The relationship between uric acid and its oxidative product allantoin ...

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suggested to be an in vivo marker for oxidative stress in humans. We measured the concentrations of uric acid and allantoin in the plasma and ureteral urine of ...
J Comp Physiol B (2006) 176:653–661 DOI 10.1007/s00360-006-0088-5

ORIGINAL PAPER

The relationship between uric acid and its oxidative product allantoin: a potential indicator for the evaluation of oxidative stress 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  Springer-Verlag 2006

Abstract Uric acid is the main nitrogenous waste product in birds but it is also known to be a potent antioxidant. Hominoid primates and birds lack the enzyme urate oxidase, which oxidizes uric acid to allantoin. Consequently, the presence of allantoin in their plasma results from non-enzymatic oxidation. In humans, the allantoin to uric acid ratio in plasma increases during oxidative stress, thus this ratio has been suggested to be an in vivo marker for oxidative stress in humans. We measured the concentrations of uric acid and allantoin in the plasma and ureteral urine of whitecrowned sparrows (Zonotrichia leucophrys gambelii) at rest, immediately after 30 min of exercise in a hop/ hover wheel, and after 1 h of recovery. The plasma allantoin concentration and the allantoin to uric acid ratio did not increase during exercise but we found a

positive relationship between the concentrations of uric acid and allantoin in the plasma and in the ureteral urine in the three activity phases. In the plasma, the slope of the regression describing the above positive relationships was significantly higher immediately after activity. We suggest that the slope indicates the rate of uric acid oxidation and that during activity this rate increases as a result of higher production of free radicals. The present study demonstrates that allantoin is present in the plasma and in the ureteral urine of white-crowned sparrows and therefore might be useful as an indicator of oxidative stress in birds.

Communicated by I.D. Hume

Introduction

E. Tsahar (&) Æ Z. Arad Department of Biology, Technion—Israel Institute of Technology, Haifa 32000, Israel e-mail: [email protected] e-mail: [email protected]

Uric acid is the main nitrogen waste product of birds (Wright 1995), but it is also a potent antioxidant (Ames et al. 1981). The importance of uric acid as an antioxidant has been recognized in humans for years (Ames et al. 1981). More recently this role has been postulated for 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 reptilian species 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 the potential risk of gout, caused by high plasma concentrations of uric acid (Becker 1993; Alderman et al. 1999), the plasma concentration of uric acid in humans is maintained at high levels presumably because of its

I. Izhaki Department of Biology, University of Haifa at Oranim, K. Tivon 36006, Israel e-mail: [email protected] C. G. Guglielmo Division of Biological Sciences, University of Montana, Missoula, MT 59802, USA Present address: C. G. Guglielmo Department of Biology, University of Western Ontario, London, ON, Canada N6A 5B7 e-mail: [email protected]

Keywords Oxidative stress Æ Antioxidation Æ Free radicals Æ Allantoin Æ Uric acid Æ White-crowned sparrow

