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Uric acid and allantoin levels in Down syndrome: antioxidant and oxidative stress mechanisms?
Clinica Chimica Acta 341 (2004) 139 – 146 www.elsevier.com/locate/clinchim
Uric acid and allantoin levels in Down syndrome: antioxidant and oxidative stress mechanisms? Ingrid Zˇitnˇanova´ a,*, Peter Koryta´r a, Okezie I. Aruoma b, Ma´ria Sˇustrova´ c, Iveta Garaiova´ a, Jana Muchova´ a, Tere´zia Kalnovicˇova´ a, ˇ uracˇkova´ a Siegfried Pueschel d, Zdenˇka D a
Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava, Slovak Republic b Department of Neuroinflammation, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Charring Cross Hospital Campus, Fulham Palace Road, London W6 8RF, England, UK c Institute of Preventive and Clinical Medicine, Slovak Centre for Down Syndrome, Bratislava, Slovakia d Child Development Center, Rhode Island Hospital, Brown University School of Medicine, Providence, RI 02903, USA Received 28 July 2003; received in revised form 13 November 2003; accepted 24 November 2003
Abstract Background: Down syndrome (DS) is a chromosomal abnormality (trisomy 21) leading to mental retardation, to the characteristic change of individual’s phenotype and to the pathological features of Alzheimer disease. Patients with DS have elevated ratio of superoxide dismutase to (catalase plus glutathione peroxidase) with respect to controls in all age categories suggesting that oxidative imbalance contributes to the clinical manifestation of accelerated aging. Results: We report that persons with DS have elevated uric acid levels compared with controls, 348.56 F 22.78 versus 284.00 F 20.86 Amol/ l ( p = 0.018). The levels of hypoxanthine and xanthine in DS children (6.35 F 0.31 and 1.02 F 0.23 Amol/l) were significantly lower than in controls (7.83 F 0.59 and 2.43 F 0.66 Amol/l). This result suggests increased conversion of hypoxanthine and xanthine to uric acid with subsequent free radical-dependent oxidation of uric acid to allantoin, mechanisms potentiated by the oxidative stress in DS. Allantoin is a nonenzymatic oxidative product of uric acid in human. In DS individuals, the levels of allantoin were significantly higher than those in healthy controls (18.58 F 2.27 and 14.07 F 1.07 Amol/l, respectively, p = 0.03). Conclusions: Our data supported the presumption of increased oxidative stress in DS. D 2004 Elsevier B.V. All rights reserved. Keywords: Down syndrome; Oxidative stress; Purine metabolism; Uric acid; Allantoin
1. Introduction Down syndrome (DS) is a chromosomal abnormality (trisomy 21) leading to mental retardation, to the * Corresponding author. Tel.: +421-7-59357-414; fax: +421-759357-557. E-mail address: [email protected] (I. Zˇitnˇanova´). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.11.020
characteristic change of individual’s phenotype and to the pathological features of Alzheimer disease [1 –3]. Chronic oxidative stress has been implicated in patients with DS [4 –6]. In the early 1980s, it was reported that DS and gout could coexist in DS patients [7]. The latter disease is characterised by increased plasma concentration of uric acid. Uric acid in the human body was traditionally considered to be a
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soluble end product of purine metabolism without any physiological value. On the one hand, higher concentrations of uric acid can form urate crystals which can
be deposited in tissues or joints where they may lead to inflammation process and prooxidant effects. On the other hand, uric acid is regarded to be an important
Table 1 Allantoin levels in tissues and body fluids of patients and healthy controls found in different laboratories Grootveld and Halliwell [16]
Sample type
Allantoin (AM) F S.D.
