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The relationship between uric acid and its oxidative product allantoin: a potential indicator for the evaluation of oxidative stress in birds

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The relationship between uric acid and its oxidative product allantoin: a potential indicator for the evaluation of oxidative stress in birds
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  Abstract Uric acid is the main nitrogenous wasteproduct in birds but it is also known to be a potentantioxidant. Hominoid primates and birds lack theenzyme urate oxidase, which oxidizes uric acid toallantoin. Consequently, the presence of allantoin intheir plasma results from non-enzymatic oxidation. Inhumans, the allantoin to uric acid ratio in plasma in-creases during oxidative stress, thus this ratio has beensuggested to be an in vivo marker for oxidative stress inhumans. We measured the concentrations of uric acidand allantoin in the plasma and ureteral urine of white-crowned sparrows ( Zonotrichia leucophrys gambelii ) atrest, immediately after 30 min of exercise in a hop/hover wheel, and after 1 h of recovery. The plasmaallantoin concentration and the allantoin to uric acidratio did not increase during exercise but we found apositive relationship between the concentrations of uric acid and allantoin in the plasma and in the ureteralurine in the three activity phases. In the plasma, theslope of the regression describing the above positiverelationships was significantly higher immediately afteractivity. We suggest that the slope indicates the rate of uric acid oxidation and that during activity this rateincreases as a result of higher production of free radi-cals. The present study demonstrates that allantoin ispresent in the plasma and in the ureteral urine of white-crowned sparrows and therefore might be usefulas an indicator of oxidative stress in birds. Keywords Oxidative stress Æ Antioxidation Æ Free radicals Æ Allantoin Æ Uric acid Æ White-crowned sparrow Introduction Uric acid is the main nitrogen waste product of birds(Wright1995), but it is also a potent antioxidant (Ameset al.1981). The importance of uric acid as an antiox-idant has been recognized in humans for years (Ameset al.1981). More recently this role has been postulatedfor birds as well (Iqbal et al.1999; Klandorf et al.1999, 2001; Simoyi et al.2002; Lin et al.2004; Stinefelt et al. 2005). Hominoid primates, birds and most reptilianspecies lack the enzyme urate oxidase (Moriwaki et al.1999; Oda et al.2002). Hence, in these taxa uric acid is the end product of purine catabolism. In spite of thepotential risk of gout, caused by high plasma concen-trations of uric acid (Becker1993; Alderman et al.1999), the plasma concentration of uric acid in humansis maintained at high levels presumably because of its Communicated by I.D. HumeE. Tsahar ( & ) Æ Z. AradDepartment of Biology, Technion—Israel Instituteof Technology, Haifa 32000, Israele-mail: elat@techunix.technion.ac.ile-mail: zarad@techunix.technion.ac.ilI. IzhakiDepartment of Biology, University of Haifa at Oranim,K. Tivon 36006, Israele-mail: izhaki@research.haifa.ac.ilC. G. GuglielmoDivision of Biological Sciences, University of Montana,Missoula, MT 59802, USA Present address : C. G. GuglielmoDepartment of Biology, University of Western Ontario,London, ON, Canada N6A 5B7e-mail: cguglie2@uwo.caJ Comp Physiol B (2006) 176:653–661DOI 10.1007/s00360-006-0088-5  123 ORIGINAL PAPER The relationship between uric acid and its oxidative productallantoin: a potential indicator for the evaluation of oxidativestress in birds Ella Tsahar Æ Zeev Arad Æ Ido Izhaki Æ Christopher G. Guglielmo Received: 1 February 2006/Revised: 17 April 2006/Accepted: 18 April 2006/Published online: 17 May 2006 Ó Springer-Verlag 2006  beneficial role as an antioxidant (Ames et al.1981;Kirschbaum2001; Hediger2002; Enomoto et al.2002). The plasma concentrations of uric acid and its salts inbirds are exceptionally high compared with other ver-tebrates (Lumeij and Remple1991), sometimes wellabove the limit of its theoretical solubility (for sodiumurate: 0.6 mM at 43 ° C; Lumeij and Remple1991). Theantioxidative properties of uric acid and its high con-centrations in birds’ plasma have been suggested as apossible mechanism to protect birds from oxidativedamage (Klandorf et al.2001; Simoyi et al.2002,2003). Indeed, increased plasma uric acid concentration