Progress 10/01/03 to 08/30/09
Outputs
OUTPUTS: 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 our studies have demonstrated that it functions as an antioxidant to reduce oxidative stress. Study 1: Despite the importance of this enzyme, the tissue distribution of XOR in physiologically normal chickens is not well known. In this first study, we analyzed XOR activity in extracts of 8 tissues from broilers at 7 and 10 wk of age. Study 2: The purpose was to determine the effects of allopurinol (AL) on xanthine oxidoreductase (XOR)activity and uric acid (UA) levels in chickens. Thirty 5-week-old broilers were divided into three groups and fed 0 (control), 25 (AL25) or 50 (AL50) mg AL per kg of body mass for 5 weeks. Chicks were weighed twice weekly and leukocyte oxidative activity (LOA) and plasma purine levels were determined weekly in five birds per group. Chicks were sacrificed after 2 or 5 weeks, and samples from tissues were taken for analysis of XOR activity. Results from these studies have been disseminated to the scientific community through conferences and refereed publications. PARTICIPANTS: Collaborators: 1: Dr. Knox Van Dyke Department of Pharmacology and Toxicology Robert C Byrd WVU School of Medicine West Virginia University Morgantown, WV 26506-9223 2. Dr. Giovanni Casotti cDepartment of Biology West Chester University West Chester, PA 19383 3. Dr. William Radke University of Central Oklahoma, Edmond, OK, 73034 4.Dr. Brian Dorr USDA Wildlife Services National Wildlife Research Center Mississippi Field Station P.O. Drawer 6099 Mississippi State University, Mississippi 39762 5. Dr. Jim Anderson West Virginia University Wildlife and Fisheries Resources, PO Box 6125, Morgantown, WV 26506 6. Dr. Ken Blemings West Virginia University Division of Animal and Veterinary Sciences PO Box 6108, Morgantown, WV 26506 TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Study 1: No differences in XOR activity due to age were found in any tissue. Liver and kidney showed the greatest activity, that in the kidney being about 89 per cent of the activity in the liver. Enzyme activity in intestine and pancreas was about 60 and 37 per cent 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 per cent of those 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 showed that XOR distribution in chickens differs from that in mammals, in which the highest levels have been found in liver and intestine. Study 2: Plasma UA concentrations were lower and xanthine and hypoxanthine concentrations were greater in AL25 and AL50 birds compared to controls, whereas no differences were detected in allantoin concentrations. By week 5, body mass was reduced to 84.0 and 65.1% of that in controls for AL25 and AL50 broilers, respectively, and LOA was 4.1 times greater in AL25 compared to control birds. Liver XOR activity was increased by 1.1 and 1.2 times in AL25 and AL50 birds, but there was no change in XOR activity in the pancreas and intestine. These results suggest that AL effect on XOR activity is tissue dependent. In conclusion, these studies have established the importance of uric acid and its regulation on oxidative stress in chickens. Factors that reduce uric acid can be associated with increased oxidative stress while those that increase uric acid reduce oxidative stress. Ultimately these studies will contribute to a greater understanding of animal health.
Publications
- Cleveland, B.M., Leonard, S., Klandorf, H., and Blemings, K. (2009) Urate oxidase knockdown decreases oxidative stress in a murine hepatic cell line. Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity 2: 93-98.
- Carro, M.D., Falkenstein, E., Blemings, K.P. and Klandorf, H. 2009. Determination of xanthine oxidoreductase activity in broilers: effect of pH and temperature of the assay and distribution in tissues. Poultry Science 88: 2406-14. Carro, M.D., Falkenstein, E., Radke, W.J. and Klandorf, H. Effects of allopurinol on uric acid concentrations, xanthine oxidoreductase activity and oxidative stress in broiler chickens. (2009). Comparative Biochemistry and Physiology - Part C: Toxicology & Pharmacology .
- Carro, M., Falkenstein, E., Van Dyke, K. and H. Klandorf (2009). Effects of allopurinol on plasma uric acid concentrations and leukocyte oxidative activity in broilers. FASEB J. 23: 617.7.