Alla/UA (%)
Controls Rheumatoid patients
Serum Serum Synovial fluid
18.6 F 3.8 36.1 F 6.3 20.9 F 7.3
4.7 F 1.6 10.5 F 3.8 11.8 F 5.6
Arterial blood Venous blood
Allantoin + allantoate 9.3 F 1.1 8.7 F 0.9
Serum
Allantoin (AM) 15.7 F 7.9
Becker [11]
Kock et al. [28]
Kock et al. [29] Healthy male adults Healthy female adults Patients with angina pectoris Patients with cerebral insult Patients with myocardial infarction Ogihara et al. [17] Controls Willson’s disease patients before therapy Willson’s disease patients after therapy Sasaki et al. [30] Hemodialysed patients Aruoma [18] Healthy male athletes before exercise Healthy male athletes after exercise Hellsten et al. [31] Healthy males before exercise Healthy males after exercise
Moison et al. [32] Preterm babies
Serum
Plasma
Plasma
Plasma
Muscle Plasma Muscle Plasma
Allantoin (AM) 16.7 F 8.3 15.8 F 7.2 17.8 F 6.8 14.9 F 9.1 18.4 F 7.7 Allantoin (AM) 6.5 F 0.8 11.0 F 1.8 4.3 F 0.5 Allantoin (AM) 42.6 F 37.7 Allantoin (AM) 22.1 18.1
Plasma
Mikami et al. [19] Healthy controls Athletes
Serum
Alla/UA (%) 6.2 4.2
Allantoin 0.003 F 0.007 Amol/g 11.9 F 2.6 Amol/l 0.1 F 0.014 Amol/g Doubled Alla/UA (%) 4.7 F 2.5 147 F 186
Plasma Aspirates
Ogihara et al. [33] Chronic lung disease babies (CLD) Non-CLD babies
Alla/UA (%) 2.3 F 0.4 15.9 F 8.6 1.5 F 0.3
Alla/UA (%) 41.2 F 15.8 11.7 F 9.9 Allantoin (Amol/l) 28 F 2.2 65 F 8.8
Alla/UA (%) 4.6 F 0.4 14.4 F 2.6
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antioxidant in human adult plasma. It can directly react with free radicals [8] and may interfere with Fenton’s chemistry by chelating catalytic metal ions [9– 11]. Although some studies reported elevated levels of uric acid in DS individuals [12,13], some speculations exist about the pathogenetic mechanisms leading to the increase in uric acid levels in DS patients. Accumulation of uric acid can also be a result of dysfunction in its renal excretion [13,14]. Puukka et al. [15] found that the levels of erythrocyte adenosine deaminase (AD) and adenine phosphoribosyl transferase activities correlated with increased plasma urate concentrations in DS individuals. AD is coded by a gene on chromosome 21, which is trisomic in patients with DS. The increased gene dose for AD can cause the increased activity of the enzyme, leading to the increased production of uric acid in children with DS. Another plausible explanation for impairment of purine metabolism in DS patients is the imbalance in the antioxidant enzyme defence system. The overexpression of the enzyme superoxide dismutase (SOD) (the gene for this enzyme is localised on the chromosome 21) can lead to increased oxidative stress [6] caused by increased ratio of SOD/(catalase + glutathione peroxidase). Human cells lack the enzyme uricase, which in some species can oxidise uric acid further. However, if uric acid is exposed to the system producing oxygen radical species, various oxidative products may be formed (allantoin, allantoate, glyoxylate, urea, oxalate) [16]. Allantoin is a nonenzymatic oxidative product of urate metabolism which can be measured by HPLC [14,16]. Measurement of uric acid and allantoin in tissues and body fluids (particularly in serum and synovial fluids) are largely advocated as markers of oxidative stress in vivo (Table 1). Increased levels of allantoin have been reported in Willson disease [17]. Exhaustive exercise can also lead to increased plasma allantoin concentration [18,19]. The aim of our study was to compare the levels of purine metabolism products (uric acid, hypoxanthine and xanthine) in plasma of DS patients with those of healthy controls and to examine levels of allantoin in both groups. If there is an increased free radical attack on urate
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in DS individuals, the logical product of this attack would be allantoin.
2. Material and methods 2.1. Patients For this study, we used blood of 16 children with DS (average age 10.06 F 1.04 years) and 16 agematched healthy controls (11.94 F 0.97). In all persons with DS, the diagnosis was confirmed by cytogenetic analysis. All patients in this study are regularly examined at the Center for Down syndrome at the Institute for Preventive and Clinical Medicine in Bratislava, Slovakia. These individuals did not have complicating medical conditions. They did not suffer from chronic diseases such as diabetes mellitus, acute respiratory diseases, renal insufficiency or any serious heart defects. Individuals suffering from mentioned diseases were excluded from our study. The studies were performed according to the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects or their guardians and the study was approved by The Ethics Committee of Derer’s University Hospital in Bratislava. 2.2. Sample preparation Human blood was collected into heparinized tubes. The plasma was obtained by blood centrifugation at 1500 g for 10 min and stored at 80 jC until required. Before analysis, plasma samples were thawed at room temperature and deproteinized with 1/25 volume of 4.6 mol/l HClO4 then centrifuged for 10 min at 7000 g and ultrafiltered through a 0.2-Am filter (Millipore). 2.3. Chemicals Allantoin, hypoxanthine, xanthine, uric acid were obtained from Sigma Chemical (St. Louis, MO, USA) and used for preparation of standard solutions. Methanol was purchased from Merck (Germany) and was of HPLC-grade. All other chemicals were purchased from Lachema (Brno, Czech Republic) and were of analytical grade.
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2.4. Preparation of the stock standards Allantoin was dissolved in deionized water. Hypoxanthine and xanthine were dissolved in 2 mmol/ l KOH and uric acid was dissolved in 2 mmol/l KOH followed by a short heating in a boiling water bath and by ultrasound. Calibration curves for hypoxanthine, xanthine and allantoin standards were constructed covering the range from 25 to 500 pmol. Uric acid was injected to produce a calibration curve from 0.5 to 100 nmol. The calibration was based on the areas calculated with CSW 1.7DLL (DataApex) software.
Fig. 2. Uric acid levels in plasma of patients with Down syndrome (DS) and healthy controls (C). Data are means F S.E.M. (n = 16). *Denotes p < 0.05 (significantly different).
