Determination of xanthine oxidoreductase activity in broilers: Effect of pH and temperature of the assay and distribution in tissues

MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Determination of xanthine oxidoreductase activity in broilers: Effect of pH and temperature of the assay and distribution in tissues M. D. Carro,*†1 E. Falkenstein,* K. P. Blemings,* and H. Klandorf* *Division of Animal and Nutritional Sciences, West Virginia University, Morgantown 26506-6108; and †Departamento de Produccion Animal, Universidad de Leon, 24007 Leon, Spain found in the liver. Traces of enzyme activity were also detected in 3 out of 10 brain samples, and no activity was found in the plasma. Our results show that XOR distribution in chickens differs from that in mammals, in which the highest levels have been found in liver and intestine. An additional objective was the evaluation of the effect of pH (7.2, 7.7, 8.2, and 8.7) and temperature (25 and 41°C) on the enzyme activity in liver and kidney samples. Temperature had a similar effect on both tissues, with the activity at 25°C being about 30% of that measured at 41°C. At 41°C, the enzyme activity in liver and kidney decreased quadratically as pH decreased from 8.7 to 7.2. The highest activity in kidney was measured at pH 8.2, although there were no differences between enzyme activities at pH 8.7 or 8.2 in the liver. Our results indicate that the optimum pH of the enzyme in chicken liver and kidney is around 8.2.

Key words: xanthine dehydrogenase, xanthine oxidase, broiler, pH, temperature 2009 Poultry Science 88:2406–2414 doi:10.3382/ps.2009-00278

INTRODUCTION

(3) xanthine + NAD+ + H2O ↔ uric acid

The enzyme xanthine oxidoreductase (XOR) catalyzes the 2 last steps in purine catabolism, forming uric acid from hypoxanthine and xanthine. Xanthine oxidoreductase exists in 2 distinct functional but interconvertible forms: xanthine oxidase (XO; EC 1.1.3.22), which catalyzes reactions 1 and 2, and xanthine dehydrogenase (XD; EC 1.1.1.204), which catalyzes reaction 3 (Terada et al., 1990):

+ NADH + H+.

(1) xanthine + 2 O2 + H2O ↔ uric acid + 2 O2− + 2 H+ (2) xanthine + O2 + H2O ↔ uric acid + H2O2

©2009 Poultry Science Association Inc. Received June 2, 2009. Accepted July 15, 2009. 1 Corresponding author: [email protected]

The XOR of mammalian tissues is predominantly an XD, which can be converted into XO either irreversibly by proteolysis or in a reversible manner by the oxidation of sulfhydryl residues (Corte and Stirpe, 1972). Richert and Westerfeld (1951) were the first to report that the XD was not autoxidizable in birds and in this respect differs from the more common XO form of mammalian tissues. However, a minor but consistent XO activity has been reported in chicken liver and kidney (Remy et al., 1951; Strittmatter, 1965; Nishino et al., 1989). These results indicate that under specific conditions, avian tissues might contain some XO activity, although most studies on the avian enzyme have focused on the XD form with little attention allotted to XO activity. Previous studies have investigated the effects of specific variations of diet on chicken XD activity (Scholz and Featherson, 1968; Itoh et al., 1978) as well as the developmental patterns of XD in the embryo and the posthatching period (Lee and Fisher, 1971). These stud-

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ABSTRACT Xanthine oxidoreductase (XOR) is the enzyme responsible for the synthesis of uric acid, which exists primarily in the dehydrogenase form in birds. Uric acid is the major end product of the metabolism of nitrogen-containing compounds in birds and it functions as an antioxidant to reduce oxidative stress. Despite the importance of this enzyme, the tissue distribution of XOR in physiologically normal chickens is not well known. In this study, we analyzed XOR activity in extracts of 8 tissues from broilers at 7 and 10 wk of age. No differences in XOR activity due to the age were found in any tissue. Liver and kidney showed the greatest activity, that in the kidney being about 89% of the activity in the liver. Enzyme activity in intestine and pancreas was about 60 and 37% of that in the liver. All breast muscle, heart, and lung samples showed enzyme activity, but values were only 3.0, 1.2, and 0.6% of those

XANTHINE OXIDOREDUCTASE ACTIVITY IN BROILER TISSUES

MATERIALS AND METHODS Broilers and Experimental Procedure Ten 5-wk-old broiler chickens (Ross × Cobb; males) were obtained from West Virginia Agricultural and Forestry Experiment Station (Morgantown), weighed, and

individually banded. Broilers were floor-reared for 5 wk and given a standard grower diet and drinking water ad libitum. The diet was based on corn, soybean meal, soybean oil, defluorinated phosphate, limestone, salt, NB3000 (Nutra Blend LLC, Neosho, MO), methionine (99%), and Coban 90 (Elanco Animal Health, Greenfield, IN) in the proportions of 64.4, 28.1, 4.43, 1.68, 0.73, 0.12, 0.31, 0.10, and 0.08 g/100 g of DM. The diet contained 2,877 Mcal/kg of DM, 18.1% CP, and 0.82% Ca. Recommendations for space, temperature, light, and husbandry were followed (Aviagen, 2009), and the experimental protocol was approved by the West Virginia University Animal Care and Use Committee. Broilers were weighed twice weekly and feed intake was monitored 3 times a week throughout the experimental period. Each week, a blood sample was obtained from the wing vein of 5 broilers and placed into heparinized tubes for measurement of plasma concentrations of uric acid, allantoin, xanthine, and hypoxanthine. After 2 wk, 5 birds were killed by cervical fracture and samples from liver, kidney, pancreas, lung, heart, small intestine (proximal duodenum), breast muscle, and brain were taken and placed into sterile bags. The duodenum was rinsed with sterile saline solution (NaCl 0.9%; 4°C) to eliminate digesta content. Samples were immediately frozen in liquid nitrogen, transported to the laboratory, and stored at −80°C until analyzed for XOR activity. The remaining broilers were killed at 10 wk of age and the same protocol for obtaining tissues was followed.