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beneficial role as an antioxidant (Ames et al. 1981; Kirschbaum 2001; Hediger 2002; Enomoto et al. 2002). The plasma concentrations of uric acid and its salts in birds are exceptionally high compared with other vertebrates (Lumeij and Remple 1991), sometimes well above the limit of its theoretical solubility (for sodium urate: 0.6 mM at 43C; Lumeij and Remple 1991). The antioxidative properties of uric acid and its high concentrations in birds’ plasma have been suggested as a possible mechanism to protect birds from oxidative damage (Klandorf et al. 2001; Simoyi et al. 2002, 2003). Indeed, increased plasma uric acid concentration in chickens reduced leukocyte oxidative activity (Simoyi et al. 2002; Machı´n et al. 2004), while decreased concentrations were associated with increased leukocyte oxidative activity (Klandorf et al. 2001). Allantoin, the oxidative product of uric acid, has been proposed as a potential biomarker for in vivo free radical reactions (Grootveld and Halliwell 1987; Lagendijk 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 in most other mammalian species) and birds (Moriwaki et al. 1999) implies that the presence of allantoin in their plasma results only from the non-enzymatic oxidation of uric acid. In humans, the plasma concentration of allantoin and/or the allantoin:uric acid ratio increases during oxidative stress situations such as chronic lung diseases (James et al. 2003), Down syndrome (Zitnanova et al. 2004), brain ischemia (Marklund et al. 2000), rheumatoid arthritis (YardimAkaydin et al. 2004) and chemotherapy treatment (Durken et al. 2000). Exercise in humans and other mammals is also associated with increased formation of free radicals, which can damage cells and tissues (Wetzstein et al. 1998; Ji 1999; Liu et al. 1999; Mastaloudis et al. 2001; Muradian et al. 2002; Chevion et al. 2003). Interestingly, the allantoin concentration and the allantoin:uric acid ratio in human plasma and muscle tissue were found to increase during intensive exercise, demonstrating the presumptive antioxidative function of uric acid (Hellsten et al. 1997, 2001; Mikami et al. 2000a, b). The effect of exercise on the production of free radicals in birds is not yet clear. The basal and maximal rate of H2O2 production, oxygen consumption and free radical leak in the respiratory chain in heart, brain and lung mitochondria of birds are lower than in mammals of similar size (Herrero and Barja 1997; Barja 1998). Also, birds express only the dehydrogenase form of the enzyme xanthine oxidoreductase which does not produce free radicals (Sato et al. 1995; Moriwaki et al. 1999). Hence, theoretically, they should suffer less

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J Comp Physiol B (2006) 176:653–661

from oxidative stress during activity. Allantoin has been 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 concentration changes as in mammals. As uric acid is the end product of protein catabolism in birds, its concentration in the plasma is affected by the protein turnover rate. It has been shown that the concentration of uric acid increases in the plasma after intensive flight (Gannes et al. 2001; Klaassen et al. 2000), where it is associated with protein degradation. If uric acid functions as an antioxidant this could be a beneficial mechanism to lower oxidative stress at that time. The aim of this study was to evaluate the effect of exercise on the relationship between the concentrations of uric acid and allantoin in the plasma and urine of white-crowned sparrows. Specifically, we measured the concentrations of uric acid and allantoin in the plasma and in the ureteral urine of white-crowned sparrows at rest, immediately after 30 min of exercise in a hop/hover wheel and after 1 h of recovery. If birds follow the human model we would expect that the allantoin to uric acid ratio would increase during and following activity as a result of a higher rate of uric acid oxidation caused by increased production of free radicals. We also expected that the allantoin:uric acid ratio in the ureteral urine would be highest during recovery and lowest at rest, as we expected the birds to defend uric acid concentration in the plasma during activity and hence excrete less uric acid during this phase.

Material and methods Care and maintenance Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii), were caught near Mabton, Washington in September 2003 (n = 20). The birds were held in an outdoor aviary at the University of Montana Research Station at Fort Missoula for 10 months before the experiments, and maintained on a mixed seed diet (40% white millet, 40% cracked corn and 20% sunflower; Purina Mills, Regional recipe). Four days before the initiation of experiments the birds were transferred to individual cages (40 · 45 · 45 cm) in a room at 22C, and kept on a light:dark cycle similar to the natural cycle (17L:7D in July). Seeds and water were provided ad libitum. Possession of birds and experimental protocols were approved by the U.S. Fish and Wildlife Service, the Montana Department of Fish, Wildlife and Parks, and the University of Montana Institutional Animal Care and Use Committee.