2.5. Uric acid, hypoxanthine, xanthine analysis The HPLC was performed on a Watrex chromatograph system equipped with 100 4 mm guard column packed with Separon SGX C18, 7 Am (Watrex), 250 4 mm analytical column packed with Nucleosil 120-C18, 5 Am (Watrex). For the determination of uric acid, hypoxanthine and xanthine UV detection was performed at 254 nm (DeltaChrome UVD 200). A 100 Al of plasma ultrafiltrate was directly injected onto a column. The mobile phase was 0.05 mol/l potassium phosphate buffer, pH = 4.6 and 5% methanol at a flow rate of 0.8 ml/min. The quantification of uric acid, hypoxanthine and xanthine was based on the peak areas and compared with standards.
Fig. 1. Analytical steps in the allantoin determination. Full details in the text.
Fig. 3. Hypoxanthine (HX) and xanthine (X) levels in healthy controls (C) and in patients with DS. (DS) (n = 16 for HX and 13 for X). Data are means F S.E.M. *Denotes p < 0.05 (significantly different).
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Fig. 4. Allantoin levels in plasma of patients with DS (DS) and healthy controls (C). Data are means F S.E.M. (n = 16). *Denotes p < 0.05 (significantly different).
2.6. Determination of allantoin Allantoin analysis was carried out essentially as described by Grootveld and Halliwell [16] with a slight modification (Fig. 1). Plasma samples and standards were treated in the same way. A 100 Al of plasma ultrafiltrate was injected onto a C18 Nucleosil column. The mobile phase was 0.05 mol/ l potassium phosphate buffer, pH = 4.6 and 5% methanol at a flow rate of 0.8 ml/min, wavelength 254 nm. A fraction covering the retention time range (2.9 – 5.2 min) where allantoin elutes (but not uric acid) was collected and evaporated to dryness
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at 40 jC under vacuum. The residue was reconstituted in 100 Al of 0.12 mol/l NaOH and incubated for 20 min at 100 jC. After adding 100 A1 of 1 mol/l HCl and 10 Al of 3 mmol/l dinitrophenylhydrazine in 1 mol/l HCl, the heating at 100 jC continued for 5 min. After cooling the sample, 100 Al volume was injected onto a Nucleosil C18 column. The mobile phase was 55% (v/v) 30 mmol/l sodium citrate/27.7 mmol/l sodium acetate buffer, pH = 4.75 and 45% methanol at a flow rate of 0.8 ml/min. The detection was performed at the wavelength 360 nm. The plasma allantoin concentration was quantified by comparing the peak area of glyoxylate 2,4-dinitrophenylhydrazone with values from the standard curve. The recovery of three different concentrations of added allantoin to plasma averaged 94%. The mean coefficient of variation for analysis of nine replicate plasma samples was 4.8%. 2.7. Statistical analysis Data are expressed as means F S.E.M. Student’s unpaired t-test was used to compare the statistical differences of detected levels of uric acid, hypoxanthine, xanthine and allantoin in both experimental groups (DS and control). Differences among means were considered statistically significant by the criterion of probability value < 0.05.
Fig. 5. Correlation between uric acid levels and age in children with DS (DS) and controls (C). Correlation coefficient for patients with DS is r = 0.68 ( p = 0.01) and for controls r = 0.46 ( p>0.05).
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3. Results
4. Discussion
3.1. Uric acid, xanthine, hypoxanthine and allantoin levels in Down syndrome
Down syndrome is a disease with developmental brain abnormalities resulting in early mental and precocious, age-dependent Alzheimer-type neurodegeneration [3,23]. Trisomic cells are more sensitive to oxidative stress. This sensitivity may be due to the imbalance in the hydrogen peroxide metabolism or another unknown factor [6]. The cells tend to compensate this negative effect by increasing glutathione peroxidase (GPx) activity while that of catalase (CAT) remains unchanged. Thus, the ratio of SOD to (CAT + GPx) is important from the point of view of sensitivity to ROS, rather than in absolute quantities of individual antioxidants. There are specific synergic interactions between antioxidant enzymes and therefore a comprehensive understanding of the antioxidative system should be based on knowledge of activities and mutual interactions of enzymes, involved in free radical detoxification [6]. The peroxynitrite-dependent DNA damage involves a marked increase in the products (hypoxanthine and xanthine) arising from the deamination of guanine and adenine. Antioxidants such as ergothioneine inhibit the formation of hypoxanthine (a precursor of xanthine) and xanthine and this may have many implications for inflammatory conditions characterised by overproduction of uric acid (the oxidation product of xanthine) [22,24]. The conversion of hypoxanthine to xanthine
In this study, the levels of uric acid (UA), hypoxanthine (HX), xanthine (X) and allantoin (Alla) in plasma of children with DS (n = 16) and in age-matched healthy control group (n = 16) were determined. DS patients had significantly elevated levels of uric acid (348.35 F 22.78 Amol/l versus controls 284.00 F 20.86 Amol/l, p = 0.018) (Fig. 2). These results are consistent with previous reports [11,20,21]. The levels of both hypoxanthine and xanthine in children with DS were lower than those in controls (6.35 F 0.31 and 1.02 F 0.23 Amol/ for DS and 7.83 F 0.59 Amol/l, p = 0.018 and 2.43 F 0.66 Amol/l, p = 0.030 for controls). Xanthine levels in both experimental groups were lower than hypoxanthine in all sets (Fig. 3). Indeed, xanthine and hypoxanthine are products of free radical-dependent deamination of guanine and adenine [22]. Down syndrome patients had significantly higher allantoin levels (18.58 F 2.27 Amol/l) when compared with controls (14.07 F 1.07 Amol/l) ( p = 0.03) (Fig. 4). We determined the correlation between uric acid levels in plasma of DS individuals and the age of patients (Figs. 5 and 6).
