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Animal Health Trust, Lanwades Park, Kentford, Suffolk, CB8 7UU, UK, * Equine Studies Group, WALTHAM Centre for Pet Nutrition, Waltham-on-the-Wolds, Leicestershire, LE14 4RT, UK, Department of Bioengineering, Imperial College London, SW7 2BX, UK, and ** School of Health and Life Sciences, King's College London, SE1 9NN, UK
3 To whom correspondence should be addressed. E-mail: chris.deaton{at}aht.org.uk.
KEY WORDS: • ascorbic acid • oxidative stress • exercise
Pulmonary epithelial lining fluid has a vast antioxidant capacity. In horses, ascorbic acid is quantitatively the major nonenzymatic antioxidant, which is likely to reflect horses' ability to synthesize this antioxidant (1). Horses suffering from recurrent airway obstruction (RAO)4 demonstrate oxidative stress, the severity of which is related to the degree of neutrophilic airway inflammation (1,2). RAO-affected horses in remission have lower concentrations of ascorbic acid in bronchoalveolar lavage fluid (BALF) compared with healthy non-RAO–affected horses despite the resolution of cytological airway inflammation (3). This low antioxidant capacity may render RAO-affected horses in remission more susceptible to oxidative challenge than healthy non-RAO–affected horses.
Exercise can induce the production of reactive oxygen species by electron leak from the mitochondrial electron-transport chain, ischemia-reperfusion, and autooxidation of catecholamines (4). In humans, evidence exists for a beneficial effect of antioxidant supplementation on exercise-induced oxidative damage [reviewed in (5)] and on pulmonary function in patients with obstructive lung diseases [reviewed in (6)].
Antioxidant supplementation was previously demonstrated to decrease the exercise-induced increase in plasma uric acid concentration in RAO-affected horses (7); however, the effects of supplementation and exercise on the pulmonary and systemic status of ascorbic acid were not evaluated. The aims of the present study were, therefore, to investigate the effects of a supplement containing a combination of different antioxidants on plasma- and BALF-antioxidant concentrations and on exercise-induced alterations in systemic and pulmonary oxidative stress in RAO-affected horses in remission.
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MATERIALS AND METHODS |
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 TOP MATERIALS AND METHODS RESULTSDISCUSSIONLITERATURE CITED |
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Five RAO-affected horses in remission from their disease were studied [4 geldings and 1 mare; age, 16 ± 4 y; body wt, 460 ± 51 kg (means ± SD)]. Remission was defined as <20 neutrophils/µL of BALF. The experimental protocol was approved by the Ethics Committee of the Animal Health Trust and conformed to the Animals (Scientific Procedures) Act 1986. Horses were stabled continuously throughout the study on shredded-paper bedding and were fed 9 kg of haylage (Marksway Horsehage, ryegrass, Marksway and Son, UK) and 3 kg of feed/d in two feeds that contained (in percentages) 10.0 protein, 4.0 fat, 15.0 fiber, and 9.0 ash. Water was provided ad libitum.
Study design
One week before the start of the study, each horse performed an incremental exercise test on a high-speed treadmill to determine maximum oxygen uptake (VO2max) (1). The horses were studied in a crossover design with each animal acting as its own control. The horses underwent a 4-wk lead-in period, after which three horses received an oral antioxidant supplement (Winergy Ventil - ate®, Effem Equine, Milton Keynes, UK), and two were fed a placebo for 4 wk. After a 4-wk washout period, the groups were reversed. The investigators were unaware of the identity of each supplement. The placebo comprised (in percentages) 6.0 protein, 2.5 fat, 21.0 fiber, and 5.0 ash. The antioxidant supplement was produced from high-fiber materials, cereal grains, dried forages, sugar-production product and byproducts, brewers yeast, oil, fats, and tubers and roots products and byproducts, and contained (in percentages) 13.0 protein, 10.0 fat, 10.0 fiber, and 8.0 ash. Vitamin E (10 g of 
-tocopherol acetate/kg of supplement), vitamin C (57 g of calcium ascorbyl monophosphate/kg of supplement), and selenium (4 mg/kg of supplement) were included in the antioxidant supplement. The total daily intake amounts of vitamin E, vitamin C, and selenium on the placebo diet were 1.1 mg/kg of body wt, <0.95 mg/kg of body wt (below the limit of detection), and 1.9 µg/kg of body wt, respectively; and 6.0 mg/kg of body wt, 10 mg/kg of body wt, and 5.1 µg /kg of body wt, respectively, for the antioxidant diet. During the study, horses were exercised 3 d/wk on the treadmill at a 3° incline (5 min at 1.7 m/s, 5 min at 3.7 m/s, 2 min at 90% of VO2max, and 5 min at 1.7 m/s). For the remaining 4 d, they walked for 30min on a horsewalker.