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Experimental design To avoid the possible adverse effect of consecutive bleedings (taking over 10% of blood volume within 5 days) while taking three blood samples from all birds (resting, exercising and recovery), birds were randomly divided into two groups. We first took blood and urine samples (‘‘resting’’ samples) from the first group. These birds were left to recover for 5 days, and were then exercised (‘‘exercise’’ followed by ‘‘recovery’’ samples). The second group was first exercised (‘‘exercise’’ followed by ‘‘recovery’’ samples) then left to 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 without food and a third hour without food and water. Birds were exercised one at a time. Food was withdrawn 2 h before exercise. Birds were then placed in the wheel chamber for 15 min before exercise. During this period, we covered the wheel chamber with black cloth to let the birds settle, and measured minimal VO2 for 2 min (see below). We turned the wheel by hand for 30 min at a speed of 30–40 rpm. Blood and ureteral urine samples were taken immediately ( < 5 min) after the termination of exercise. The birds were then returned to the cages for 1 h, during which time they had water available (but not food), and thereafter sampled again (‘‘recovery’’ samples). The birds were weighed before and after exercise to the nearest 0.01 g. Urine and blood samples Ureteral urine samples were collected by briefly inserting a closed-ended, perforated cannula, custommade of polyethylene tubing (PE160, Intramedic, MD, USA), into the bird’s cloaca (Goldstein and Braun 1986). We collected blood (~ 160 ll) in heparinized microhematocrit tubes by puncturing the brachial vein with a 26-gauge needle. Plasma was separated from cells after centrifugation at 2,000 g for 6 min. Samples were immediately frozen at –196C in liquid N2 and were analyzed within 20 days. VO2 measurements Oxygen consumption and CO2 production were measured in an open-flow running wheel chamber, modified for hopping/hovering birds (Chappell et al. 1999). The internal wheel dimensions were approximately 16 cm in width and 27 cm in diameter (volume of 9,156 cm3). The flow rate of dried (Drierite), CO2-free (Ascarite) air was regulated and measured at 3.5 l min–1 by a STP-corrected mass flow controller/meter (Sierra

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Instruments 840-L, Monterey, CA, USA). Approximately 150 ml min–1 of air exiting the respirometer wheel was sub-sampled, dried and passed through a CO2 analyzer (Sable Systems CA-2A, Las Vegas, NV, USA). The excurrent air was then redried, scrubbed of CO2 and passed through an O2 analyzer (Sable Systems FC-1B). Gas analyzers were calibrated with N2 and a standard, certified dry gas mixture (77.10% N2, 20.90% O2, 2.00% CO2; NORCO, Missoula, MT, USA). Analog outputs were converted to digital format by an analog/digital converter (Sable Systems UI2) and collected by a PC computer. The 2 min minimum (average of lowest 2 min of VO2 measured, before exercising), the 2 min maximum (average for highest 2 min of VO2 measured) and the 30 min mean VO2 during exercise, were calculated using Datacan V software (Sable Systems). Uric acid and allantoin assays Uric acid was measured by the endpoint assay (WAKO Diagnostics UA 20R/30R), modified for a micro-plate spectrophotometer (Biotek Powerwave X340, Winooski, VT, USA), as follows: a 5 ll sample or standard was mixed with 300 ll of the pre-warmed (to 37C) reagent, incubated for 10 min at 37C, shaken and the absorbance read at 550 nm (700 nm reference). Plasma was run undiluted whereas urine was diluted 30–80 times with LiOH. LiOH dissolves the urate precipitates and any trapped ions (Roxburgh and Pinshow 2002; Tsahar et al. 2005). A uric acid standard (3 mM) was prepared in 0.1 M glycine buffer, pH 9.3. Because the uric acid assay uses the enzyme uricase to convert uric acid to allantoin and then measures total allantoin, we subtracted the allantoin concentration from that of uric acid for each sample. Plasma (70 ll) and urine (100 ll) were analyzed for allantoin. Allantoin was determined by the Rimini–Schryver reaction described by Young and Conway (1942) (see also Poffer et al. 2002). In this reaction allantoin is first hydrolyzed under weak alkaline conditions at 100C to allantoic acid, which is then further degraded to urea and glyoxylic acid in a weak acid solution. The glyoxylic acid then reacts with phenylhydrazine hydrochloride to produce a phenylhydrazone of the acid. This product forms an unstable chromophore with potassium ferricynide. The color is read in a spectrophotometer (Beckman DU640) at 522 nm, 20 min after the reaction. To eliminate the possibility that uric acid might interfere with the specificity of the reaction, we ran the assays using standards of uric acid (0.2–5 mM). Uric acid did not absorb light at 522 nm and hence we concluded that it does not interfere with the reaction. All chemicals