Fig. 6. Correlation between allantoin and age levels in children with DS (DS) and in controls (C). Correlation coefficient for patients with DS is r = 0.52 ( p = 0.05) and for controls r = 0.23 ( p>0.05).
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and then xanthine to uric acid in a reaction catalysed by xanthine oxidase increases the burden of superoxide radical (O2S ) in vivo. Indeed, endogenously produced nitric oxide (NOS) will react with O2S to produce ONOO , a reaction that occurs with a high rate constant. ONOO may increase the oxidation of xanthine dehydrogenase to xanthine oxidase [25], an enzyme widely reported to be activated in oxidative burst [26] which uses hypoxanthine and xanthine as substrates [22]. Dismutation of the superoxide anion will produce hydrogen peroxide. We suggest that antioxidants that can modulate production of xanthine and hypoxanthine in vivo will modulate accumulation of uric acid in Down syndrome. The major finding of our work is a significant increase of uric acid and allantoin levels in plasma of children with DS. On the other hand, levels of hypoxanthine and xanthine were significantly lower than those in healthy individuals. Hyperuricemia in children with DS has been determined also by other authors [11] who attribute this increase to the elevated activity of erythrocyte adenosine deaminase and adenine phosphoribosyltransferase activities. Nishida et al. [14] suggest that glomerular dysfunction could also contribute to hyperuricemia in DS individuals. However, we determined significantly elevated levels of allantoin in plasma of children with DS which implicates the role of increased oxidative stress. Under physiological conditions, urate is a final degradation product of purine bases, because human lacks the enzyme uricase converting the uric acid to allantoin. However, during increased oxidative stress, reactive oxygen species (ROS) can contribute to the formation of allantoin from uric acid. Allantoin is one from a number of uric acid oxidation products [27]. Oxidation of urate to allantoin implies that urate is a scavenger of ROS. Increased oxidative stress in patients with DS plays a role also in premature aging. There is a positive correlation between uric acid levels in plasma of DS individuals and the age of patients (Figs. 5 and 6). The higher the age of a child with DS is, the higher levels of uric acid in plasma are (correlation coefficient r = 0.682, p = 0.01). In control subjects, this correlation is not so obvious (r = 0.459, p>0.05). Similar correlation was found also between allantoin levels and age of individuals with DS (r = 0.52, p = 0.05 for DS patients and r = 0.23, p>0.05 for controls).
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Our data support findings about increased oxidative stress in DS individuals. They indicate that allantoin and uric acid levels in children with DS may be considered as biomarkers of oxidative stress in neurological disorders when carefully evaluated with appropriate controls. Acknowledgements This work has been partially supported by the grant agency VEGA, Ministry of Education, Slovak Republic (1/8303/01 and 1/9243/02) and grant of the U.S. – Slovak Science and Technology Program (005-96-12). Authors wish to thank Mrs. L’ubica Chandogova´ and Ly´dia Mikova´ for their technical assistance. References [1] Wolozin B, Scicutella A, Davies P. Proc Natl Acad Sci U S A 1988;85:6202 – 6. [2] Kolata G. Science 1985;230:1152 – 3. [3] Wisniewski KE, Kida E, Brown TW. In: Rondal JA, Perera J, Nadel I, Comblain A, editors. Down’s syndrome. Psychological, Psychobiological, and Socio-Educational Perspectives. London: Whurr Publishers; 1996. p. 3 – 19. [4] Sinet PM. Ann NY Acad Sci 1982;396:83 – 94. [5] de Haan JB, Wolvetang EJ, Cristiano F, Iannello R, Bladier C, Kelner MJ, et al. Adv Pharmacol 1997;38:379 – 402. [6] Muchova´ J, Sˇustrova´ M, Garaiova´ I, Lipta´kova´ A, Blazˇ´ıcˇek P, Kvasnicˇka P, et al. Free Radic Biol Med 2001;31:499 – 508. [7] Ciompi ML, Bazzichi LM, Bertolucci D, Mazzoni MR, Barbieri P, Mencacci S, et al. Clin Rheumatol 1984;3:229 – 33. [8] Aruoma OI, Halliwell B. FEBS Lett 1989;244:76 – 80. [9] Pecha´nˇ I. Clin Biochem Metab 1995;3:207 – 10. [10] Ames BN, Cathcart R, Schwiers E, Hochstein P. Proc Natl Acad Sci U S A 1981;78:6858 – 62. [11] Becker BF. Free Radic Biol Med 1993;14:615 – 31. [12] Mertz ET, Fuller RW, Concon JM. Science 1963;141:535 – 6. [13] Kaufman JM, O’Brien WM. N Engl J Med 1967;276:953 – 6. [14] Nishida Y, Akaoka I, Kobayashi M, Maruki K, Oshima Y. J Rheumatol 1979;6:103 – 7. [15] Puukka R, Puukka M, Leppilampi M, Linna SL, Kouvalainen K. Clin Chim Acta 1982;126:275 – 81. [16] Grootveld M, Halliwell B. Biochem J 1987;243:803 – 8. [17] Ogihara H, Ogihara T, Miki M, Yasuda H, Mino M. Pediatr Res 1995;37:219 – 26. [18] Aruoma OI. In: Reilly T, Orme M, editors. The Clinical Pharmacology of Sport and Exercise. Amsterdam: Elsevier Science; 1997. p. 71 – 82. [19] Mikami T, Kita K, Tomita S, Qu G, Tasaki Y, Ito A. Free Radic Res 2000;32:235 – 44.
