years)
69.78 (10.05)
68.62 (9.41)
Plasma hypoxanthine (pmol/L)
4.43 (2.54)
6.26 (4.02)
Plasma xanthine (prnol/L)
3.77 (2.76)
2.19 (1.28)
Plasma oxypurinol
--_______-
33.54 (8.00)
1.36 (1.03)
0.78 (0.57)
4.16 (2.96)
1.82 (1.31)
(pmol/L)
Aqueous humor hypoxanthine
(pmol/L)
Aqueous humor xanthine (pmohL> Aqueous humor oxypurinol
(pmol/L)
-__________-
30.68 (6.86)
Vol. 59, No.s 25126, 1996
Purine in Human Aqueous Humor
PL389
Since there were no gender differences in mean values, subjects of both sexes were grouped together. In both allopurinol treated and untreated patients, aqueous humor hypoxanthine levels were significantly lower than those of xanthine and of plasma purine bases. Aqueous humor xanthine levels were significantly (p=O.O48) higher in untreated patients than in allopurinol treated. In the latter group, oxypurinol levels were higher than those of the other purine bases; moreover allopurinol concentration was not significantly different in the two fluids.
Discussion Uric acid has been detected in human aqueous humor obtained during glaucoma or cataract surgery (2). The present paper shows that hypoxanthine and xanthine are purine nucleotide degradation products present in detectable amounts in the human aqueous humor from cataractous eyes. Aqueous humor xanthine levels were not significantly different from those of plasma; aqueous humor hypoxanthine concentrations were lower than those of xanthine and than plasma oxypurines. Although mean values for normal hypoxanthine and xanthine plasma levels reported in literature vary widely (5), those obtained in the present study are in the high range of the values observed when blood samples were centrifuged immediately after collection (5-7). In order to examine the transport of oxypurines through the blood-aqueous barrier, allopurinol was administered. Allopurinol represents a competitive inhibitor of xanthine oxidase and along with its major matabolite and oxidation product, oxypurinol, it produces pseudoirreversible inactivation of the enzyme (8). Furthermore this result confirms the previous finding that oxypurinol is freely diffusible through a dialysis membrane and not protein-bound (9). Allopurinol and oxypurinol are structural analogues of hypoxanthine and xanthine, respectively (10). Since the half-clearance time of oxypurinol is far more prolongued than that of allopurinol(1 l), oxypurinol was the only drug present in plasma in assayable amounts. In patients treated with allopurinol, oxypurinol was also detectable in aqueous humor. where its levels were comparable with those of plasma, suggesting that oxypurines are transported through the blood-aqueous humor barrier. Although patients were treated with allopurinol for only three days leading to a non significant increase in plasma oxypurine levels, the lower xanthine levels observed in aqueous humor of these patients as compared to untreated ones suggests the absence of xanthine oxidase activity in the human eye. Before conversion into uric acid by xanthine oxidase, the end products of purine nucleotide degradation are hypoxanthine for adenine nucleotides and xanthine for guanine nucleotides (12). The higher aqueous humor xanthine concentration over that of hypoxanthine could indicate a predominance of guanine nucleotide catabolism over adenine nucleotide breakdown in the cataractous eye. However this finding could be also attributed to a high salvage of hypoxanthine through the hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzed reaction (13,14). Although HPRT has been observed in all cells, the highest activity seems to be present in the human brain (15,16). In conclusion oxypurines are transported trough the bloodaqueous humor barrier and the ratio between aqueous humor and plasma concentrations of hypoxanthine is significantly lower than that of xanthine, suggesting that, at least in cataractous eye, there is either a predominance of guanine nucleotide catabolism or an increased salvage of hypoxanthine.
Acknowledeements Thanks are due to Dr. Franca Daniele, MD for her help in preparing the manuscript. References 1. 2. 3. 4. 5, 6. 7.