Tissue Preparation and XO and XD Assay Tissues were prepared as described by Terada et al. (1990). Frozen tissue samples (0.5 g) were homogenized in 4 mL of ice-cold 0.1 M Tris buffer (pH = 8.2) using a Polytron PT 2100 (Kinematika AG, Littau, Switzerland) for 20 s at 19,000 rpm. The Tris buffer contained 10 mM EDTA to inactivate metalloproteases, 1 mM dithiothreitol (DTT) to prevent oxidative conversion of XD to XO, and 0.5 mg/L of leupeptin and 1 mM phenylmethylsulfonyl fluoride as protease inhibitors. The homogenate was centrifuged at 14,000 × g at 4°C for 30 min. One milliliter of the supernatant fraction was immediately chromatographed on Sephadex G-25 (PD-10 desalting colums; GE Healthcare, Piscataway, NJ) and equilibrated with 1.5 mL of the buffer (4°C) used in the homogenization procedure to remove low molecular weight inhibitors. The eluates were stored on ice and assayed for XO and XD activities within 1 to 2 h after homogenizing the tissue. Analysis of XD and XO activities was based on the method described by Terada et al. (1990) with modifications in the temperature and incubation time. The XD and XO activities were assayed by measuring the formation of uric acid when xanthine was incubated with the eluates. Preliminary assays of all tissues from 2 birds, in which the amount of uric acid produced was measured at 15, 30, 45, 60, and 90 min of incubation,

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ies have focused on liver, pancreas, intestine, and kidney tissues, but to our knowledge, there is no information on XD distribution in other chicken tissues. In addition, these studies were conducted with chickens up to 6 wk old, with no information on XOR activity in older birds. In mammalian tissues, XOR activity is widely distributed, with the highest levels being measured in the liver and intestine (Al-Khalidi and Chaglassian, 1965; Parks and Granger, 1986; Berry and Hare, 2004). Although it is well established that XOR can act as a source of superoxide and hydrogen peroxide radicals, XOR also generates uric acid (Harrison, 2002). Most mammals produce uricase, an enzyme that converts uric acid into allantoin for excretion. However, birds, reptiles, higher primates, and humans lack uricase, and consequently, plasma uric acid concentrations are markedly higher. In birds, uric acid formation assumes much greater importance than in mammals because it is the end product of both purine metabolism and the deamination that occurs in protein catabolism (Stevens, 1996), and this may affect XOR tissue distribution. Uric acid also functions as a protective agent against oxidative stress, thus limiting the tissue damage associated with reactive oxygen species (Holmes and Austad, 1995). The objective of this study was to determine the XOR activity in liver, kidney, intestine, pancreas, breast muscle, heart, lung, and brain tissues in broilers aged 7 and 10 wk. The most common method used to determine XOR activity is the spectrophotometric assay, in which the rate of formation of uric acid from hypoxanthine or xanthine is measured (Stirpe and Della Corte, 1969). However, comparing absolute values of XOR activity measured in different studies is difficult because of the temperature and buffer variations in the measurements (Nishino et al., 1989). In most studies involving mammalian tissues, XOR activity has been measured at either 25°C (Rajagopalan and Handler, 1967; Wajner and Harkness, 1989) or 37°C (Terada et al., 1990; Saksela and Raivio, 1996). In studies involving avian tissues, the activity of XOR has been measured at 25°C (Strittmatter, 1965; Nishino et al., 1989; Sato et al., 1995), 30°C (Bruguera et al., 1988), 38°C (Remy et al., 1955), or 41°C (Scholz and Featherston, 1968). Moreover, a relatively wide range of pH, ranging in most studies from 7.0 to 8.5 (Wajner and Harkness, 1989), has been used to measure XOR activity despite the clear pH dependence of the enzyme shown in human liver (Wajner and Harkness, 1989) and chicken liver (Remy et al., 1955). A second objective of this study was thus to determine the effects of temperature and pH on XOR activity from broiler tissues.

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Plasma Assays Uric acid, allantoin, xanthine, and hypoxanthine in plasma were determined by HPLC based on the method described by Lux et al. (1992). Heparinized blood samples were centrifuged at 2,000 × g, 4°C for 15 min. Plasma was collected and 0.5 mL was mixed with 0.5 mL of 5 mM potassium dihydrogen phosphate mobile phase buffer (pH 3.1), vortexed, and transferred into prewashed filter units (Millipore Ultrafree-MC 30000 MWCO; Millipore, Billerica, MA). The mixture was centrifuged at 3,800 × g for 20 min and filtered again through 0.2-µm syringe filters (Fisher Scientific Co., Pittsburgh, PA). Samples were stored at −20°C until analyzed. Ten microliters of each sample was injected into a Waters Breeze HPLC system (Waters Corporation,

Milford, MA), which comprised a pump (Waters 1525 Binary HPLC Pump with a Waters 717 Plus Autosampler), a variable wavelength absorbance detector (Waters 2487 Dual Absorbance Detector), a cartridge guard column (YMC ODS-AQ, 120 Å, 250 × 4.6 mm inside diameter), and a reverse-phase analytical column (C18 YMC ODS-AQ, 120 Å, 5 µm, 250 × 4.6 mm inside diameter). The mobile phase was aqueous 5 mM potassium dihydrogen phosphate containing 5 mmol/L of 1-heptanesulfonic acid (ion-pairing reagent) and adjusted to pH 3.1 using orthophosphoric acid. The flow rate was 1.0 mL/min, and detection was at 270 nm for uric acid, xanthine, and hypoxanthine, and at 205 nm for allantoin. Uric acid, xanthine, hypoxanthine, and allantoin peaks were identified based on retention times of standards injected separately. Data collection and integration were performed using the Breeze Software (Waters Corporation). Plasma samples were also analyzed for XO and XD activities following the procedure described above.