inchickens reduced leukocyte oxidative activity (Simoyiet al.2002; Machı´ n et al.2004), while decreased con-centrations were associated with increased leukocyteoxidative activity (Klandorf et al.2001).Allantoin, the oxidative product of uric acid, hasbeen proposed as a potential biomarker for in vivo freeradical reactions (Grootveld and Halliwell1987; La-gendijk et al.1995; Ogihara et al.1998; Benzie et al. 1999; Yardim-Akaydin et al.2004). The absence of  urate oxidase activity in humans (while it is present inmost other mammalian species) and birds (Moriwakiet al.1999) implies that the presence of allantoin intheir plasma results only from the non-enzymatic oxi-dation of uric acid. In humans, the plasma concentra-tion of allantoin and/or the allantoin:uric acid ratioincreases during oxidative stress situations such aschronic lung diseases (James et al.2003), Down syn-drome (Zitnanova et al.2004), brain ischemia(Marklund et al.2000), rheumatoid arthritis (Yardim-Akaydin et al.2004) and chemotherapy treatment(Durken et al.2000).Exercise in humans and other mammals is alsoassociated with increased formation of free radicals,which can damage cells and tissues (Wetzstein et al.1998; Ji1999; Liu et al.1999; Mastaloudis et al.2001; Muradian et al.2002; Chevion et al.2003). Interest- ingly, the allantoin concentration and the allantoin:uricacid ratio in human plasma and muscle tissue werefound to increase during intensive exercise, demon-strating the presumptive antioxidative function of uricacid (Hellsten et al.1997,2001; Mikami et al.2000a,b). The effect of exercise on the production of freeradicals in birds is not yet clear. The basal and maximalrate of H 2 O 2 production, oxygen consumption and freeradical leak in the respiratory chain in heart, brain andlung mitochondria of birds are lower than in mammalsof similar size (Herrero and Barja1997; Barja1998). Also, birds express only the dehydrogenase form of theenzyme xanthine oxidoreductase which does not pro-duce free radicals (Sato et al.1995; Moriwaki et al.1999). Hence, theoretically, they should suffer lessfrom oxidative stress during activity. Allantoin hasbeen detected in the plasma of birds (Poffers et al.2002; Simoyi et al.2003), however, it is not known if  exercising birds undergo a similar pattern of concen-tration changes as in mammals.As uric acid is the end product of protein catabolisminbirds,itsconcentrationintheplasmaisaffectedbytheprotein turnover rate. It has been shown that the con-centration of uric acid increases in the plasma afterintensiveflight(Gannesetal.2001;Klaassenetal.2000), where it is associated with protein degradation. If uricacidfunctionsasanantioxidantthiscouldbeabeneficialmechanism to lower oxidative stress at that time.The aim of this study was to evaluate the effect of exercise on the relationship between the concentra-tions of uric acid and allantoin in the plasma and urineof white-crowned sparrows. Specifically, we measuredthe concentrations of uric acid and allantoin in theplasma and in the ureteral urine of white-crownedsparrows at rest, immediately after 30 min of exercisein a hop/hover wheel and after 1 h of recovery. If birdsfollow the human model we would expect that theallantoin to uric acid ratio would increase during andfollowing activity as a result of a higher rate of uric acidoxidation caused by increased production of free rad-icals. We also expected that the allantoin:uric acid ratioin the ureteral urine would be highest during recoveryand lowest at rest, as we expected the birds to defenduric acid concentration in the plasma during activityand hence excrete less uric acid during this phase. Material and methods Care and maintenanceGambel’s white-crowned sparrows (Z onotrichia leu-cophrys gambelii ), were caught near Mabton, Wash-ington in September 2003 ( n = 20). The birds wereheld in an outdoor aviary at the University of MontanaResearch Station at Fort Missoula for 10 months be-fore the experiments, and maintained on a mixed seeddiet (40% white millet, 40% cracked corn and 20%sunflower; Purina Mills, Regional recipe). Four daysbefore the initiation of experiments the birds weretransferred to individual cages (40 · 45 · 45 cm) in aroom at 22 ° C, and kept on a light:dark cycle similar tothe natural cycle (17L:7D in July). Seeds and waterwere provided ad libitum. Possession of birds andexperimental protocols were approved by the U.S. Fishand Wildlife Service, the Montana Department of Fish,Wildlife and Parks, and the University of MontanaInstitutional Animal Care and Use Committee. 