T.O.ROGNUM, O.D.SAUGSTAD, S.OYASAETER, B.OLAISEN, Pediatrics 82 615-618 (1988) k.W~I..AM, K.M.LIU, R.W, YEE, P.LEE, Current Eye Research 2 645-646 (198211983) G.P.J.M.GERRITS, A.A.M.HAAGEN, R.A. DE ABREU, et al. clin. Chem. 3 1439-1457 (1988) H.A. SIMMONDS & R.A. HARKNESS, J . Chrom. XZ 369-381 (1981) R. BOULIEU, C. BORY, P. BALTASSAT, C. GONNET, Anal. Biochem.129 398-404 (1983) J. G.PUIG, F.A.MATEOS, M.E.MIRANDA, R.J.TORRES, E.DE MIGUEL, C.P.DE AYALA, A. GIL, Adv. Exp. Med. Biol. 124 69-72 (1994). R.A. HARKNESS. GM. MC CREANOR ,D. SIMPSON , IR. MAC FADYEN , J. Inherited
PL-390
Purine in Human Aqueous Humor
Vol. 59, No.s 25/26, 1996
Metab. fj 407-410 (1986) R.W. RUNDLES, Arch. Intern. Med. 145 1492-1405 (1985) G.B.ELION, A. KOVENSKY, G.H. HITCHINGS, L. METZ, R.W. RUNDLES, Biochem. Pharmaco1.u 863-880 (1966) 10. G.B. ELION, Ann. Rheum. Dis. 25 608-614 (1966) 11. G.B ELION, F.M. BENEZRA, T.D BEARDMORE, W.N. KELLEY, Adv.Exp.Med. Biol. 122A 263-267 (1980) 12. M.A. BECKER, B. J. ROESSLER . The Metabolic and Molecular Bases of Inherited rsease II, C.R.SCRIVER A.L.BEAUDET W.S.SLY D.VALLE (Eds), 1655-1677 (1995) 13. f GIACOMELLO C SAL&NO J Biol Ciem 253 8038-6044 (1978) 14. c: SALERNO, A. &ACOMELL~; J:Biol: Chek. uh 3671-3673 (1981). 15. F.M. ROSENBLOOM, W.N. KELLEY, J.MILLER, J.F. HENDERSON, J.E.SEEGMILLER, J.A.M.A. 2112175-177 (1967) 16.W.N.KELLEY, M.L.GREENE, F.M.ROSENBLOOM et al., Ann.Intern.Med. a 155-206 (1969)
8. 9.
PI1 SOOZ4-3205(96)00579-6
ELSEVIER
Ph%RMACOLOGY LElTER.3 Accelemted Communication
HYPOXANTHINE
LEVELS IN HUMAN AQUEOUS FROM CATARACTOUS EYES
AND XANTHINE
G.Spoto, L.Mastropasqua,
F.Gizzi, A.Arduini, P.Del Gallo, M.Ciancaglini, A.Giacomello.
Institutes of Biochemical
Science , Ophthalmology, Medical Physiophathology, “G. D’Annunzio” 66013 Chieti - Italy
HUMOR
P.E.Gallenga,
University
(Submitted April 30, 1996, accepted May 14, 1996, received in final form October
8, 1996)
Abstract: Purine nucleotide degradation products have been determined by HPLC in aqueous humor obtained during cataract surgery and from plasma of 22 patients (12 women). Uric acid, cytosine, guanosine monophosphate, uracyl, guanine, adenosine, adenosine monophosphate, thyminc. adenine, inosine, cyclic guanosine monophosphate, hypoxanthine and xanthine were evaluated. Uric acid and the last two were the only compounds detectable in measurable amounts in aqueous humor and in plasma of all patients. Aqueous humor xanthine levels were not significantly different from plasma; aqueous humor hypoxanthine concentrations were lower than those of xanthine and then plasma oxypurine levels. In 8 patients, treated with allopurinol, oxypurinol concentrations in aqueous humor and in plasma were comparable suggesting that oxypurines are transported through the bloodaqueous humor barrier. Key Wwd.s: hypoxanthine,
xanthine, cataracts,
aqueous humor
Introduction Few studies on purine metabolism in the human eye have been carried out. Elevated levels of hypoxanthine in vitreous humor from victims of infant sudden death syndrome have been reportled (1) and high uric acid concentrations in human aqueous humor of some glaucomatous eyes have been described (2). However data concerning the levels of nucleobases and nucleosides in human aqueaus humor are lacking. Purpose of the present paper was to determine the main purine compounds present in human aqueous humor from cataractous eyes and to compare their concentrations with those of plasma; the distribution of oxypurinol across the blood-aqueous barrier, after allopurinol administration, has been also studied.