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[20] Peeters MA, Megarbane A, Cattaneo F, Rethore MO, Lejeune J. J Intellect Disabil Res 1993;37:491 – 505. [21] Watts RW, Perera YS, Allsop J, Newton C, Platts-Mills TA, Webster AD. Clin Exp Immunol 1979;36:355 – 63. [22] Aruoma OI, Spencer JPE, Mahmood N. Food Chem Toxicol 1999;37:1043 – 53. [23] Dierssen M, Vallina IF, Baamonde C, et al. Brain Res Dev Brain Res 1996;95:122 – 4. [24] Aruoma OI, Whiteman M, England TG, Halliwell B. Biochem Biophys Res Commun 1997;231:389 – 91. [25] Houston M, Chumley P, Radi R, Rubbo H, Freeman BA. Arch Biochem Biophys 1998;355:1 – 8. [26] Parks DA, Granger DN. Acta Physiol Scand 1986;548: 87 – 99.
[27] Kaur H, Halliwell B. Chem Biol Interact 1990;73:235 – 47. [28] Kock R, Delvoux B, Greiling H. Eur J Clin Chem Clin Biochem 1993;31:303 – 10. [29] Kock R, Delvoux B, Sigmund M, Greiling H. Eur J Clin Chem Clin Biochem 1994;32:837 – 42. [30] Sasaki K, Motomiya Y, Oyama N, Hirao Y, Okajima E, Uji Y, et al. Nippon Jinzo Gakkai Shi 1996;38:164 – 70. [31] Hellsten Y, Tullson PC, Richter EA, Bangsbom J. Free Radic Biol Med 1997;22:169 – 74. [32] Moison RM, de Beaufort AJ, Haasnoot AA, Dubbelman TMAR, van Zoeren-Grobben D, Berger HM. Free Radic Biol Med 1997;23:226 – 34. [33] Ogihara T, Kim HS, Hirano K, et al. Biol Neonate 1998; 73:24 – 33.
Uric acid and allantoin levels in Down syndrome: antioxidant and oxidative stress mechanisms? Ingrid Zˇitnˇanova´ a,*, Peter Koryta´r a, Okezie I. Aruoma b, Ma´ria Sˇustrova´ c, Iveta Garaiova´ a, Jana Muchova´ a, Tere´zia Kalnovicˇova´ a, ˇ uracˇkova´ a Siegfried Pueschel d, Zdenˇka D a
Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava, Slovak Republic b Department of Neuroinflammation, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Charring Cross Hospital Campus, Fulham Palace Road, London W6 8RF, England, UK c Institute of Preventive and Clinical Medicine, Slovak Centre for Down Syndrome, Bratislava, Slovakia d Child Development Center, Rhode Island Hospital, Brown University School of Medicine, Providence, RI 02903, USA Received 28 July 2003; received in revised form 13 November 2003; accepted 24 November 2003
Abstract Background: Down syndrome (DS) is a chromosomal abnormality (trisomy 21) leading to mental retardation, to the characteristic change of individual’s phenotype and to the pathological features of Alzheimer disease. Patients with DS have elevated ratio of superoxide dismutase to (catalase plus glutathione peroxidase) with respect to controls in all age categories suggesting that oxidative imbalance contributes to the clinical manifestation of accelerated aging. Results: We report that persons with DS have elevated uric acid levels compared with controls, 348.56 F 22.78 versus 284.00 F 20.86 Amol/ l ( p = 0.018). The levels of hypoxanthine and xanthine in DS children (6.35 F 0.31 and 1.02 F 0.23 Amol/l) were significantly lower than in controls (7.83 F 0.59 and 2.43 F 0.66 Amol/l). This result suggests increased conversion of hypoxanthine and xanthine to uric acid with subsequent free radical-dependent oxidation of uric acid to allantoin, mechanisms potentiated by the oxidative stress in DS. Allantoin is a nonenzymatic oxidative product of uric acid in human. In DS individuals, the levels of allantoin were significantly higher than those in healthy controls (18.58 F 2.27 and 14.07 F 1.07 Amol/l, respectively, p = 0.03). Conclusions: Our data supported the presumption of increased oxidative stress in DS. D 2004 Elsevier B.V. All rights reserved. Keywords: Down syndrome; Oxidative stress; Purine metabolism; Uric acid; Allantoin
1. Introduction Down syndrome (DS) is a chromosomal abnormality (trisomy 21) leading to mental retardation, to the * Corresponding author. Tel.: +421-7-59357-414; fax: +421-759357-557. E-mail address: [email protected] (I. Zˇitnˇanova´). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.11.020
characteristic change of individual’s phenotype and to the pathological features of Alzheimer disease [1 –3]. Chronic oxidative stress has been implicated in patients with DS [4 –6]. In the early 1980s, it was reported that DS and gout could coexist in DS patients [7]. The latter disease is characterised by increased plasma concentration of uric acid. Uric acid in the human body was traditionally considered to be a
140
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soluble end product of purine metabolism without any physiological value. On the one hand, higher concentrations of uric acid can form urate crystals which can
be deposited in tissues or joints where they may lead to inflammation process and prooxidant effects. On the other hand, uric acid is regarded to be an important
Table 1 Allantoin levels in tissues and body fluids of patients and healthy controls found in different laboratories Grootveld and Halliwell [16]
Sample type
Allantoin (AM) F S.D.