654 J Comp Physiol B (2006) 176:653–661  123  Experimental designTo avoid the possible adverse effect of consecutivebleedings (taking over 10% of blood volume within5 days) while taking three blood samples from all birds(resting, exercising and recovery), birds were randomlydivided into two groups. We first took blood and urinesamples (‘‘resting’’ samples) from the first group.These birds were left to recover for 5 days, and werethen exercised (‘‘exercise’’ followed by ‘‘recovery’’samples). The second group was first exercised(‘‘exercise’’ followed by ‘‘recovery’’ samples) then leftto recover for 5 days before being sampled for the‘‘resting’’ data. To match the conditions of ‘‘exercise’’samples, ‘‘resting’’ samples were taken after 2 h with-out food and a third hour without food and water.Birds were exercised one at a time. Food was with-drawn 2 h before exercise. Birds were then placed inthe wheel chamber for 15 min before exercise. Duringthis period, we covered the wheel chamber with blackcloth to let the birds settle, and measured minimal VO 2 for 2 min (see below). We turned the wheel by handfor 30 min at a speed of 30–40 rpm. Blood and ureteralurine samples were taken immediately (< 5 min) afterthe termination of exercise. The birds were then re-turned to the cages for 1 h, during which time they hadwater available (but not food), and thereafter sampledagain (‘‘recovery’’ samples). The birds were weighedbefore and after exercise to the nearest 0.01 g.Urine and blood samplesUreteral urine samples were collected by brieflyinserting a closed-ended, perforated cannula, custom-made of polyethylene tubing (PE160, Intramedic, MD,USA), into the bird’s cloaca (Goldstein and Braun1986). We collected blood ( ~ 160 l l) in heparinizedmicrohematocrit tubes by puncturing the brachial veinwith a 26-gauge needle. Plasma was separated fromcells after centrifugation at 2,000 g for 6 min. Sampleswere immediately frozen at –196 ° C in liquid N 2 andwere analyzed within 20 days.VO 2 measurementsOxygen consumption and CO 2 production were mea-sured in an open-flow running wheel chamber, modifiedfor hopping/hovering birds (Chappell et al.1999). Theinternal wheel dimensions were approximately 16 cmin width and 27 cm in diameter (volume of 9,156 cm 3 ).The flow rate of dried (Drierite), CO 2 -free (Ascarite)air was regulated and measured at 3.5 l min –1 bya STP-corrected mass flow controller/meter (SierraInstruments 840-L, Monterey, CA, USA). Approxi-mately 150 ml min –1 of air exiting the respirometerwheel was sub-sampled, dried and passed through aCO 2 analyzer (Sable Systems CA-2A, Las Vegas, NV,USA). The excurrent air was then redried, scrubbed of CO 2 and passed through an O 2 analyzer (Sable Sys-tems FC-1B). Gas analyzers were calibrated with N 2 and a standard, certified dry gas mixture (77.10% N 2 ,20.90% O 2 , 2.00% CO 2 ; NORCO, Missoula, MT,USA). Analog outputs were converted to digital for-mat by an analog/digital converter (Sable Systems UI-2) and collected by a PC computer. The 2 min mini-mum (average of lowest 2 min of VO 2 measured, be-fore exercising), the 2 min maximum (average forhighest 2 min of VO 2 measured) and the 30 min meanVO 2 during exercise, were calculated using Datacan Vsoftware (Sable Systems).Uric acid and allantoin assaysUric acid was measured by the endpoint assay (WAKODiagnostics UA 20R/30R), modified for a micro-platespectrophotometer (Biotek Powerwave X340, Winoo-ski, VT, USA), as follows: a 5 l l sample or standardwas mixed with 300 l l of the pre-warmed (to 37 ° C)reagent, incubated for 10 min at 37 ° C, shaken and theabsorbance read at 550 nm (700 nm reference). Plasmawas run undiluted whereas urine was diluted 30–80times with LiOH. LiOH dissolves the urate precipi-tates and any trapped ions (Roxburgh and Pinshow2002; Tsahar et al.2005). A uric acid standard (3 mM) was prepared in 0.1 M glycine buffer, pH 9.3. Becausethe uric acid assay uses the enzyme uricase to converturic acid to allantoin and then measures total allantoin,we subtracted the allantoin concentration from that of uric acid for each sample. Plasma (70 l l) and urine(100 l l) were analyzed for allantoin. Allantoin wasdetermined by the Rimini–Schryver reaction describedby Young and Conway (1942) (see also Poffer et al.2002). In this reaction allantoin is first hydrolyzed un-der weak alkaline