- Settle, T., Moritz, J., Leonard, S., Klandorf, H., Falkenstein, E. and Carro, M.D. 2009. The effects of a phytogenic feed additive versus and antibiotic feed additive on oxidative stress in broiler chicks FASEB J. 23: 617.2.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The long-term goal of this research project is to establish models of oxidative stress in birds and determine the efficacy of manipulating plasma uric acid concentrations to modulate oxidative stress and thus increase our understanding of how oxidants exert their specific affects on tissues. Studies have shown that there is a positive correlation between age and the accumulation of pentosidine in the breast skin of birds. However, previous research has only analyzed skin from deceased birds. The major objective of these studies was to determine if pentosidine concentrations can be determined from skin biopsy collections of living birds. PARTICIPANTS: Brian Dorr USDA, Dept. of Agriculture Wildlife Services National Wildlife Research Center Mississippi Field Station P.O. Drawer 6099 Mississippi State University 39762 TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Allopurinol is a xanthine oxidase inhibitor that has been recently proposed as a therapy to reduce oxidative stress in different human tissues. The effects of xanthine oxidase on oxidative stress are complex, since it forms uric acid (a potent antioxidant) but also catalyzes the formation of reactive oxygen species. These studies investigated the effects of allopurinol on plasma uric acid (PUA) concentrations and oxidative activity in broilers. Feeding allopurinol decreased feed intake and significantly reduced body weight compared to that measured in the control group. As expected, allopurinol reduced PUA at each wk although there was no effect of allopurinol on measurements of oxidative stress over the first 3 wk of study, but at wk 4 oxidative stress measurements were greater than that measured in control birds. The general lack of effect of allopurinol on measurements of oxidative stress would indicate that the increase in oxidative stress caused by the reduction of PUA could have been overcome by a reduction in the formation of reactive oxidative species. Patagial tags have been placed on many wild birds without serious side effects, which makes the patagium a potential site for skin collection in living birds. Patagial and breast skin samples from deceased Double-crested Cormorants (Phalacrocorax auritus), Monk Parakeets (Myiopsitta monachus), and living Black Vultures (Coragyps atratus) were provided by USDA Wildlife Services in Gainesville, FL and Starkville, MS. These samples were analyzed for pentosidine concentrations according to the procedure published by Iqbal et al. Initial comparisons of Double-crested Cormorants and Monk Parakeets revealed that there were no significant differences in pentosidine concentrations between the breast and patagium for either species. These results indicates that age curves generated from the analysis of breast skin can be reliably used to age birds with skin collected from the patagium. The collection of patagial skin from Black Vultures indicated that skin samples can be collected safely from living birds.
Publications
- Refereed publications: Seaman, C., Moritz, J., Falkenstein, E. Van Dyke, K., Casotti, G. and Klandorf, H. (2008). Inosine ameliorates the effects of hemin-induced oxidative stress in broilers. Comp. Biochem. Physiol. 151:670-675.
- Abstracts: Settle T., J. Moritz, S. Leonard, E. Falkenstein, M. Carro and H.Klandorf (2008). The effects of a phytogenic feed additive versus an antibiotic feed additive on oxidative stress in broiler chicks. Poultry Sci.
- Cooey, C., Anderson, J. Dorr, B. and Klandorf, H. (2008). Pentosidine use as a method of aging living birds. Berryman Symposium Proceedings.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The long-term goal of this research proposal is to establish a model of oxidative stress in poultry and determine the efficacy of elevated plasma uric acid concentrations to function as a protectant against oxidative stress and thus limit tissue damage caused by oxidants. Raising plasma uric acid levels through feed manipulation can lead to increased resistance to oxidative stress and increased animal well-being. One of the products of oxidative stress in the tissues of animals is the advanced glycation endproduct pentosidine (Ps). Recent studies have shown that a linear correlation exists between the age of individual birds and the accumulation of Ps in their tissues. However, previous research has only analyzed skin from deceased birds. The major objective of this study was to determine if pentosidine concentrations can be determined from skin biopsy samples of living birds. Patagial tags have been placed on many wild birds without serious side effects, which makes the
patagium a potential site for skin collection in living birds. Patagial (n=63) and breast (n=113) skin samples from 113 deceased Double-crested Cormorants, patagial (n=105) and breast (n=105) skin samples from 105 deceased Monk Parakeets, and patagial (n=30) skin samples from 30 living Black Vultures were provided by USDA Wildlife Services in Gainesville, FL and Starkville, MS. These samples were analyzed for pentosidine concentrations according to the procedure published by Iqbal et al. (1999). Initial comparisons of 25 Double-crested Cormorants and 38 Monk Parakeets revealed that there was no significant differences in pentosidine concentrations between the breast and patagium for either species (p = 0.4944 and p = 0.3440 respectively). The collection of patagial skin from Black Vultures indicated that skin samples can be collected safely from living birds. In order to determine the healing rate of skin biopsy sites, approximately 12 Double-crested Cormorants will be trapped and
held in captivity. Skin samples will be taken from the patagium using a 6 mm biopsy punch. The healing rate of the sampled area will be monitored daily.