Alla/UA (%)
Controls Rheumatoid patients
Serum Serum Synovial fluid
18.6 F 3.8 36.1 F 6.3 20.9 F 7.3
4.7 F 1.6 10.5 F 3.8 11.8 F 5.6
Arterial blood Venous blood
Allantoin + allantoate 9.3 F 1.1 8.7 F 0.9
Serum
Allantoin (AM) 15.7 F 7.9
Becker [11]
Kock et al. [28]
Kock et al. [29] Healthy male adults Healthy female adults Patients with angina pectoris Patients with cerebral insult Patients with myocardial infarction Ogihara et al. [17] Controls Willson’s disease patients before therapy Willson’s disease patients after therapy Sasaki et al. [30] Hemodialysed patients Aruoma [18] Healthy male athletes before exercise Healthy male athletes after exercise Hellsten et al. [31] Healthy males before exercise Healthy males after exercise
Moison et al. [32] Preterm babies
Serum
Plasma
Plasma
Plasma
Muscle Plasma Muscle Plasma
Allantoin (AM) 16.7 F 8.3 15.8 F 7.2 17.8 F 6.8 14.9 F 9.1 18.4 F 7.7 Allantoin (AM) 6.5 F 0.8 11.0 F 1.8 4.3 F 0.5 Allantoin (AM) 42.6 F 37.7 Allantoin (AM) 22.1 18.1
Plasma
Mikami et al. [19] Healthy controls Athletes
Serum
Alla/UA (%) 6.2 4.2
Allantoin 0.003 F 0.007 Amol/g 11.9 F 2.6 Amol/l 0.1 F 0.014 Amol/g Doubled Alla/UA (%) 4.7 F 2.5 147 F 186
Plasma Aspirates
Ogihara et al. [33] Chronic lung disease babies (CLD) Non-CLD babies
Alla/UA (%) 2.3 F 0.4 15.9 F 8.6 1.5 F 0.3
Alla/UA (%) 41.2 F 15.8 11.7 F 9.9 Allantoin (Amol/l) 28 F 2.2 65 F 8.8
Alla/UA (%) 4.6 F 0.4 14.4 F 2.6
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antioxidant in human adult plasma. It can directly react with free radicals [8] and may interfere with Fenton’s chemistry by chelating catalytic metal ions [9– 11]. Although some studies reported elevated levels of uric acid in DS individuals [12,13], some speculations exist about the pathogenetic mechanisms leading to the increase in uric acid levels in DS patients. Accumulation of uric acid can also be a result of dysfunction in its renal excretion [13,14]. Puukka et al. [15] found that the levels of erythrocyte adenosine deaminase (AD) and adenine phosphoribosyl transferase activities correlated with increased plasma urate concentrations in DS individuals. AD is coded by a gene on chromosome 21, which is trisomic in patients with DS. The increased gene dose for AD can cause the increased activity of the enzyme, leading to the increased production of uric acid in children with DS. Another plausible explanation for impairment of purine metabolism in DS patients is the imbalance in the antioxidant enzyme defence system. The overexpression of the enzyme superoxide dismutase (SOD) (the gene for this enzyme is localised on the chromosome 21) can lead to increased oxidative stress [6] caused by increased ratio of SOD/(catalase + glutathione peroxidase). Human cells lack the enzyme uricase, which in some species can oxidise uric acid further. However, if uric acid is exposed to the system producing oxygen radical species, various oxidative products may be formed (allantoin, allantoate, glyoxylate, urea, oxalate) [16]. Allantoin is a nonenzymatic oxidative product of urate metabolism which can be measured by HPLC [14,16]. Measurement of uric acid and allantoin in tissues and body fluids (particularly in serum and synovial fluids) are largely advocated as markers of oxidative stress in vivo (Table 1). Increased levels of allantoin have been reported in Willson disease [17]. Exhaustive exercise can also lead to increased plasma allantoin concentration [18,19]. The aim of our study was to compare the levels of purine metabolism products (uric acid, hypoxanthine and xanthine) in plasma of DS patients with those of healthy controls and to examine levels of allantoin in both groups. If there is an increased free radical attack on urate
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in DS individuals, the logical product of this attack would be allantoin.