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were purchased from Sigma (Sigma Chemical, St. Louis, MO, USA). Osmolarity Plasma osmolarity was measured in a freeze-point depression osmometer (Osmette II, Precision Systems, Natick, Massachusetts, USA). Statistical analysis Least-squares linear regression was used to test for relations between the concentrations of uric acid and allantoin in the plasma and in the ureteral urine, and the relation between plasma concentrations of uric acid and allantoin immediately after exercise and maximum VO2. A repeated measures ANOVA was used to compare the plasma and ureteral urine concentrations of uric acid and allantoin and plasma osmolarity among the three activity phases. We used a linear model to assess the effect of the three activity phases (resting, exercising and recovery) on the relations between uric acid and allantoin concentrations in the plasma and in the ureteral urine. The linear model used in the analysis was: y = b0 + b1 x1 + b2 x2 + b3 x1x2 + b4 x3 + b5 x1x3 + , where y is the dependent variable (allantoin concentration), x1 is uric acid concentration, and x2 and x3 are dummy variables that represent the effect of phase. The recovery phase was randomly chosen as the reference for the intercept (b0) and the slope (b1) comparisons, b2 is the difference between the exercising and the recovery intercepts, b3 is the difference between the exercising and the recovery slopes, b4 is the difference between the resting and the recovery intercepts, and b5 is the difference between the resting and the recovery slopes. Data are reported as means ± SE. Significance was accepted at P < 0.05.

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The average lowest 2 min VO2 measured before exercising (163.8 ± 7.9 ml h–1) was significantly lower than the 2 min maximal VO2 (311.7 ± 10.2 ml h–1) during exercise (paired t13 = 14.29, P < 0.0001), and 2 min maximum VO2 ranged between 257 and 405 ml h–1. The average VO2 for the 30 min exercising was 233 ± 14.3 ml h–1. The plasma concentrations of uric acid and allantoin measured immediately after exercise, but not at recovery, were positively correlated with the maximum VO2 (Fig. 1). The plasma concentration of uric acid measured at the recovery was correlated with body mass loss (r = 0.7, P = 0.004, n = 15). Respiratory exchange ratio (VO2/VCO2) measured during maximum activity was 0.87 ± 0.02. Uric acid and allantoin in the plasma We had 14 birds with full data sets (three activity phases) of plasma samples (volume was insufficient in some of the samples taken), hence the following statistical analysis was applied only for these birds (n = 14). The plasma concentration of uric acid differed significantly among the three activity phases; the highest concentration was measured after 1 h of recovery and the lowest in the resting phase (withinsubjects repeated measures ANOVA: F2,26 = 7.24, P = 0.003, Table 1). The plasma concentration of uric acid in the recovery phase was significantly higher than that of the resting and exercising phases, while that of the exercising and resting phases did not differ one from the other (Fig. 2). However, when the percent body mass loss was added as a covariant, there was no effect of the activity phase on the uric acid plasma

Results Body mass loss and metabolic rate during exercise During the 30 min exercise period in the running wheel, the birds lost 2.99 ± 0.32% of their initial body mass (23.7 ± 0.46 g). Percent of body mass loss was correlated with the average VO2 for 30 min exercise (r = 0.618, P = 0.006, n = 18). Plasma osmolarity did not 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 measures ANOVA: F2,14 = 0.209, P = 0.8).