Patients: The study population consisted of 22 subjects (12 women). admitted at the Division of Ophthalmology of the University of Chieti for cataract extraction. Eight (8) patientsl(4 males) were subjected to oral allopurinol(300 mg/day) for 3 days before ocular surgery. Local ethical committee approval was obtained and patients gave their informed consent to participate in the study. Venous blood and aqueous humor were obtained at the time of surgery. Specimens were centrifuged within 15 min after collection, and plasma was used for compound determinations. Materials: Purine and pyrimidine compounds were purchased Mannheim or Sigma. All other reagents were of analytical grade from Merck.
from Boehringer-
Cor=pondence: Dr. Giuseppe Spoto, Institute of Biochemical Sciences, University “Gabhele D’ Annunnzio”, via dei Vestini 31- 66013 Chieti, Italy. Fax: +39 871 355356; E-mail: [email protected]
Vol. 59, No.s 25/26,1996
Pa&e in Human Aqueous Humor
PL-388
Extraction procedure: Aqueous humor and plasma samples were deproteinized by adding 8 M perchloric acid at l/20 of the sample volume. After mixing, the samples were kept in crushed ice for 20 min and then centrifuged (9000xg) for 5 minutes at room temperature. The excess pemhloric acid was cold precipitated as potassium perchlorate by adding a mixture containing 1 mol of potassium hydroxide and 4 M K2HP04 (this adjusted the pH range of the sample to 6.0-6.7). The neutralized extract was again centrifuged and filtered through a nylon-66 filter, 0.22 pm (Rainin corporation). The clear filtrate obtained was used directly for HPLC assay or stored at -80 “C. Chromatographic apparatus. HPLC system was from Beckman and consisted in a two 11OA pumps, a variable wavelength spectrophotometer Spectroflow 783 (Kratos Analytical) measuring at 254 nm and an autosampler Promis (Spark Holland). Chromatographic conditions. Purine and pyrimidine bases and nucleosides determinations were done as previously described (3). The column used was a 5ym Spherisorb S5 0DS2, (250 x 4 mm) with a pre-column. The mobile phase employed consisted in 47 mM KH2PO4 pH 4.5. The flow rate was 0.7 ml/mm; detection was performed at 254 nm. Peak identities were confirmed by coelution with standards and by disappearance of the peak after incubation with the appropriate enzymes (4). Quantitative comparisons were carried out using standard solutions of known concentrations. Statistical Analysis. Statistical analysis was conducted using version 5 of the Systat software, (Systat Inc, Evanston,
IL).
Results Uric acid, cytosine, guanosine monophosphate, uracyl, guanine, adenosine, adenosine monophosphate, thymine, adenine, inosine, cyclic guanosine monophosphate, hypoxanthine and xanthine were evaluated. Uric acid and the last two were the only compounds detectable in measurable amounts in aqueous humor and in plasma of all patients. In subjects treated with allopurinol, oxypurinol was also assayable, while allopurinol was found in very small amounts in both fluids. The means (SD) of the measured variables are presented in the Table. TABLE. Means (SD) of the measured variables in patients treated and untreated with allopurinol Patients untreated with allopurinol
Patients treated with allopurinol
Variable
(n=14)
(n=8)
Age (years)
69.78 (10.05)
68.62 (9.41)
Plasma hypoxanthine (pmol/L)
4.43 (2.54)
6.26 (4.02)
Plasma xanthine (prnol/L)
3.77 (2.76)
2.19 (1.28)
Plasma oxypurinol
--_______-
33.54 (8.00)
1.36 (1.03)
0.78 (0.57)
4.16 (2.96)
1.82 (1.31)
(pmol/L)
Aqueous humor hypoxanthine
(pmol/L)
Aqueous humor xanthine (pmohL> Aqueous humor oxypurinol
(pmol/L)
-__________-
30.68 (6.86)
Vol. 59, No.s 25126, 1996
Purine in Human Aqueous Humor
PL389
Since there were no gender differences in mean values, subjects of both sexes were grouped together. In both allopurinol treated and untreated patients, aqueous humor hypoxanthine levels were significantly lower than those of xanthine and of plasma purine bases. Aqueous humor xanthine levels were significantly (p=O.O48) higher in untreated patients than in allopurinol treated. In the latter group, oxypurinol levels were higher than those of the other purine bases; moreover allopurinol concentration was not significantly different in the two fluids.