Statistical Analyses The XO and XD activities in the different tissues were analyzed as a mixed model ANOVA using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC). Tissue was considered a fixed effect and bird a random effect. Changes with time in plasma variables were analyzed as repeated measurements conducted on the same bird. Effects were declared significant at P < 0.05, and Tukey’s multiple comparison test was used to asses differences among means. In the analysis of pH effects on XO and XD activities, nonorthogonal polynomial contrasts were used to test for linear and quadratic effects of pH. Correlations between enzyme activities in the different tissues were determined by Pearson correlation analysis using the PROC CORR procedure of SAS.

RESULTS Growth rates and feed intake measurements of birds were within established guidelines (Aviagen, 2009). Mean live BW were 2.17 ± 0.088 and 4.81 ± 0.231 kg at 7 and 10 wk of age, respectively.

Effects of pH and Temperature on XO and XD Activities Preliminary analysis of the tissues investigated showed that liver and kidney had the greatest enzyme activity. For this reason, they were selected for analyzing the effects of pH and temperature on enzyme activity. The influence of pH (8.7, 8.2, 7.7, and 7.2) on enzyme activity was investigated in 10 liver and 10 kidney samples at 41°C. As shown in Table 1, XO and XD activities were highly dependent on pH. The XO activity in liver and kidney increased linearly (P < 0.001) and total

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showed that the activity observed at 30 min was an adequate criterion for determining enzyme activities. The activity was linear up to 60 min of incubation, but after 90 min, a decrease in enzyme activities was observed in the liver and kidney. Each eluate (200 µL) was incubated with 100 µM xanthine in the presence of ambient oxygen (XO activity) or with 100 µM xanthine and 0.67 mM NAD+ (XO + XD activity) in total reaction volumes of 3 mL, with 0.1 M Tris buffer (pH = 8.2). Reaction mixtures were incubated at 41°C for 30 min, and absorbance was measured at 294 nm in a Beckman Spectrophotometer DU 640 (Beckman Instruments, Fullerton, CA). The amount of uric acid produced was determined from the difference between the absorbance values at 30 and 0 min, as compared with external standards of known uric acid concentration. Blanks containing xanthine or xanthine plus NAD+ were incubated along with the sample tubes to correct for nonenzymatic oxidation of xanthine. Each assay was performed in duplicate. One unit of activity was defined as the production of 1 nmol of uric acid per min at 41°C and pH 8.2 using 100 µM xanthine as substrate in the presence (XO + XD activity) or absence of 0.67 mM NAD+ (XO activity). Xanthine dehydrogenase activity was calculated as the difference between total (XO + XD) and XO activity. Specific activities were expressed in terms of units per milligram of protein in the eluate. The protein content in the eluate was assessed by Bio-Rad Protein Assay (Bio-Rad Chemical Division, Hercules, CA) according to the method of Bradford (1976) and using serum albumin as standard. The pH and temperature dependence of XO and XD activities were studied in liver and kidney tissues. The tissues were homogenized as described above, but the pH of the Tris buffer was adjusted with HCl to pH 7.2, 7.7, 8.2, or 8.7 before incubating the eluates at 41°C for 30 min. The analysis of temperature effects was conducted at pH = 8.2, and the eluates were incubated at 25 or 41°C for 30 min.

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XANTHINE OXIDOREDUCTASE ACTIVITY IN BROILER TISSUES Table 1. Effect of pH (8.7, 8.2, 7.7, and 7.2) on xanthine oxidase (XO), xanthine dehydrogenase (XD), total enzyme activity (XO + XD), and percentage of XO (XO%) in samples from liver and kidney of broilers1 Mean activities, nmol of uric acid/min per mg of protein Tissue

pH

Liver

  SEM   Effect of pH (P = )    Linear    Quadratic Kidney

XD a

8.7 8.2 7.7 7.2

d

64.0 61.0c 53.3b 40.1a 0.95

67.7 67.0c 62.4b 56.3a 0.68

5.28a 8.86a 14.5b 28.3c 1.557

<0.001 0.021

<0.001 <0.001

<0.001 <0.001

<0.001 0.002

50.7b 55.0c 47.5b 36.2a 1.19

53.2b 58.8c 52.7b 41.8a 1.05

4.64a 6.38a 10.3b 13.5c 0.890

<0.001 <0.001

<0.001 <0.001

<0.001 0.411

<0.001 0.217

c

XO%

3.68 5.98a 9.11b 16.3c 0.992

2.54a 3.75b 5.23c 5.57c 0.346

8.7 8.2 7.7 7.2

XO + XD

a–d

For each tissue, means within a column lacking a common superscript differ (P < 0.05). All analyses were conducted at 41°C. Values are the mean of 10 samples.

1

and XD activities decreased quadratically (P < 0.001) as pH decreased from 8.7 to 7.2. The greatest total and XD activities (P < 0.05) in kidney were found at pH 8.2, but in liver samples, no differences (P > 0.05) were found between XD activities at pH 8.7 or 8.2. In liver samples, XO activity accounted for 5.28% of total activity at pH 8.7, but this value increased linearly (P < 0.001) as pH decreased, reaching 28.3% at pH 7.2. In kidney samples, XO activity accounted for 4.64% of total enzyme activity at pH 8.7, but its contribution increased up to 13.5% at pH 7.2. The influence of temperature on enzyme activity was investigated in the same liver and kidney samples at a pH of 8.2 (Table 2). Xanthine oxidase activity was not detected in 15 out of 20 samples when the assay was performed at 25°C, whereas XO activity was detected in all samples at 41°C and accounted for 8.96 and 6.38% of XO + XD activity for liver and kidney, respectively. Xanthine dehydrogenase activity at 25°C

was 32.0 and 29.5% of that found at 41°C for liver and kidney samples, respectively. The negative effect of the low temperature on XD activity was fairly consistent in kidney samples, as the values at 25°C ranged from 26.7 to 32.8% of those measured at 41°C. The response in liver samples was more variable, the values at 25°C ranging from 19.6 to 54.2% of those measured at 41°C.