2. Material and methods 2.1. Patients For this study, we used blood of 16 children with DS (average age 10.06 F 1.04 years) and 16 agematched healthy controls (11.94 F 0.97). In all persons with DS, the diagnosis was confirmed by cytogenetic analysis. All patients in this study are regularly examined at the Center for Down syndrome at the Institute for Preventive and Clinical Medicine in Bratislava, Slovakia. These individuals did not have complicating medical conditions. They did not suffer from chronic diseases such as diabetes mellitus, acute respiratory diseases, renal insufficiency or any serious heart defects. Individuals suffering from mentioned diseases were excluded from our study. The studies were performed according to the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects or their guardians and the study was approved by The Ethics Committee of Derer’s University Hospital in Bratislava. 2.2. Sample preparation Human blood was collected into heparinized tubes. The plasma was obtained by blood centrifugation at 1500 g for 10 min and stored at 80 jC until required. Before analysis, plasma samples were thawed at room temperature and deproteinized with 1/25 volume of 4.6 mol/l HClO4 then centrifuged for 10 min at 7000 g and ultrafiltered through a 0.2-Am filter (Millipore). 2.3. Chemicals Allantoin, hypoxanthine, xanthine, uric acid were obtained from Sigma Chemical (St. Louis, MO, USA) and used for preparation of standard solutions. Methanol was purchased from Merck (Germany) and was of HPLC-grade. All other chemicals were purchased from Lachema (Brno, Czech Republic) and were of analytical grade.
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2.4. Preparation of the stock standards Allantoin was dissolved in deionized water. Hypoxanthine and xanthine were dissolved in 2 mmol/ l KOH and uric acid was dissolved in 2 mmol/l KOH followed by a short heating in a boiling water bath and by ultrasound. Calibration curves for hypoxanthine, xanthine and allantoin standards were constructed covering the range from 25 to 500 pmol. Uric acid was injected to produce a calibration curve from 0.5 to 100 nmol. The calibration was based on the areas calculated with CSW 1.7DLL (DataApex) software.
Fig. 2. Uric acid levels in plasma of patients with Down syndrome (DS) and healthy controls (C). Data are means F S.E.M. (n = 16). *Denotes p < 0.05 (significantly different).
2.5. Uric acid, hypoxanthine, xanthine analysis The HPLC was performed on a Watrex chromatograph system equipped with 100 4 mm guard column packed with Separon SGX C18, 7 Am (Watrex), 250 4 mm analytical column packed with Nucleosil 120-C18, 5 Am (Watrex). For the determination of uric acid, hypoxanthine and xanthine UV detection was performed at 254 nm (DeltaChrome UVD 200). A 100 Al of plasma ultrafiltrate was directly injected onto a column. The mobile phase was 0.05 mol/l potassium phosphate buffer, pH = 4.6 and 5% methanol at a flow rate of 0.8 ml/min. The quantification of uric acid, hypoxanthine and xanthine was based on the peak areas and compared with standards.
Fig. 1. Analytical steps in the allantoin determination. Full details in the text.
Fig. 3. Hypoxanthine (HX) and xanthine (X) levels in healthy controls (C) and in patients with DS. (DS) (n = 16 for HX and 13 for X). Data are means F S.E.M. *Denotes p < 0.05 (significantly different).
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Fig. 4. Allantoin levels in plasma of patients with DS (DS) and healthy controls (C). Data are means F S.E.M. (n = 16). *Denotes p < 0.05 (significantly different).
2.6. Determination of allantoin Allantoin analysis was carried out essentially as described by Grootveld and Halliwell [16] with a slight modification (Fig. 1). Plasma samples and standards were treated in the same way. A 100 Al of plasma ultrafiltrate was injected onto a C18 Nucleosil column. The mobile phase was 0.05 mol/ l potassium phosphate buffer, pH = 4.6 and 5% methanol at a flow rate of 0.8 ml/min, wavelength 254 nm. A fraction covering the retention time range (2.9 – 5.2 min) where allantoin elutes (but not uric acid) was collected and evaporated to dryness
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at 40 jC under vacuum. The residue was reconstituted in 100 Al of 0.12 mol/l NaOH and incubated for 20 min at 100 jC. After adding 100 A1 of 1 mol/l HCl and 10 Al of 3 mmol/l dinitrophenylhydrazine in 1 mol/l HCl, the heating at 100 jC continued for 5 min. After cooling the sample, 100 Al volume was injected onto a Nucleosil C18 column. The mobile phase was 55% (v/v) 30 mmol/l sodium citrate/27.7 mmol/l sodium acetate buffer, pH = 4.75 and 45% methanol at a flow rate of 0.8 ml/min. The detection was performed at the wavelength 360 nm. The plasma allantoin concentration was quantified by comparing the peak area of glyoxylate 2,4-dinitrophenylhydrazone with values from the standard curve. The recovery of three different concentrations of added allantoin to plasma averaged 94%. The mean coefficient of variation for analysis of nine replicate plasma samples was 4.8%. 2.7. Statistical analysis Data are expressed as means F S.E.M. Student’s unpaired t-test was used to compare the statistical differences of detected levels of uric acid, hypoxanthine, xanthine and allantoin in both experimental groups (DS and control). Differences among means were considered statistically significant by the criterion of probability value < 0.05.