conditions at 100 ° C to allantoic acid,which is then further degraded to urea and glyoxylicacid in a weak acid solution. The glyoxylic acid thenreacts with phenylhydrazine hydrochloride to producea phenylhydrazone of the acid. This product forms anunstable chromophore with potassium ferricynide. Thecolor is read in a spectrophotometer (Beckman DU-640) at 522 nm, 20 min after the reaction. To eliminatethe possibility that uric acid might interfere with thespecificity of the reaction, we ran the assays usingstandards of uric acid (0.2–5 mM). Uric acid did notabsorb light at 522 nm and hence we concluded that itdoes not interfere with the reaction. All chemicals J Comp Physiol B (2006) 176:653–661 655  123  were purchased from Sigma (Sigma Chemical, St.Louis, MO, USA).OsmolarityPlasma osmolarity was measured in a freeze-pointdepression osmometer (Osmette II, Precision Systems,Natick, Massachusetts, USA).Statistical analysisLeast-squares linear regression was used to test forrelations between the concentrations of uric acid andallantoin in the plasma and in the ureteral urine, andthe relation between plasma concentrations of uric acidand allantoin immediately after exercise and maximumVO 2 . A repeated measures ANOVA was used tocompare the plasma and ureteral urine concentrationsof uric acid and allantoin and plasma osmolarity amongthe three activity phases. We used a linear model toassess the effect of the three activity phases (resting,exercising and recovery) on the relations between uricacid and allantoin concentrations in the plasma andin the ureteral urine. The linear model used in theanalysis was: y = b 0 + b 1 x 1 + b 2 x 2 + b 3 x 1  x 2 + b 4 x 3 + b 5 x 1  x 3 +  , where y is the dependent variable(allantoin concentration), x 1 is uric acid concentration,and x 2 and x 3 are dummy variables that represent theeffect of phase. The recovery phase was randomlychosen as the reference for the intercept ( b 0 ) and theslope ( b 1 ) comparisons, b 2 is the difference betweenthe exercising and the recovery intercepts, b 3 is thedifference between the exercising and the recoveryslopes, b 4 is the difference between the resting andthe recovery intercepts, and b 5 is the difference be-tween the resting and the recovery slopes. Data arereported as means ± SE. Significance was accepted at P  < 0.05. Results Body mass loss and metabolic rate during exerciseDuring the 30 min exercise period in the runningwheel, the birds lost 2.99 ± 0.32% of their initial bodymass (23.7 ± 0.46 g). Percent of body mass loss wascorrelated with the average VO 2 for 30 min exercise( r  = 0.618, P  = 0.006, n = 18). Plasma osmolarity didnot differ among the three phases (rest: 321.3 ±12.4 mOsm; exercise: 329.2 ± 13.1 mOsm; recovery:329.9 ± 8.9 mOsm; within-subjects repeated measuresANOVA: F  2,14 = 0.209, P  = 0.8).The average lowest 2 min VO 2 measured beforeexercising (163.8 ± 7.9 ml h –1 ) was significantly lowerthan the 2 min maximal VO 2 (311.7 ± 10.2 ml h –1 )during exercise (paired t  13 = 14.29, P  < 0.0001), and2 min maximum VO 2 ranged between 257 and405 ml h –1 . The average VO 2 for the 30 min exercisingwas 233 ± 14.3 ml h –1 . The plasma concentrations of uric acid and allantoin measured immediately afterexercise, but not at recovery, were positively correlatedwith the maximum VO 2 (Fig.1). The plasma concen-tration of uric acid measured at the recovery was cor-related with body mass loss ( r  = 0.7, P  = 0.004, n = 15). Respiratory exchange ratio (VO 2 /VCO 2 )measured during maximum activity was 0.87 ± 0.02.Uric acid and allantoin in the plasmaWe had 14 birds with full data sets (three activityphases) of plasma samples (volume was insufficient insome of the samples taken), hence the following sta-tistical analysis was applied only for these birds( n = 14). The plasma concentration of uric acid dif-fered significantly among the three activity phases; thehighest concentration was measured after 1 h of recovery and the lowest in the resting phase (within-subjects repeated measures ANOVA: F  2,26 = 7.24, P  = 0.003, Table1). The plasma concentration of uricacid in the recovery phase was significantly higher thanthat of the resting and exercising phases, while that of the exercising and resting phases did not differ onefrom the other (Fig.2). However, when the percentbody mass loss was added as a covariant, there was noeffect of the activity phase on the uric acid plasma Fig. 1 The plasma concentrations