PARTICIPANTS: Drs. Michael Avery & Brian Dorr: USDA Wildlife Services in Gainesville, FL and Starkville, MS. Funding from my USDA contract permitted my graduate student Crissa Cooey to visit Dr. Avery in Florida and collect Monk parakeet skin samples.
Impacts
The results from these studies will be used to determine whether this sampling technique can be applied to wild-caught birds sampled and released on the same day. The patagium may represent a potentially less invasive source of skin sample for biologists interested in sampling live birds. Accurate age information could aid species recovery programs and provide insights into longevity now understood entirely from banding programs and captive birds.
Publications
- Cleveland, B.M., Leonard, S. Klandorf, H. Blemings, K. (2007). Reducing urate oxidase mRNA alters the oxidative stress response in a mouse hepatic cell line. FASEB J.
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Progress 01/01/06 to 12/31/06
Outputs
The long-term goal of this research proposal is to establish a model of oxidative stress in poultry and determine the efficacy of elevated plasma uric acid concentrations to function as a protectant against oxidative stress and thus limit tissue damage caused by oxidants. Raising plasma uric acid levels through feed manipulation can lead to increased resistance to oxidative stress and increased animal well-being. Studies in our laboratory have demonstrated the role of hemin as a model for generating oxidative stress. The addition of inosine, a precursor of uric acid, to the diet of birds injected daily with hemin was found to dramatically reduced oxidative stress. Further, pretreatment of birds with inosine prior to hemin injection prevented the resultant increase in measurements of oxidative stress. One of the products of oxidative stress in the tissues of animals is the advanced glycation endproduct pentosidine (Ps). Recent studies suggest that a linear correlation
exists between the age of individual birds and the accumulation of Ps in their tissues. Current studies are investigating Ps concentrations in skin samples of black vultures. Biopsy punches of 4, 6 and 8mm size were obtained from birds at the time of collection and assayed for Ps concentration. Results from this study suggest that precise measurements of Ps concentration can be determined at each fragment size. However, skin samples of 6mm in size were found to be more accurate for the determination of Ps concetration. Current studies are comparing measurements of Ps in skin samples from the patagium versus breast. The patagium may represent a potentially less invasive source of skin sample for biologists interested in sampling live birds. Accurate age information could aid species recovery programs and provide insights into longevity now understood entirely from banding programs and captive birds.
Impacts
The results from our studies suggest that uric acid has a concentration dependent affect on scavenging free radicals. The ability of uric acid to limit reactive species oxidation of biological components gives it a crucial antioxidant role in vivo. By slowing the accumulation of reactive species-mediated markers of tissue injury these results suggest that uric acid protects birds from reactive species-mediated tissue damage, which thus contributes to avian longevity. Measurement of Ps in skin samples has been established as a methodology to provide an age estimate in birds. Accurate age information could aid species recovery programs and provide insights into longevity now understood entirely from banding programs and captive birds.
Publications
- 4. Sieman, C., Falkenstein, E., and Klandorf, H. (2005) Inosine ameliorates the effects of hemin induced oxidative stress in broilers. Poultry Science
- 1. Fallon, J.A., Cochrane, R.L., Dorr, B. and H. Klandorf. (2006). Interspecies comparison of pentosidine accumulation in birds. Auk 123 (1): 148-152.
- 2. Fallon, J.A., Radke, W.J., and H. Klandorf. (2006). Stability of pentosidine concentration in museum prepared study skins. Auk 123 (3): 870-876.