Distribution of XO and XD Activities in Tissues The distribution of XO and XD activities in tissues from broilers of 7 and 10 wk of age are shown in Tables 3 and 4, respectively. No activity was measured in any plasma sample. In tissues from 7-wk-old broilers, XD and total enzyme activities were highest in kidney and liver, as compared with pancreas and intestine, and low in the remainder of the tissues. The same pattern of enzyme distribution was observed in tissues from 10-wk-

Table 2. Effect of temperature (41°C vs. 25°C) on the analysis of xanthine oxidase (XO), xanthine dehydrogenase (XD), total enzyme activity (XO + XD), and percentage of XO (XO%) in samples from liver and kidney of broilers1 Mean activities, nmol of uric acid/min per mg of protein Tissue Liver   SEM  P = Kidney   SEM  P = a,b

Temperature, °C

XO

XD b

XO + XD b

41 25

5.98 0.12a 0.362 <0.001

61.0 19.7a 2.05 <0.001

67.0 19.8a 1.91 <0.001

8.86b 0.31a 0.385 <0.001

41 25

3.75b 0.04a 0.232 <0.001

55.1b 16.3a 1.05 <0.001

58.1b 16.3a 1.08 <0.001

6.38b 0.24a 0.349 <0.001

For each tissue, means within a column lacking a common superscript differ (P < 0.001). All analyses were conducted at pH = 8.2. Values are the mean of 10 samples.

1

  XO%

b

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  SEM   Effect of pH (P = )    Linear    Quadratic

XO

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Table 3. Xanthine oxidase (XO), xanthine dehydrogenase (XD), total enzyme activity (XO + XD), and percentage of XO (XO%) in tissues of 7-wk-old broilers1 Mean activities (range), nmol of uric acid/min per mg of protein Tissue Liver Kidney Intestine Pancreas Breast muscle Heart Brain Lung SEM P=

XO

XD

5.73 (3.24 to 3.64 (2.33 to 4.16 (2.04 to 4.13 (2.51 to 0.71 (0.17 to ND2 (—)a ND (—)a ND (—)a 0.415 <0.001

c

7.53) 4.74)b 5.29)bc 5.71)bc 1.06)a

XO + XD d

57.6 (46.4 to 64.7) 54.1 (48.5 to 57.8)d 31.5 (22.7 to 37.4)c 19.4 (16.5 to 35.8)b 1.56 (1.06 to 2.16)a 0.91 (0.38 to 1.74)a 0.51 (ND to 0.79)a 0.16 (0.22 to 0.87)a 2.226 <0.001

XO% d

63.3 (52.6 to 72.2) 57.7 (53.2 to 60.9)d 35.7 (26.5 to 42.4)c 23.5 (11.7 to 31.4)b 2.27 (1.66 to 3.13)a 0.91 (0.38 to 1.74)a 0.51 (ND to 0.79)a 0.16 (0.22 to 0.87)a 2.071 <0.001

9.09 (5.22 to 11.9)b 6.34 (4.06 to 7.87)ab 11.6 (7.59 to 12.5)b 19.1 (11.9 to 28.8)c 30.1 (10.3 to 38.9)d ND (—)a ND (—)a ND (—)a 2.312 <0.001

a–d

Means within a column lacking a common superscript differ (P < 0.05). All analyses were conducted at 41°C and pH = 8.2. Values are the mean of 5 samples. 2 ND = not detected. 1

DISCUSSION The avian XOR has been reported to occur mostly in the XD form (Richert and Westerfeld, 1951; Harrison, 2002), although some XO activity has been measured in some studies (Remy et al., 1951; Strittmatter, 1965; Rajagopalan and Handler, 1967; Nishino et al., 1989). Nishino (1994) indicated that XD is converted to XO during purification, either irreversibly by proteolysis or reversibly by sulfhydryl oxidation of the protein molecule. In the present study, the buffer used for the homogenization of tissues and incubation of eluates contained DTT to prevent oxidative conversion of XD to XO and phenylmethylsulfonyl fluoride and leupeptin to inhibit proteases. Despite these precautions, XO activity was detected in liver, kidney, intestine, and pancreas. In samples from heart, brain, and lung, the activity was probably too low to detect any XO activity. The XO activity measured in liver, kidney, intestine, and pancreas might reflect the conversion of XD to the XO form as a consequence of the freezing and purifica-

Table 4. Xanthine oxidase (XO), xanthine dehydrogenase (XD), total enzyme activity (XO + XD), and percentage of XO (XO%) in tissues of 10-wk-old broilers1 Mean activities (range), nmol of uric acid/min per mg of protein Tissue Liver Kidney Intestine Pancreas Breast muscle Heart Brain Lung SEM P= a–e

XO 6.23 (4.12 to 4.15 (3.02 to 4.82 (2.46 to 5.21 (2.51 to 0.52 (1.46 to ND2 (—)a ND (—)a ND (—)a 0.583 <0.001

XD c

9.41) 5.17)b 7.69)bc 5.71)bc 8.44)a

XO + XD d

63.5 (55.6 to 78.4) 56.3 (44.0 to 67.6)d 39.1 (21.6 to 47.2)c 21.0 (16.0 to 28.5)b 1.14 (0.55 to 1.87)a 0.61 (0.09 to 1.43)a 0.30 (ND to 0.32)a 0.09 (0.19 to 0.44)a 2.767 <0.001