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Fig. 1 The plasma concentrations of uric acid (filled circles) and allantoin (open circles) of exercising white-crowned sparrows increased as functions of maximum VO2 (uric acid: y = 0.004x – 0.85; r = 0.39, F1,16 = 10.38, P = 0.0053; allantoin: y = 0.0013x – 0.21, r = 0.24, F1,16 = 4.87, P = 0.043)

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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 phases Rest

Exercise

Mean ± SE Plasma uric acid (mM) Plasma allantoin (mM) Plasma AUR Ureteral urine uric acid (mM) Ureteral urine allantoin (mM) Ureteral urine AUR

0.34 0.15 0.50 50.30 4.95 0.29

± ± ± ± ± ±

0.035a 0.01ns 0.08ns 22.22ns 2.16ns 0.1ns

Range

Mean ± SE

0.08–0.51 0.09–0.22

0.39 0.15 0.39 83.42 4.08 0.07

3.43–270 0.56–28

± ± ± ± ± ±

0.037a 0.02ns 0.02ns 24.78ns 0.66ns 0.02ns

Recovery Range 0.19–0.75 0.08–0.34 18.89–340.2 1.18–8.52

Mean ± SE 0.49 0.17 0.37 33.51 3.97 0.15

± ± ± ± ± ±

0.048b 0.01ns 0.03ns 6.73ns 0.83ns 0.04ns

Range 0.22–0.93 0.09–0.22 9.35–81.74 0.16–8.66

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)

concentration (within-subjects repeated measures ANOVA: F2,24 = 0.057, P = 0.94). The allantoin concentration did not differ among the activity phases (within-subjects repeated measures ANOVA: F2,26 = 1.105, P = 0.34, Table 1, Fig. 2). Adding the body mass loss as a covariant had no effect on the result. No significant differences were found in the allantoin to uric acid ratio among the three activity phases (within-subject repeated measures ANOVA: F2,26 = 2.32, P = 0.12). When body mass loss is added as a covariant the ratio significantly differs among the activity phases (within-subjects repeated measures ANOVA: F2,24 = 9.28, P = 0.001). Plasma concentrations of allantoin and uric acid were positively correlated in all three activity phases (rest: r = 0.48, P = 0.006; exercise: r = 0.7, P = 0.0002;

Fig. 2 The plasma concentration of uric acid (filled bars) of white-crowned sparrows in the recovery phase was significantly different from that during the resting and exercising phases (Bonferroni Test, P < 0.05). No significant differences were found in the concentration of allantoin (open bars) among the three activity phases. Data are presented as means ± SE (n = 14). Different letters above bars denote significant differences (P < 0.05). ns not significant, P > 0.05

recovery: r = 0.5, P = 0.004). The slope of the regression of plasma allantoin as a function of uric acid in the exercise phase was significantly higher than the slopes of the resting and recovery phases, while the intercept of the exercising phase was significantly lower (Fig. 3). No significant difference was found between the slopes and intercepts of the resting and recovery phases. Uric acid and allantoin in the ureteral urine We had 12 birds with full data sets of ureteral urine samples (the urine volume was insufficient in some of the samples taken), hence the following statistical analysis was applied only for these birds (n = 12). The concentrations of allantoin and uric acid in the ureteral urine were significantly related in the resting and exercising activity phases (r = 0.73, P = 0.0004; r = 0.41, P = 0.024, respectively) but not in the recovery phase (r = 0.3, P = 0.07). There were no significant differences among the slopes of the regression lines and among the intercepts of the three phases (P > 0.05). No significant differences were found in the concentrations of uric acid and allantoin among the three activity phases (within-subjects repeated measures ANOVA: F2,22 = 1.59, P = 0.23; F2,22 = 1.69, P = 0.85, respectively). No significant differences were found in the allantoin to uric acid ratio among the three activity phases (within-subjects repeated measures ANOVA: F2,22 = 2.57, P = 0.099). Adding body mass loss as a covariant did not affect the results. The concentrations of uric acid and allantoin in ureteral urine are summarized in Table 1.

