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1 Division of Animal and Veterinary Sciences, West Virginia University, Morgantown 26506-6108; and 2 Department of Pharmacology and Toxicology, Robert C. Byrd West Virginia University School of Medicine, West Virginia University, Morgantown, West Virginia 26506
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Birds have high metabolic rates, body temperatures, and plasma glucose concentrations yet physiologically age at a rate slower than comparably sized mammals. These studies were designed to test the hypothesis that the antioxidant uric acid protects birds against oxidative stress. Mixed sex broiler chicks (3 wk old) were fed diets supplemented or not with purines (0.6 mol hypoxanthine or inosine). Study 1 consisted of 18 female Cobb × Cobb broilers that were fed purines for 7 days, whereas study 2 consisted of 12 males in a 21-day trial. Study 3 involved 30 mixed sex broilers that were fed 40 or 50 mg allopurinol/kg body mass (BM) for 21 days, a drug that lowers plasma uric acid (PUA). PUA and leukocyte oxidative activity (LOA) were determined weekly for all studies. For study 2, pectoralis major shear force, relative kidney and liver sizes (RKS and RLS), and plasma glucose concentrations were also determined. In study 1, PUA concentration was increased three- and twofold (P < 0.001) in birds fed inosine or hypoxanthine, respectively, compared with control birds. LOA of birds supplemented with inosine was lower (P < 0.05) than that of control or hypoxanthine birds. In study 2, PUA concentrations were increased fivefold (P < 0.001) in birds fed inosine and twofold (P < 0.001) in birds fed hypoxanthine compared with control birds at day 21. RKS (g/kg BM) was greater (P < 0.001) for chicks fed purine diets compared with control chicks. Muscle shear value was lower (P < 0.05) in chicks fed purine diets. PUA concentration was decreased (P < 0.001) in birds consuming allopurinol diets, whereas LOA was increased (P < 0.01) in study 3. These studies show that PUA concentrations can be related to oxidative stress in birds, which can be linked to tissue aging.
purines; allopurinol
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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BIRDS HAVE GREAT LONGEVITY for their body size (6), despite the fact that they also have much higher metabolic rates, body temperatures, and blood glucose concentration compared with mammals (13). Theoretically, birds should sustain proportionately greater damage from processes such as macromolecular damage mediated by oxidants and the glycation and glycoxidation of proteins and nucleic acids (11), processes that have been proposed to be involved in much of the physiological deterioration that causes senescence. This suggests that birds have evolved mechanisms to alleviate the oxidative insult. For example, a 20-g mouse that lives 3 years experiences about one-twentieth the oxidative burden of a 20-g canary that lives 20 years (6). Therefore, birds have mechanisms either to reduce the rate of production of oxidants per unit of energy expended and/or that they have enhanced antioxidant defenses.
Recent studies revealed that urate is one of the strongest determinants of serum lipid resistance to oxidation (9, 12, 21). Hellstein and co-workers (5) proposed that uric acid is a potent scavenger of oxidants in human and animal tissues. However, very high uric acid levels in mammals result in gout, a disease in which uric acid crystals are deposited in joints. Uric acid is bound to a protein in the nephrons of avian kidneys, resulting in a soluble compound, hence birds can tolerate very high uric acid levels (2). The role of uric acid as an oxidant scavenger is not limited to vertebrates. Souza and co-workers (20) presented evidence that uric acid in blood-sucking insects (Rhodnius prolixus) constituted the major oxidant scavenging system against heme-induced oxidations. The present investigation was designed to test the hypothesis that uric acid, a naturally occurring product of purine metabolism, plays a critical role in regulating oxidative stress and this can be linked to reduction of deleterious processes of oxidatively induced aging.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Study 1
The purpose of this trial was to determine the effect of inosine and hypoxanthine on plasma uric acid (PUA) concentration and leukocyte oxidative activity (LOA) of broilers. Eighteen 3-wk-old Cobb × Cobb female chicks were randomly assigned to three treatments replicated three times with two chicks per pen. Birds were fed inosine- or hypoxanthine-supplemented (0.6 mol/kg) diets for 7 days. Control diet contained an equal weight of cornstarch. Feed and water were offered ad libitum. LOA and PUA concentration were determined.Study 2
The purpose of this study was to look at the effects of inosine and hypoxanthine in greater depth. Twelve 3-wk-old Cobb × Cobb male chicks were placed at random into three pens, each representing a dietary treatment. Treatments here were similar to those of study 1. Chickens were given free access to feed and water throughout the 3-wk-long experiment. PUA and glucose concentrations, LOA, relative kidney size (RKS), relative liver size (RLS), and pectoralis major shear force were determined.Muscle shear force. Frozen pectoralis major muscles were thawed and cooked to an internal temperature of 72°C on a smokeless indoor grill (Farberware, Bronx, NY). Cooked muscle was cooled to room temperature and processed for shear force measurement on the same day. Shear values (SV), reported in kilograms, were determined on a TA-HDi Texture Analyzer (Texture Technologies, Scarsdale, NY). A Stable Micro Systems apparatus was attached to a 50-kg load cell, and tests were performed at a crosshead speed of 127 mm/min.