Means within a column lacking a common superscript differ (P < 0.05). All analyses were conducted at 41°C and pH = 8.2. Values are the mean of 5 samples. 2 ND = not detected. 1

XO% e

69.8 (59.8 to 86.0) 60.4 (47.8 to 72.5)d 43.9 (24.1 to 54.3)c 26.2 (20.1 to 36.9)b 1.66 (0.82 to 2.57)a 0.61 (0.09 to 1.43)a 0.30 (ND to 0.32)a 0.09 (0.19 to 0.44)a 3.109 <0.001

8.78 (5.30 to 12.5)bc 6.87 (5.83 to 7.97)b 10.9 (6.04 to 16.7)c 19.3 (7.29 to 29.2)d 30.9 (21.9 to 39.2)e ND (—)a ND (—)a ND (—)a 1.356 <0.001

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old broilers. Xanthine oxidase was detected in liver, kidney, intestine, pancreas, and breast muscle from all birds, but no XO activity was detected in the heart, brain, or lung. In 7 out of 10 birds, no enzyme activity was detected in the brain. No activity was measured in any plasma sample. In all tissues analyzed, the predominant form of the enzyme was XD, with the XO accounting for 6.6, 8.9, 11.2, 19.2, and 30.5% of total enzyme activity in kidney, liver, intestine, pancreas, and breast muscle, respectively (mean values for all samples). No significant (P = 0.157 to 0.969) differences were found when XO, XD, and total enzyme activities of 7-wk-old broilers were compared with activities measured in 10-wk-old broilers. As shown in Table 5, plasma concentrations of allantoin, xanthine, and hypoxanthine did not change (P = 0.790, 0.592, and 0.552, respectively) over the 5-wk experimental period. In contrast, uric acid concentrations in plasma were higher (P < 0.05) at 8 to 10 wk compared with that at 6 wk of age.

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XANTHINE OXIDOREDUCTASE ACTIVITY IN BROILER TISSUES Table 5. Mean plasma concentrations of uric acid, allantoin, xanthine, and hypoxanthine in broilers 6 to 10 wk of age (n = 5) Age, wk Item Uric acid, µM Allantoin, µM Xanthine, µM Hypoxanthine, µM Allantoin:uric acid, µmol/µmol

6

7 a

390 216 7.89 3.56 0.55

8 ab

412 225 7.03 3.22 0.55

9 bc

469 228 6.47 4.58 0.49

10 bc

465 225 7.83 4.67 0.48

c

487 240 6.74 4.08 0.49

SEM

P=

14.8 14.6 0.761 0.716 0.032

<0.001 0.790 0.592 0.553 0.485

a–c

Means within a row lacking a common superscript differ (P < 0.05).

Effects of pH and Temperature on XO and XD Activities The observed decrease in liver and kidney XO activity with increasing pH contrasts with previous observations in mice. Krenitsky and Tuttle (1978) reported that the dehydrogenase-derived oxidase activity in mouse intestine decreased from 37 to 25 units (nmol of uric acid produced per min per mL of extract at 37°C), whereas the dehydrogenase-associated oxidase activity decreased only slightly (from 5.0 to 4.9 units) as pH decreased from 9.0 to 7.4. The results from our study suggest that a greater proportion of XD was converted to the XO form as pH decreased, or that some XO activity was present in the samples and it was better detected at a low pH. Saksela and Raivio (1996) pointed out that even if proteases are included in the homogenization buffer, preparations from mammalian tissues typically contain at least 10 to 15% of their total enzyme activity in the XO form. To our knowledge, there are no studies investigating the possible conversion of avian XD to XO during the incubation with buffer in the enzyme assay. In rat liver, Kooij et al. (1994) observed that conversion of XD to XO occurred after 2 to 3 h of incubation of samples with buffer at 37°C, whereas McKelvey et al. (1988) analyzed the conversion of XD to XO in rat liver and kidney and concluded that posthomogenization proteolysis did not occur, even when samples were homogenized without DTT. If the same conclusion is applicable to the chicken samples, our results might indicate that conversion of XD to XO took place during freezing or tissue homogenization. However, when

eluates from liver and kidney were frozen at −20°C and reanalyzed after 2 wk, no significant changes in XO or XD activities were detected (results not shown), indicating that no proteolysis or conversion of XD to XO occurred during this period. In agreement with our results, other investigators have observed a pH dependence on XOR activity (Remy et al., 1955; Krenitsky and Tuttle, 1978). Remy et al. (1955) found that enzyme activity in chicken liver increased as pH rose from 5.0 to 8.0, with a fairly broad optimum pH between 8.0 and 8.5, and Krenitsky and Tuttle (1978) reported that XOR activity in the mouse intestine increased up to an optimal pH of 8.5 and then decreased at higher pH values. In our study, no significant differences in total enzyme activity were detected between pH 8.7 and 8.2 in liver, but the activity in kidney was lower at pH 8.7 compared with that at pH 8.2. These results would indicate that the optimum pH of the enzyme in chicken liver and kidney is around 8.2. This suggests that under physiological conditions, the enzyme operates at less than its maximal activity determined in vitro, but the biological significance of this is unknown. In addition, it should be noticed that enzyme activity was measured in tissue homogenates, which represent a pool of XOR from different cell types in organs. Therefore, as stated by Saksela et al. (1998), data derived from these sources do not necessarily reflect the in vivo conditions at the cellular level. In the study on temperature effects on enzyme activity, we used 41°C, the body temperature of chickens, and 25°C because the enzyme assay has been performed at room temperature in many studies. Temperature exerted a comparable effect on both tissues, but XO activity was more strongly influenced compared with XD activity. Xanthine oxidase activity was found in all samples when the analysis was conducted at 41°C and represented 8.96 and 6.38% of XO + XD enzyme activity in liver and kidney, respectively. These results are in agreement with those from Remy et al. (1951), who found that XO activity in chicken liver ranged from 4.0 to 10.0% of the enzyme activity estimated with methylene blue as the acceptor when the assay was performed at 38°C and pH 7.4. In contrast, lower XO activity has been measured in other studies conducted at 25°C and pH 7.5 or 7.8. Rajagopalan and Handler (1967) reported that XO activity of chicken liver was about 2.5% of the activity measured with NAD+ or methylene blue as