Fig. 5. Correlation between uric acid levels and age in children with DS (DS) and controls (C). Correlation coefficient for patients with DS is r = 0.68 ( p = 0.01) and for controls r = 0.46 ( p>0.05).
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3. Results
4. Discussion
3.1. Uric acid, xanthine, hypoxanthine and allantoin levels in Down syndrome
Down syndrome is a disease with developmental brain abnormalities resulting in early mental and precocious, age-dependent Alzheimer-type neurodegeneration [3,23]. Trisomic cells are more sensitive to oxidative stress. This sensitivity may be due to the imbalance in the hydrogen peroxide metabolism or another unknown factor [6]. The cells tend to compensate this negative effect by increasing glutathione peroxidase (GPx) activity while that of catalase (CAT) remains unchanged. Thus, the ratio of SOD to (CAT + GPx) is important from the point of view of sensitivity to ROS, rather than in absolute quantities of individual antioxidants. There are specific synergic interactions between antioxidant enzymes and therefore a comprehensive understanding of the antioxidative system should be based on knowledge of activities and mutual interactions of enzymes, involved in free radical detoxification [6]. The peroxynitrite-dependent DNA damage involves a marked increase in the products (hypoxanthine and xanthine) arising from the deamination of guanine and adenine. Antioxidants such as ergothioneine inhibit the formation of hypoxanthine (a precursor of xanthine) and xanthine and this may have many implications for inflammatory conditions characterised by overproduction of uric acid (the oxidation product of xanthine) [22,24]. The conversion of hypoxanthine to xanthine
In this study, the levels of uric acid (UA), hypoxanthine (HX), xanthine (X) and allantoin (Alla) in plasma of children with DS (n = 16) and in age-matched healthy control group (n = 16) were determined. DS patients had significantly elevated levels of uric acid (348.35 F 22.78 Amol/l versus controls 284.00 F 20.86 Amol/l, p = 0.018) (Fig. 2). These results are consistent with previous reports [11,20,21]. The levels of both hypoxanthine and xanthine in children with DS were lower than those in controls (6.35 F 0.31 and 1.02 F 0.23 Amol/ for DS and 7.83 F 0.59 Amol/l, p = 0.018 and 2.43 F 0.66 Amol/l, p = 0.030 for controls). Xanthine levels in both experimental groups were lower than hypoxanthine in all sets (Fig. 3). Indeed, xanthine and hypoxanthine are products of free radical-dependent deamination of guanine and adenine [22]. Down syndrome patients had significantly higher allantoin levels (18.58 F 2.27 Amol/l) when compared with controls (14.07 F 1.07 Amol/l) ( p = 0.03) (Fig. 4). We determined the correlation between uric acid levels in plasma of DS individuals and the age of patients (Figs. 5 and 6).
Fig. 6. Correlation between allantoin and age levels in children with DS (DS) and in controls (C). Correlation coefficient for patients with DS is r = 0.52 ( p = 0.05) and for controls r = 0.23 ( p>0.05).
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and then xanthine to uric acid in a reaction catalysed by xanthine oxidase increases the burden of superoxide radical (O2S ) in vivo. Indeed, endogenously produced nitric oxide (NOS) will react with O2S to produce ONOO , a reaction that occurs with a high rate constant. ONOO may increase the oxidation of xanthine dehydrogenase to xanthine oxidase [25], an enzyme widely reported to be activated in oxidative burst [26] which uses hypoxanthine and xanthine as substrates [22]. Dismutation of the superoxide anion will produce hydrogen peroxide. We suggest that antioxidants that can modulate production of xanthine and hypoxanthine in vivo will modulate accumulation of uric acid in Down syndrome. The major finding of our work is a significant increase of uric acid and allantoin levels in plasma of children with DS. On the other hand, levels of hypoxanthine and xanthine were significantly lower than those in healthy individuals. Hyperuricemia in children with DS has been determined also by other authors [11] who attribute this increase to the elevated activity of erythrocyte adenosine deaminase and adenine phosphoribosyltransferase activities. Nishida et al. [14] suggest that glomerular dysfunction could also contribute to hyperuricemia in DS individuals. However, we determined significantly elevated levels of allantoin in plasma of children with DS which implicates the role of increased oxidative stress. Under physiological conditions, urate is a final degradation product of purine bases, because human lacks the enzyme uricase converting the uric acid to allantoin. However, during increased oxidative stress, reactive oxygen species (ROS) can contribute to the formation of allantoin from uric acid. Allantoin is one from a number of uric acid oxidation products [27]. Oxidation of urate to allantoin implies that urate is a scavenger of ROS. Increased oxidative stress in patients with DS plays a role also in premature aging. There is a positive correlation between uric acid levels in plasma of DS individuals and the age of patients (Figs. 5 and 6). The higher the age of a child with DS is, the higher levels of uric acid in plasma are (correlation coefficient r = 0.682, p = 0.01). In control subjects, this correlation is not so obvious (r = 0.459, p>0.05). Similar correlation was found also between allantoin levels and age of individuals with DS (r = 0.52, p = 0.05 for DS patients and r = 0.23, p>0.05 for controls).
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