Discussion The present study demonstrates that allantoin, the oxidative product of uric acid, is present in the plasma

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and in the ureteral urine of white-crowned sparrows. Because birds do not posses the enzyme urate oxidase, the presence of allantoin in their plasma indicates a direct oxidation of uric acid. Strong oxidizers such as hypochlorous acid and hydroxyl radicals rapidly oxidize uric acid into allantoin and other products (Kaur and Halliwell 1990). This oxidation inactivates the free radical by electron transfer from uric acid to the free radical before the free radicals oxidize a potential molecular, hence uric acid functions as an antioxidant (Simic et al. 1989). Contrary to our prediction, no differences were found in the concentration of allantoin or in the allantoin to uric acid ratio in the plasma and in the ureteral urine among the three activity phases. When body mass loss was added as a covariant to the analysis, the ratio was significantly different, but contrarily to what we expected, the lowest ratio was found at the recovery phase, which might indicate the effect of protein catabolism during exercise on the plasma uric acid concentration. However, we did find a positive correlation between uric acid and allantoin concentrations in the plasma, and that the slope of this correlation was higher immediately after exercise compared with the resting and recovery phases. To the best of our knowledge, this is the first time that a positive correlation between the concentrations of uric

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acid and allantoin in the plasma is found in an in vivo system (Mikami et al. 2000b). Humans are similar to birds in that they do not posses the enzyme urate oxidase. However, it is not surprising that such a correlation was not found previously in humans, where this system was intensively studied (Hellstein-Westing et al. 1994; Hellstein et al. 1997, 2001; Mikami et al. 2000a, b), as the plasma concentration of uric acid in humans is tightly regulated within a relatively narrow range. In contrast, its concentration in birds’ plasma is evidently extremely variable. The variability in avian uric acid concentration in plasma allowed us to reveal such a correlation (Fig. 4). One potential explanation for the linear relation between uric acid and allantoin is that a certain percentage of uric acid is constantly being oxidized. Factors that might affect the fraction of uric acid oxidized could include the concentrations of free radicals and of other antioxidants in the system. We hypothesize that the slope of the relation between the plasma concentrations of uric acid and allantoin measures the relative intensity of the uric acid oxidation reaction, namely, the higher the slope the greater the fraction of uric acid oxidized to allantoin per unit of time. Thus, immediately after exercise, the rate of oxidation was higher, as indicated by the higher slope of the regression. We suggest that this increase in the slope of allantoin against uric acid concentration in the plasma of the white-crowned sparrows could result from higher production of free radicals during exercise. 0.5 Allantoin concentration in plasma (mM)

Fig. 3 The plasma concentrations of uric acid and allantoin of the white-crowned sparrows were significantly correlated in all activity phases (resting open diamond; exercise open square; recovery open triangle). No significant differences were found between the slopes and between the intercepts of the resting and the recovery phases (t = 0.244, P = 0.8; t = 0.023, P = 0.98, respectively). Thus, data of the resting and recovery phases were pooled and presented by the dashed line (y = 0.087 + 0.17x). The slope of the regression in the exercise phase (y = –0.006 + 0.403x, bold line) was significantly higher from the slopes of resting and recovery phases (t = 2.87, P = 0.007), whereas the intercept of the exercise phase was significantly lower (t = –2.51, P = 0.017)

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0.4

y = 0.2965x + 0.0416 2

R = 0.649 0.3

0.2

0.1

Athletes Healthy humans

0 0

0.2 0.4 0.6 0.8 Uric acid concentration in plasma (mM)

1

1.2

Fig. 4 The plasma concentrations of uric acid and allantoin of the white-crowned sparrows were significantly correlated (F1,49 = 88.74, P < 0.0001, pooled data from all phases). Such a correlation has not been reported for humans (see insets), in which the uric acid concentration is tightly regulated within a relatively narrow range (uric acid: healthy humans: 0.166– 0.470 mM, athletes 0.319–0.507 mM; allantoin: healthy humans 0.011–0.068 mM, athletes 0.037–0.091 mM, Mikami et al. 2000b)