- 3. Kiess, A.S., Stinefelt, B.M. Gentilin, A.J., Wilson, M.E. Klandorf, H., and Blemings, K. (2005) Lysine catabolism in chickens fed at or below their lysine requirement. FASEB J. 20(5)
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Progress 01/01/05 to 12/31/05
Outputs
The long-term goal of this research proposal is to establish a model of oxidative stress in poultry and determine the efficacy of elevated plasma uric acid concentrations to function as a protectant against oxidative stress and thus limit tissue damage caused by oxidants. Raising plasma uric acid levels through feed manipulation can lead to increased resistance to oxidative stress and increased animal well-being. Studies have investigated the role of hemin as a model for generating oxidative stress. Dose response studies have determined optimum levels of hemin (20 mgkg) while current studies will determine the effect of adding inosine to the diet of birds with moderate oxidative stress. One of the products of oxidative stress in the tissues of animals is the advanced glycation endproduct pentosidine (Ps). Recent studies suggest that a linear correlation exists between the age of individual birds and the accumulation of Ps in their tissues. These investigations
examined Ps concentrations in skin tissue of cormorants and captive raised Ruffed grouse. Skin samples were taken from the breast of cormorants and Ruffed grouse of known ages, ranging from a few days to ten years. Ps concentrations were determined using reverse phase HPLC. Fluorescent detection (excitation 310nm, emission 385nm) revealed concentrations of Ps/mg collagen to increase linearly with age (p<.0001). No significant sex differences were detected. A second study examined variations in Ps concentrations of six Ruffed grouse prepared as study skins. Samples taken at three-month intervals over the course of a year revealed no significant variation in Ps concentrations within individual birds although Ps concentration differed slightly depending on site selection. Stability of Ps after study skin preparation would enable age estimation of museum specimens. Current studies are investigating Ps concentrations in skin samples of black vultures. Biopsy punches of 4, 6 and 8mm size
were obtained from birds at the time of collection and stored for Ps determination. Accurate age information could aid species recovery programs and provide insights into longevity now understood entirely from banding programs and captive birds.
Impacts
The results from our studies suggest that uric acid has a concentration dependent effect on scavenging free radicals. The ability of uric acid to limit reactive species oxidation of biological components gives it a crucial antioxidant role in vivo. By slowing the accumulation of reactive species-mediated markers of tissue injury these results suggest that uric acid protects birds from reactive species-mediated tissue damage and thus contributes to avian longevity.
Publications
- Fallon, J.A., Cochrane, R.L., Dorr, B. and H. Klandorf. (2006) Interspecies comparison of pentosidine accumulation in birds. Auk. (In press)
- Fallon, J.A., Radke, W.J., and H. Klandorf. (2006) Stability of pentosidine concentrations in museum prepared study skins. Auk. (In press)
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Progress 01/01/04 to 12/31/04
Outputs
Uric acid (UA) has been proposed to be the dominant antioxidant in birds. The objective of these studies was to investigate the quenching effect of varying concentrations of UA, including those found in avian plasma, on specific reactive oxygen species (ROS) and determine the ability of UA to protect DNA and cellular membranes from ROS mediated damage. Hydroxyl and superoxide radicals were detected with electron spin resonance (ESR) and their presence was reduced upon addition of UA (P < 0.05). UA inhibited hydroxyl mediated Hind III DNA damage (P<0.05). Lipid peroxidation of silica exposed RAW cell membranes was less (P < 0.02) with addition of UA to the cell incubation mixture. Our second objective was to establish models of oxidative stress in poultry. In the first study, the ability of hemin (an iron containing protein) to generate oxidative stress was investigated in broilers. Inclusion of hemin at 5, 10 and 20 mg/kg feed/bw resulted in a dose-dependent increase
in oxidative stress as measured by leukocyte oxidative activity (LOA). BW was depressed in treated animals. In a separate study the inclusion of allopurinol, (which reduces concentrations of plasma uric acid) in the diet of hemin-treated birds did not further exacerbate the increase in LOA activity.
Impacts
The results from our in vitro studies indicate that uric acid has a concentration dependent effect on scavenging hydroxyl radicals and superoxide and inhibits oxidation of DNA and lipids in cellular membranes. The ability of uric acid to prevent reactive species oxidation of biological components gives it a crucial antioxidant role in vivo, slowing the accumulation of reactive species-mediated markers of tissue injury. These results suggest that uric acid protects birds from reactive species-mediated tissue damage, and thus contributes to avian longevity.
Publications
- Machin, M., Simoyi, M.F., Blemings K.P. and Klandorf, H. (2004). Effect of dietary protein on plasma uric acid, body weight, and oxidative stress in broilers. Comp. Biochem. Physiol. 137, 381-388.
- Stinefelt, B Leonard, S., Blemings, K. Shi, X. and Klandorf, H. (2005) Free radical scavenging, DNA protection, and inhibition of lipid peroxidation provided by uric acid. Annals of Clinical & Laboratory Science 35.
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