Uric acid and glucose determination. PUA was determined using a commercially available diagnostic kit (Sigma Diagnostics, St. Louis, MO). Plasma glucose was measured using the YSI 2700 Select Biochemistry Analyzer (Yellow Springs Instrument, Yellow Springs, OH).
LOA. Chemiluminescence techniques are functional assays to quantify the release of oxidants from cells or tissues (15, 22). Luminol-based chemiluminescence (LBCL) was used to estimate LOA as described by Iqbal and co-workers (8). A Berthold luminometer (Multi-Biolumat LB 9505C model, Berthold Australia, Bundoora, Victoria, Australia) was used for this procedure, and results were reported as integrated light units.
Study 3
The purpose of this study was to determine the effect of lowering PUA concentration on LOA. Thirty 3-wk-old Cobb × Cobb mixed sex broiler chicks were randomly assigned to three treatments each with two replications of five chicks per pen. Diets were supplemented with 40 or 50 mg allopurinol measured per kilogram body mass (BM) and fed to chicks for 3 wk. Allopurinol lowers PUA and can be used to treat hyperuricemia (gout). Control diet was supplemented with cornstarch in place of allopurinol. Feed and water were offered ad libitum. PUA concentration and LOA were determined weekly.Statistics
Statistical analyses were performed with the SAS software version 7 (17) using the general linear models procedure to perform analysis of variance. Sampling error (birds within pen) was used to test hypotheses in study 2. Multiple comparisons were performed between treatment means to determine significant differences. Differences were considered significant at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Study 1
PUA concentration.
Birds fed inosine-supplemented diets had greater (P < 0.001) PUA concentration compared with birds fed cornstarch- (control) or hypoxanthine-supplemented diets (Fig.
1). Inosine was more effective in raising
PUA concentration compared with hypoxanthine.
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LOA.
Birds fed inosine-supplemented diets had the least (P < 0.05) LOA compared with birds receiving cornstarch- or
hypoxanthine-supplemented diets (Fig. 2).
PUA concentration was inversely correlated with LOA according to the
linear regression relationship: Y = 
0.9x + 2.1 with an r2 of 0.85.
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Study 2
PUA concentration.
After 1 wk of being fed inosine or hypoxanthine, birds from both groups
had significantly greater PUA concentrations than birds fed the control
diet (Fig. 3). By week 2, PUA
concentration in birds fed the inosine-supplemented diet had increased
fivefold compared with birds receiving the control diet
(P < 0.001). Compared with birds fed
hypoxanthine-supplemented diet, inosine supplemented birds had two
times higher (P < 0.001) PUA concentration (Fig. 3).
Throughout the experiment, birds fed inosine consistently showed the
highest PUA concentration.
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LOA.
LBCL-measured oxidative activity increased progressively in control
birds for the duration of treatment. After 1 wk of treatment, birds fed
inosine- or hypoxanthine-supplemented diets exhibited little response
to treatment (Fig. 4). By week
2, LOA of hypoxanthine-supplemented birds was three times lower
than that of birds receiving control diet (P < 0.001).
Birds fed inosine-supplemented diet, which consistently had the highest
PUA concentration throughout the study, had twofold lower
(P < 0.01) LOA than birds fed
hypoxanthine-supplemented diet. PUA concentration was inversely
correlated with LOA according to the linear regression relationship:
Y = 4.8x + 5.5 with an r2 of 0.95.
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Relative kidney and liver sizes.
Differences in RKS between birds fed control diet and birds fed
inosine- or hypoxanthine-supplemented diets were significant (P < 0.001) (Table 1).
Birds fed inosine-supplemented diet had higher (P < 0.05) RKS compared with birds fed hypoxanthine-supplemented diet. There
were no significant differences (P > 0.05) in RLS (Table 1).
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Pectoralis major shear values. Average SV of pectoralis major muscles of birds fed control diet, inosine-, or hypoxanthine-supplemented diets were 3.9, 2.8, and 2.7 kg, respectively. Muscles of birds fed control diet had higher (P < 0.01) SV compared with those of birds fed inosine- or hypoxanthine-supplemented diets (Table 1), signifying that meat from birds fed inosine- or hypoxanthine-supplemented diets had fewer crosslinks than that from birds consuming control diet.
Plasma glucose concentration.
There were no consistent differences in plasma glucose concentrations
between the treatments (Table 2).
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Study 3
PUA.
Throughout the experiment, birds fed control diet had the highest
(P < 0.01) PUA concentration compared with birds
consuming allopurinol-containing diets (Fig.
5). The lowest PUA concentrations were
recorded from birds consuming diets supplemented with 50 mg
allopurinol.