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The highest concentration of plasma uric acid of the white-crowned sparrows in the present study was measured in the recovery phase, 1 h after exercise. The osmolarity of the plasma did not differ among the activity phases, suggesting that the change in uric acid and allantoin concentrations were not the result of dehydration. An increased plasma concentration of uric acid has been detected previously after long and short flights in several bird species (Jenni-Eiermann and Jenni 1991; George and John 1993; Shmueli et al. 2000; Gannes et al. 2001; Jenni-Eiermann et al. 2002), and is commonly used as an indicator of protein oxidation (Jenni-Eiermann and Jenni 1991; Gannes et al. 2001). The correlation between plasma uric acid concentration at the recovery phase and body mass loss might suggest that the increased concentration of uric acid resulted from protein degradation. We speculate that one of the consequences of increased protein catabolism during prolonged exercise may be an improved antioxidative defense resulting from the higher uric acid concentration. A similar pattern of increased plasma uric acid in the recovery phase, rather than immediately after exercise, was found in mammalian species including humans (Po¨so¨ et al. 1994; Ra¨sa¨nen et al. 1995; Liu et al. 1999; Mastaloudis et al. 2001). Unlike birds, mammals do not oxidize proteins into uric acid, and post-exercise accumulation of uric acid in mammalian plasma is the result of degradation of purine nucleotides (e.g. ATP, AMP, IMP, Hellsten et al. 1999). This similar pattern suggests that birds and mammals might share similar regulatory mechanisms of purine catabolism during exercise. The concentrations of uric acid and allantoin could also be affected by the rate of uric acid and allantoin clearance, as the glomerular filtration rate (GFR) of birds might change during flight (Giladi et al. 1997). In avian kidneys, urate is excreted primarily as the result of its active secretion in proximal tubuli, rather than of its glomerular filtration (Goldstein and Skadhauge 2000). The mechanism of allantoin excretion in birds has not been studied so far (Poffers et al. 2002). In rats, it is freely filtered and no reabsorbtion or secretion was detected along the nephrons (Briggs et al. 1977). Hence, if allantoin excretion follows the mammalian type, and if GFR decreases during exercise while secretion does not, the excretion rate of allantoin but not of uric acid would decrease during activity. This would affect the ratio of uric acid to allantoin, both in the plasma and in the urine, during activity. We are aware of only two more studies which measured allantoin in the plasma of avian species. Simoy et al. (2003) studied allantoin to uric acid ratio in chicken and turkey plasma. This ratio was higher than

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that of the white-crowned sparrows in the present study (chicken: 1.680–1.187, turkey: 1.679–1.039). Poffer et al. (2002) studied pigeons (Columba livia domestica) and red-tailed hawks (Buteo jamaicensis) and found ratios more similar (~ 0.76 and ~ 0.28, respectively; calculated from the data) to that of the white-crowned sparrows (0.29 ± 0.02, n = 14, combined mean ± SE of all activity phases). Such differences among species could arise from many reasons, among them metabolic stress and diet. We did not find a significant effect of exercise on the allantoin concentration or the expected effect on allantoin to uric acid ratio. The system is complicated and governed by many factors, among them protein catabolism rate and the rate of nitrogenous products elimination, which both are affected by exercise intensity. It is also possible that the exercise in the present study was not intensive enough to exhibit such an effect. Yet, we cannot rule out the importance of allantoin as a marker for oxidative stress in birds. Acknowledgments We wish to thank Prof. Carlos Martı´nez delRio for his help and involvement throughout the study including his advices in the manuscript preparation and his help in the statistical analysis. Also thanks to Edwin Price, Quentin Hays, David Cerasale for their assistance in catching and maintaining the birds and in blood sampling, and for their kind hospitality, and Bradley H. Bakken for his assistance in analyzing the samples. We also thank three anonymous reviewers whose comments greatly improved this manuscript. The study was partially financed by grants to C. Martı´nez del-Rio (NSF IBN-0110416) and to C.G. Guglielmo (NSF IBN-0224954), by the Technion’s J. and A. Taub Biological Research Fund and J. S. Frankford Research Fund (to Zeev Arad), and by the Technion-University of Haifa Interuniversity Research Fund (to Zeev Arad and Ido Izhaki).

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