of uric acid (  filled circles ) andallantoin ( open circle s) of exercising white-crowned sparrowsincreased as functions of maximum VO 2 (uric acid: y = 0.004  x –0.85; r  = 0.39, F  1,16 = 10.38, P  = 0.0053; allantoin: y = 0.0013  x –0.21, r  = 0.24, F  1,16 = 4.87, P  = 0.043)656 J Comp Physiol B (2006) 176:653–661  123  concentration (within-subjects repeated measuresANOVA: F  2,24 = 0.057, P  = 0.94). The allantoin con-centration did not differ among the activity phases(within-subjects repeated measures ANOVA: F  2,26 = 1.105, P  = 0.34, Table1, Fig.2). Adding the body mass loss as a covariant had no effect on theresult. No significant differences were found in theallantoin to uric acid ratio among the three activityphases (within-subject repeated measures ANOVA: F  2,26 = 2.32, P  = 0.12). When body mass loss is addedas a covariant the ratio significantly differs among theactivity phases (within-subjects repeated measuresANOVA: F  2,24 = 9.28, P  = 0.001).Plasma concentrations of allantoin and uric acidwere positively correlated in all three activity phases(rest: r  = 0.48, P  = 0.006; exercise: r  = 0.7, P  = 0.0002;recovery: r  = 0.5, P  = 0.004). The slope of the regres-sion of plasma allantoin as a function of uric acid in theexercise phase was significantly higher than the slopesof the resting and recovery phases, while the interceptof the exercising phase was significantly lower (Fig.3).No significant difference was found between the slopesand intercepts of the resting and recovery phases.Uric acid and allantoin in the ureteral urineWe had 12 birds with full data sets of ureteral urinesamples (the urine volume was insufficient in some of the samples taken), hence the following statisticalanalysis was applied only for these birds ( n = 12). Theconcentrations of allantoin and uric acid in the ureteralurine were significantly related in the resting andexercising activity phases ( r  = 0.73, P  = 0.0004; r  = 0.41, P  = 0.024, respectively) but not in the recov-ery phase ( r  = 0.3, P  = 0.07). There were no significantdifferences among the slopes of the regression linesand among the intercepts of the three phases( P  > 0.05). No significant differences were found in theconcentrations of uric acid and allantoin among thethree activity phases (within-subjects repeated mea-sures ANOVA: F  2,22 = 1.59, P  = 0.23; F  2,22 = 1.69, P  = 0.85, respectively). No significant differences werefound in the allantoin to uric acid ratio among thethree activity phases (within-subjects repeated mea-sures ANOVA: F  2,22 = 2.57, P  = 0.099). Adding bodymass loss as a covariant did not affect the results.The concentrations of uric acid and allantoin inureteral urine are summarized in Table1. Discussion The present study demonstrates that allantoin, theoxidative product of uric acid, is present in the plasma Table 1 The concentrations of uric acid and allantoin and the allantoin to uric acid ratio (AUR) in the plasma and ureteral urine of white-crowned sparrows in the three activity phasesRest Exercise RecoveryMean ± SE Range Mean ± SE Range Mean ± SE RangePlasma uric acid (mM) 0.34 ± 0.035 a 0.08–0.51 0.39 ± 0.037 a 0.19–0.75 0.49 ± 0.048 b 0.22–0.93Plasma allantoin (mM) 0.15 ± 0.01 ns 0.09–0.22 0.15 ± 0.02 ns 0.08–0.34 0.17 ± 0.01 ns 0.09–0.22Plasma AUR 0.50 ± 0.08 ns 0.39 ± 0.02 ns 0.37 ± 0.03 ns Ureteral urine uric acid (mM) 50.30 ± 22.22 ns 3.43–270 83.42 ± 24.78 ns 18.89–340.2 33.51 ± 6.73 ns 9.35–81.74Ureteral urine allantoin (mM) 4.95 ± 2.16 ns 0.56–28 4.08 ± 0.66 ns 1.18–8.52 3.97 ± 0.83 ns 0.16–8.66Ureteral urine AUR 0.29 ± 0.1 ns 0.07 ± 0.02 ns 0.15 ± 0.04 ns Only birds that had a complete data set of the three activity phases are included in this analysis (plasma n = 14, ureteral urine n = 12)Different superscript letters within a row denote significant differences ( P  < 0.05) ns not significant ( P  > 0.05) Fig. 2 The plasma concentration of uric acid (  filled bars ) of white-crowned sparrows in the recovery phase was significantlydifferent from that during the resting and exercising phases(Bonferroni Test, P  < 0.05). No significant differences werefound in the concentration of allantoin ( open bars ) among thethree activity phases. Data are presented as means ± SE( n = 14). Different letters above bars denote significant differ-ences ( P  < 0.05). ns not significant, P  > 0.05J Comp Physiol B (2006) 176:653–661 657  123
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