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tion procedures, as has been described by others (Corte and Stirpe, 1972; Parks and Granger, 1986). However, Nishino (1994) stated that by avoiding proteolysis, the mammalian XOR can be purified as an interconvertible form, whereas Krenitsky and Tuttle (1978) reported that the inclusion of DTT in the assay mixture prevented the transformation of XD to XO when preparations from mouse small intestine were incubated at 37°C for the enzyme analysis. Krenitsky and Tuttle (1978) concluded that there were 2 XO forms in the mouse intestine: the dehydrogenase-derived oxidase (formed from the oxidation of XD) and the dehydrogenase-associated oxidase, which was not a consequence of partial proteolysis of the enzyme.

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Distribution of XO and XD Activities in Tissues Previous research has shown that distribution of XOR in animal tissues varies from species to species (AlKhalidi and Chaglassian, 1965; Muxfeldt and Schaper, 1987; Wajner and Harkness, 1989). However, most studies in chickens have focused on liver, kidney, and pancreas, with limited information on other tissues. Our results for enzyme activity in liver and kidney are similar to those previously published (Remy and Westerfeld, 1951; Hartsook et al., 1959; Nagahara et al., 1987) and confirm that both organs have the highest enzyme activity in chickens, whereas the highest levels in mammalian tissues are found in liver and intestine (Harrison, 2002). These results on XOR activity are in accordance with the observation that in birds the 2 tissues in which most uric acid formation occurs are the liver and kidney (Tinker et al., 1986). In our study, the enzyme activity in the kidney was 91 and 87% of the activity in the liver in 7- and 10-wk-old chickens, respectively. These results are in accordance with those of Nagahara et al. (1987), who demonstrated that enzyme activity in the chicken was greater in liver than in kidney, but in contrast to the findings of Hartsook et al. (1959), who observed that enzyme activity was 1.5 times greater in the kidney than in the liver. Remy and Westerfeld (1951) showed that the response of enzyme activity to dietary changes in chickens varied with the tissue investigated. When chickens were fed a diet con-

taining 35% protein, the enzyme activity in the kidney was about 42% of that in the liver, but when the dietary protein content was decreased to 21 and 14%, the activity in kidney increased up to 89 and 135% of that in the liver, respectively. These results may be related to the role of uric acid as the end product of the metabolism of nitrogen-containing compounds in birds. Other studies have shown changes in enzyme activity in response to the administration of antibiotics (Hartsook et al., 1959), enzyme inhibitors (Lee and Fisher, 1972), or different levels of protein (Pons et al., 1986; Nagahara et al., 1987). These results indicate that the relative activity of the enzyme in different tissues can be affected by different factors, which may partly explain the discrepancies observed in the various studies that investigated the tissue distribution pattern of the enzyme. Only a few studies have investigated enzyme activity in the pancreas and intestine in chickens. In our study, enzyme activity in pancreas and intestine was 37 and 56% of that in the liver in 7-wk-old chickens, and 38 and 63% in 10-wk-old chickens. Similar results have been obtained by Remy and Westerfeld (1951), who found that enzyme activity in pancreas and intestine was 24 and 58% of that in the liver when chickens were fed a diet containing 21% protein. Interestingly, when the results from the chickens were analyzed together (n = 10), a positive linear relationship was observed between the total enzyme activities measured in the liver and kidney (r = 0.58; P = 0.082), liver and pancreas (r = 0.77; P = 0.010), and kidney and pancreas (r = 0.61; P = 0.059), but no relationship was found between the enzyme activities in the intestine and the other 3 tissues (P = 0.201 to 0.921). Tinker et al. (1986) observed in cockerels that uric acid was released from muscle; they hypothesized that the liver and kidney release xanthine and hypoxanthine, which were converted to uric acid in the muscle. In support of this hypothesis, we detected XOR activity in breast muscle in our study, which was 2.7 and 2.4% of that in the liver in 7- and 10-wk-old broilers, respectively. Evidence for enzyme activity in chicken breast muscle has also been reported by Pons et al. (1986), who found that activity was about 2.0% of that in the liver in 3-d-old chickens. Further, Muxfeldt and Schaper (1987) showed that XOR activity in the heart was variable among animal species: high (rat), moderate (dog, guinea pig), and null (pig) activity. In our study, a low enzyme activity was detected in all heart samples, with values about 1.44 and 0.87% of those in the liver of 7- and 10-wk-old broilers, respectively. The lack of enzyme activity in the plasma observed in our study confirms previous observations (Al-Khalidi and Chaglassian, 1965). Al-Khalidi and Chaglassian (1965) noticed that animal species with high serum concentrations of XOR (i.e., dog and cow) also had high concentrations in the lungs, which suggested that the high serum concentration may be due to leakage of enzyme from the lung. In support of this view, lack