Discussion Uric acid has been detected in human aqueous humor obtained during glaucoma or cataract surgery (2). The present paper shows that hypoxanthine and xanthine are purine nucleotide degradation products present in detectable amounts in the human aqueous humor from cataractous eyes. Aqueous humor xanthine levels were not significantly different from those of plasma; aqueous humor hypoxanthine concentrations were lower than those of xanthine and than plasma oxypurines. Although mean values for normal hypoxanthine and xanthine plasma levels reported in literature vary widely (5), those obtained in the present study are in the high range of the values observed when blood samples were centrifuged immediately after collection (5-7). In order to examine the transport of oxypurines through the blood-aqueous barrier, allopurinol was administered. Allopurinol represents a competitive inhibitor of xanthine oxidase and along with its major matabolite and oxidation product, oxypurinol, it produces pseudoirreversible inactivation of the enzyme (8). Furthermore this result confirms the previous finding that oxypurinol is freely diffusible through a dialysis membrane and not protein-bound (9). Allopurinol and oxypurinol are structural analogues of hypoxanthine and xanthine, respectively (10). Since the half-clearance time of oxypurinol is far more prolongued than that of allopurinol(1 l), oxypurinol was the only drug present in plasma in assayable amounts. In patients treated with allopurinol, oxypurinol was also detectable in aqueous humor. where its levels were comparable with those of plasma, suggesting that oxypurines are transported through the blood-aqueous humor barrier. Although patients were treated with allopurinol for only three days leading to a non significant increase in plasma oxypurine levels, the lower xanthine levels observed in aqueous humor of these patients as compared to untreated ones suggests the absence of xanthine oxidase activity in the human eye. Before conversion into uric acid by xanthine oxidase, the end products of purine nucleotide degradation are hypoxanthine for adenine nucleotides and xanthine for guanine nucleotides (12). The higher aqueous humor xanthine concentration over that of hypoxanthine could indicate a predominance of guanine nucleotide catabolism over adenine nucleotide breakdown in the cataractous eye. However this finding could be also attributed to a high salvage of hypoxanthine through the hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzed reaction (13,14). Although HPRT has been observed in all cells, the highest activity seems to be present in the human brain (15,16). In conclusion oxypurines are transported trough the bloodaqueous humor barrier and the ratio between aqueous humor and plasma concentrations of hypoxanthine is significantly lower than that of xanthine, suggesting that, at least in cataractous eye, there is either a predominance of guanine nucleotide catabolism or an increased salvage of hypoxanthine.
Acknowledeements Thanks are due to Dr. Franca Daniele, MD for her help in preparing the manuscript. References 1. 2. 3. 4. 5, 6. 7.
T.O.ROGNUM, O.D.SAUGSTAD, S.OYASAETER, B.OLAISEN, Pediatrics 82 615-618 (1988) k.W~I..AM, K.M.LIU, R.W, YEE, P.LEE, Current Eye Research 2 645-646 (198211983) G.P.J.M.GERRITS, A.A.M.HAAGEN, R.A. DE ABREU, et al. clin. Chem. 3 1439-1457 (1988) H.A. SIMMONDS & R.A. HARKNESS, J . Chrom. XZ 369-381 (1981) R. BOULIEU, C. BORY, P. BALTASSAT, C. GONNET, Anal. Biochem.129 398-404 (1983) J. G.PUIG, F.A.MATEOS, M.E.MIRANDA, R.J.TORRES, E.DE MIGUEL, C.P.DE AYALA, A. GIL, Adv. Exp. Med. Biol. 124 69-72 (1994). R.A. HARKNESS. GM. MC CREANOR ,D. SIMPSON , IR. MAC FADYEN , J. Inherited
PL-390
Purine in Human Aqueous Humor
Vol. 59, No.s 25/26, 1996
Metab. fj 407-410 (1986) R.W. RUNDLES, Arch. Intern. Med. 145 1492-1405 (1985) G.B.ELION, A. KOVENSKY, G.H. HITCHINGS, L. METZ, R.W. RUNDLES, Biochem. Pharmaco1.u 863-880 (1966) 10. G.B. ELION, Ann. Rheum. Dis. 25 608-614 (1966) 11. G.B ELION, F.M. BENEZRA, T.D BEARDMORE, W.N. KELLEY, Adv.Exp.Med. Biol. 122A 263-267 (1980) 12. M.A. BECKER, B. J. ROESSLER . The Metabolic and Molecular Bases of Inherited rsease II, C.R.SCRIVER A.L.BEAUDET W.S.SLY D.VALLE (Eds), 1655-1677 (1995) 13. f GIACOMELLO C SAL&NO J Biol Ciem 253 8038-6044 (1978) 14. c: SALERNO, A. &ACOMELL~; J:Biol: Chek. uh 3671-3673 (1981). 15. F.M. ROSENBLOOM, W.N. KELLEY, J.MILLER, J.F. HENDERSON, J.E.SEEGMILLER, J.A.M.A. 2112175-177 (1967) 16.W.N.KELLEY, M.L.GREENE, F.M.ROSENBLOOM et al., Ann.Intern.Med. a 155-206 (1969)
8. 9.