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LOA.
The lowest (P < 0.01) LOA was recorded from birds
consuming control diet throughout the trial (Fig.
6). There was a twofold increase in LOA
in week 1 and a threefold increase in week 2 in birds consuming the diet supplemented with 50 mg allopurinol compared with birds consuming control diet. PUA concentration was inversely correlated with LOA according to the linear regression relationship: Y = 12.6x + 7.4 with an
r2 of 0.87.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Oxidative stress arises because oxygen is easily converted into toxic reactive oxygen species (ROS) (19). The predominant ROS are superoxide radical ion, hydrogen peroxide, and the hydroxyl radical ion. The latter is the most toxic and will attack and damage proteins, lipids, and DNA. The life of a cell critically depends on powerful antioxidant systems against ROS. Oxidative stress occurs whenever the production of damaging ROS exceeds the capacity of the antioxidant defenses and is involved in a large number of diseases (19). It has been shown that uric acid, a naturally occurring compound that selectively binds and inactivates peroxynitrite, inhibits the onset of clinical disease in an acute, aggressive form of mouse experimental allergic encephalomyelitis (7).
Dietary purines have been shown to have uricogenic effects in rats (3). Our study showed that inosine and hypoxanthine were uricogenic and that inosine was more effective. Intuitively, hypoxanthine may seem to be more uricogenic, considering that it is one step ahead of inosine in the purine catabolism pathway for which the end product is uric acid. This was not the case in our study. A possible explanation for this is that inosine uptake from the small intestine of chickens is greater than that of hypoxanthine because inosine has a sugar moiety.
The theory of aging (14) is based on the fact that maximum
life span (MLSP) of a given species increases as the aerobic metabolic
rate at rest decreases and body size increases (4, 14,
16). This theory of aging is largely discredited, because there
are large groups of animal species in which the MLSP is much higher
than predicted 
O2 values
(1), e.g., birds. Birds are unique because they are the
only vertebrate that was able to increase MLSP and
O2 simultaneously during evolution
(1). If free radicals are responsible for the rate of
living phenomenon and constitute an important cause of aging, then the
question of why birds show MLSPs substantially higher than predicted
from their rates of O2 consumption is raised.
In our inosine/hypoxanthine study we showed that PUA concentration is inversely correlated with oxidative activity in birds. We also showed that when PUA levels are lowered, as in the allopurinol study, oxidative stress increases dramatically. Similar effects on PUA and oxidative stress were shown in broiler chickens fed allopurinol-supplemented diets (10). This supports the reasoning that the seemingly low oxidant production per O2 consumed in birds results from a very efficient oxidant scavenging system in these species. Therefore, uric acid is one of the major antioxidants functioning in this system.
Kidney weight per unit BM was significantly higher in birds fed inosine, the treatment that resulted in the highest PUA concentration. The reason for the increase in kidney size may be due to increased processing of uric acid, because the proximal tubule of the nephron is responsible for binding uric acid to a protein that solubilizes uric acid, preventing formation of crystals.
Pectoralis major shear values of birds fed inosine- or hypoxanthine-supplemented diets were significantly lower than that of birds consuming control diet, signifying that meat from these birds was more tender as a result of fewer protein crosslinks. These studies support the view that uric acid reduces oxidative stress with the associated reduction in crosslink generation, which leads to the lowered measurement of shear force. These data demonstrate that uric acid constitutes one of the most important antioxidants in birds and is directly linked to their longevity.
Perspectives
The antioxidant nature of uric acid has only been fully recognized recently. In avian species, uric acid has even greater importance as an antioxidant because birds produce more oxidants than mammals of comparable size per oxygen molecule respired. A reduction in oxidant production leads to other benefits, such as improvement in meat quality and reduced veterinary costs. We found an increase in kidney size in birds on treatments that resulted in higher PUA. This increase in size was not pathological, because no symptoms of kidney failure or nephropathy were observed. Uric acid in birds is excreted and reabsorbed back into the blood in the kidneys, and therefore we propose that this increase in size is to accommodate the increase in uric acid production. We further propose that this is mainly related to the attachment of uric acid to proteins that become chelated to minerals that are later recovered in the ceca.Uric acid inactivates a toxic compound, peroxynitrite, which may be involved in the progressive central nervous system damage that characterizes neurodegenerative diseases. Psoriasis is a chronic inflammatory skin disease in which ROS are thought to play a critical role, and thus the disease could be related to deficient function of the antioxidant system of the organism. Urate was found to be elevated in patients with psoriasis (18). The action of urate on peroxynitrite and its elevation in patients with psoriasis suggest that urate is an important antioxidant in disease states.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Klandorf, Division of Animal and Veterinary Sciences, WVU, Morgantown, WV 26506-6108 (E-mail: Hillar.Klandorf{at}mail.wvu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00437.2001
Received 30 July 2001; accepted in final form 2 November 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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