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acceptors, whereas Strittmatter (1965) reported that enzyme activity in chicken liver and kidney with oxygen as an acceptor was 3 and 4%, respectively, of that observed with NAD+ as acceptor. In our study, XO activity was only detected in 5 out of 20 samples when the assay was performed at 25°C, accounting for less than 2% of XO + XD activity in each of them. The relatively lower XO activity observed in our study may be explained by the fact that our enzyme assays were performed at a higher pH (8.2 compared with 7.5 or 7.8 in the other studies). Decreasing the assay temperature from 41 to 25°C reduced XD activity to values about 30% of those found at 41°C, which is consistent with our expectation that enzyme activity would be higher at physiological body temperature than at room temperature. In agreement with our results, Nishino et al. (1989) observed that enzyme activity in chicken liver measured at 4°C was only about 8% of that found when the analysis was performed at 25°C. These results show the importance of taking into account the temperature of the assay when comparing results from different studies. In addition, the results illustrate clearly the importance of temperature and pH on detecting the relative contribution of XO and XD to total enzyme activity. Based on our results on liver and kidney samples, the optimal conditions to investigate the activity of the enzyme in the selected tissues are at 41°C and pH 8.2.

XANTHINE OXIDOREDUCTASE ACTIVITY IN BROILER TISSUES

ACKNOWLEDGMENTS This work was supported by Hatch grant (H393) and a grant from the Joe. C. Jackson College of Graduate Studies and Research at the University of Central Oklahoma (Edmond). M. D. Carro gratefully acknowledges support from the Spanish Ministerio de Ciencia e Innovación (PR2008-0025) and thanks Antonio J. Molina (University of León, Spain) for his useful advice on the analysis of XO and XD activities. Chicks were a generous gift from Pilgrims Pride (Moorefield, WV). Thanks

are given to the West Virginia University Animal Sciences farm crew for preparation of diets. This paper is submitted with the approval of the West Virginia Agricultural and Forestry Experiment Station (Scientific Article No. 3041).

REFERENCES Al-Khalidi, U. A. S., and T. H. Chaglassian. 1965. The species distribution of xanthine oxidase. Biochem. J. 97:318–320. Aviagen. 2009. Ross Broiler Management Manual. http://www.aviagen.com/docs/Ross%20Broiler%20Manual%202009.pdf Accessed May 20, 2009. Berry, C. E., and J. M. Hare. 2004. Xanthine oxidoreductase and cardiovascular disease: Molecular mechanisms and pathophysiological implications. J. Physiol. 555:589–606. Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. Bruguera, P., A. Lopez-Cabrera, and E. I. Canela. 1988. Kinetic mechanism of chicken liver xanthine dehydrogenase. Biochem. J. 249:171–178. Corte, E. D, and F. Stirpe. 1972. The regulation of rat liver xanthine oxidase: Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme. Biochem. J. 126:739–745. Harrison, R. 2002. Structure and function of xanthine oxidoreductase: Where are we now? Free Radic. Biol. Med. 33:774–797. Hartsook, E. W., R. V. Boucher, and T. V. Hershberger. 1959. The effect of dietary antibiotics upon hepatic and renal xanthine dehydrogenase activity in chicks. Arch. Biochem. Biophys. 82:310– 318. Holmes, D. J., and S. N. Austad. 1995. The evolution of avian senescence patterns: Implications for understanding primary aging processes. Am. Zool. 35:307–317. Itoh, R., T. Nishino, C. Usami, and K. Tsushima. 1978. An immunochemical study of the changes in chicken liver xanthine dehydrogenase activity during dietary adaptation. J. Biochem. 84:19–26. Klandorf, H., D. S. Rathore, X. Shi, and M. Iqbal. 2001. Accelerated tissue aging and increased oxidative stress in broiler chickens fed allopurinol. Comp. Biochem. Physiol. 129:93–104. Kooij, A., H. J. Schiller, M. Schijns, C. J. Van Noorden, and W. M. Frederiks. 1994. Conversion of xanthine dehydrogenase into xanthine oxidase in rat liver and plasma at the onset of reperfusion after ischemia. Hepatology 19:1488–1495. Krenitsky, T. A., and J. V. Tuttle. 1978. Xanthine oxidase activities: Evidence for two catalytically different types. Arch. Biochem. Biophys. 185:370–375. Lee, P. C., and J. R. Fisher. 1971. Regulation of xanthine dehydrogenase levels in liver and pancreas of the chick. Biochim. Biophys. Acta 237:14–20. Lee, P. C., and J. R. Fisher. 1972. Effect of allopurinol on the accumulation of xanthine dehydrogenase in liver and pancreas of chicks after hatching. Arch. Biochem. Biophys. 148:277–281. Lux, O., D. Naidoo, and C. Salonikas. 1992. Improved HPLC method for the simultaneous measurement of allantoin and uric acid in plasma. Ann. Clin. Biochem. 29:674–675. McKelvey, T. G., M. E. Höllwarth, D. N. Granger, T. D. Engerson, U. Landler, and H. P. Jones. 1988. Mechanisms of conversion of xanthine dehydrogenase to xanthine oxidase in ischemic rat liver and kidney. Am. J. Physiol. 254:G753–G760. Muxfeldt, M., and W. Schaper. 1987. The activity of xanthine oxydase in heart of pigs, guinea pigs, rabbits, rats, and humans. Basic Res. Cardiol. 82:486–492. Nagahara, N., T. Nishino, M. Kanisawa, and K. Tsushima. 1987. Effect of dietary protein on purine nucleoside phosphorylase and xanthine dehydrogenase activities of liver and kidney in chicken and pigeon. Comp. Biochem. Physiol. 88B:589–593. Nishino, T. 1994. The conversion of xanthine dehydrogenase to xanthine oxidase and the role of the enzyme in reperfusion injury. J. Biochem. 116:1–6.

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of enzyme activity in chicken plasma is in accordance with the extremely low levels of enzyme detected in the lungs in our study. Only traces of enzyme activity were detected in 3 out of 10 brain samples, which agrees with the very low or absence of XOR activity reported in the brain from different animal species (Al-Khalidi and Chaglassian, 1965; Stirpe and Della Corte, 1969). Lee and Fisher (1971) showed that enzyme activity in chicken liver and pancreas increased sharply over the first 4 d after hatching, which remained stable until the end of their study at 4 wk of age. The results of our study indicate a comparable measurement of enzyme activity in the tissues of chickens at 7 and 10 wk of age. Hartsook et al. (1959) also found similar enzyme activity in liver and kidney of chickens at 4 and 9 wk of age fed the same diet. All reported results indicate that enzyme activity increases during early development, but remains stable afterwards. Consistent with the present work, Simoyi and Klandorf (2003) reported an increase in plasma uric acid concentration in 10-wk-old turkeys compared with that observed in 8-wk-old birds fed a standard diet, and Klandorf et al. (2001) found higher plasma uric acid levels in 16-wk-old broilers compared with 22-wk-old broilers fed on the same diet. The lack of changes in plasma concentrations of allantoin, xanthine, and hypoxanthine over the course of the trial is in agreement with the results of Simoyi et al. (2003), who observed stable concentrations in turkeys from 6 to 8 wk of age. In conclusion, liver and kidney were determined to have the greatest XOR activity in chickens, followed by intestine and pancreas. The remaining analyzed tissues had low activity, whereas only trace amounts were found in some brain samples. This pattern of enzyme distribution was independent of the age of the chickens, which suggests that the enzyme concentrations are adequate to support nitrogen-containing compound excretion and to meet the antioxidant requirements of the birds as they attain adult BW. Absolute values of enzyme activity were strongly affected by pH and temperature. Decreasing the pH from 8.2 to 7.2 in the enzyme assay decreased XD and enhanced XO activity, thus increasing the contribution of the XO form to total enzyme activity. These results reflect the critical importance of measuring enzyme activity under the optimum temperature and pH conditions.

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Carro et al. Scholz, R. W., and W. R. Featherston. 1968. Effects of alterations in protein intake on liver xanthine dehydrogenase in the chick. J. Nutr. 95:1271–1277. Simoyi, F., E. Falkensteina, K. Van Dyke, K. P. Blemings, and H. Klandorf. 2003. Allantoin, the oxidation product of uric acid is present in chicken and turkey plasma. Comp. Biochem. Physiol. B 135:325–335. Simoyi, M. F., and H. Klandorf. 2003. Fructose and its effect on turkey plasma uric acid levels and productive performance. Poult. Sci. 82:478–483. Stevens, L. 1996. Avian Biochemistry and Molecular Biology. Cambridge University Press, Cambridge, UK. Stirpe, F., and E. Della Corte. 1969. The regulation of rat liver xanthine oxidase: Conversion in vitro of the enzyme activity from dehydrogenase (type D) to oxidase (type O). J. Biol. Chem. 244:3855–3863. Strittmatter, C. F. 1965. Studies on avian xanthine dehydrogenases. Properties and patterns of appearance during development. J. Biol. Chem. 240:2557–2564. Terada, L. S., J. A. Leff, and J. E. Repine. 1990. Measurement of xanthine oxidase in biological tissue. Methods Enzymol. 186:651–657. Tinker, D. A., J. T. Brosnan, and G. R. Herzberg. 1986. Interorgan metabolism of amino acids, glucose, lactate, glycerol and uric acid in the domestic fowl (Gallus domesticus). Biochem. J. 240:829–836. Wajner, M., and R. A. Harkness. 1989. Distribution of xanthine dehydrogenase and oxidase activities in human and rabbit tissues. Biochim. Biophys. Acta 991:79–84.

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Nishino, T., T. Nishino, L. M. Schopfer, and V. Massey. 1989. The reactivity of chicken liver xanthine dehydrogenase with molecular oxygen. J. Biol. Chem. 264:2518–2527. Parks, D. A., and D. N. Granger. 1986. Xanthine oxidase: Biochemistry, distribution and physiology. Acta Physiol. Scand. 548(Suppl.):87–89. Pons, A., F. J. García, A. Palou, and M. Alemany. 1986. Effect of starvation and a protein diet on the amino acid metabolism enzyme activities of the organs of domestic fowl hatchlings. Comp. Biochem. Physiol. 85B:275–278. Rajagopalan, K. V., and P. Handler. 1967. Purification and properties of chicken liver xanthine dehydrogenase. J. Biol. Chem. 242:4097–4107. Remy, C. N., D. A. Richert, R. J. Doisy, I. C. Wells, and W. W. Westerfeld. 1955. Purification and characterization of chicken liver xanthine dehydrogenase. J. Biol. Chem. 217:293–306. Remy, C. N., D. A. Richert, and W. W. Westerfeld. 1951. The determination of xanthine dehydrogenase in chicken tissues. J. Biol. Chem. 193:649–657. Remy, C. N., and W. W. Westerfeld. 1951. The effect of diet on xanthine dehydrogenase in chicken tissues. J. Biol. Chem. 193:659– 667. Richert, D. A., and W. W. Westerfeld. 1951. Xanthine oxidase in different species. Proc. Soc. Exp. Biol. Med. 76:252–254. Saksela, M., R. Lapatto, and K. O. Raivio. 1998. Xanthine oxidoreductase gene expression and enzyme activity in developing human tissues. Biol. Neonate 74:274–280. Saksela, M., and K. O. Raivio. 1996. Cloning and expression in vitro of human xanthine dehydrogenase/oxidase. Biochem. J. 315:235–239. Sato, A., T. Nishino, K. Noda, Y. Amaya, and T. Nishino. 1995. The structure of chicken liver xanthine dehydrogenase. cDNA cloning and the domain structure. J. Biol. Chem. 270:2818–2826.