Three days before the end of each supplementation period, the horses underwent bronchoalveolar lavage (BAL) of the right dorsocaudal lung. On the final day of each period, the horses performed a standard exercise test (SET) at an ambient temperature of 
20°C and relative humidity of 60%. Four hours before the SET, the horses were fed, and any food that remained 1 h later was removed. The SET consisted of 10 min at 1.7 m/s and 5 min at 3.7 m/s followed by three 2-min periods at 70, 80, and 90% of VO2max separated by 5 min at 1.7 m/s. The entire test was carried out on a 3° incline. The horses walked for 30 min postexercise at 1.7 m/s on a 0° incline. BAL was performed on the right dorsocaudal lung 1 h postexercise (i.e., 1 h after the horse completed 2 min at 90% of VO2max). Venous blood samples (20 mL) were collected 1 h before and 0, 15, and 60 min and 24 h after the SET.
Sample collection and analysis
BAL was performed using 200 mL of 0.9% saline at 37°C, and the samples were processed as described previously (1). BALF-cell pellets were homogenized mechanically for 1 min and centrifuged at 5000 x g for 2 min. Plasma, BALF, and BALF-cell pellet ascorbic acid and plasma -tocopherol were analyzed by HPLC with UV detection (8,9). The ascorbic acid redox ratio was calculated by dividing the concentration of dehydroascorbate by the total ascorbic acid concentration. HPLC with electrochemical detection was used to measure reduced and oxidized glutathione (GSH and GSSG, respectively) in BALF and red blood cell hemolysates (10). The glutathione redox ratio was calculated by dividing GSSG by the total concentration of glutathione. Plasma uric acid content was determined using Sigma kit 685-10. BALF uric acid and malondialdehyde contents were determined by HPLC with UV and fluorometric detection (11), respectively.
Statistical analyses
The majority of variables were not normally distributed as assessed by an examination of histograms, rankit plots, and the Shapiro-Wilk's normality test. After transformation of the data using either the natural logarithm or square (as appropriate), the majority of variables were still not normally distributed. Therefore, additional analysis was undertaken using nonparametric tests, and data are presented as medians and ranges (minimum to maximum values). Pairwise Wilcoxon signed-rank tests were used to compare placebos and treatments at each time point. Friedman analysis of variance was used to analyze data for the effects of exercise on systemic variables. Separate analyses were carried out for the treatment and placebo data. If significance was attained, post-hoc pairwise comparisons were performed within each treatment group using Wilcoxon signed-rank tests to compare individual SET time points to baseline values (at rest). For pulmonary variables, the effects of treatment were assessed using one-sample Wilcoxon signed-rank tests after taking the difference between pre- and postexercise measurements and, subsequently, the difference between placebo and treatment. A linear regression approach with treatment, exercise, and horse as fixed effects was used to test for an association between antioxidant concentrations. The models were built using a forward-selection approach with PROC GENMOD (SAS version 8). The fixed effects were included in all models to control for the repeated observations on horses. Model fit was checked by an examination of the residuals. All values of significance are set at P < 0.05.
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RESULTS |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Median BALF total nucleated cell count (190; range, 70–214 cells/µL) and median numbers of macrophages (91; range, 21–101 cells/µL), lymphocytes (71; range, 29–105 cells/µL), neutrophils (2; range, 0–24 cells/µL), and epithelial cells (1; range, 0–2 cells/µL) were not significantly different after antioxidant supplementation compared to the placebo treatment.
Circulatory antioxidant status
Antioxidant supplementation significantly increased median plasma ascorbic acid concentrations at rest and 0, 15, and 60 min and 24 h after the SET (Fig. 1A) compared to placebo administration (P < 0.05). The plasma concentration of ascorbic acid increased (P = 0.04) immediately postexercise compared to at rest when supplemented with the antioxidant diet or the placebo. Plasma concentrations of dehydroascorbate (0.8; range, 0.3–1.4 µmol/L) and uric acid (17; range, 16–21 µmol/L) and the ascorbic acid redox ratio values (0.10; range, 0.03–0.10) at rest were not altered by antioxidant supplementation or the SET. Median concentration of plasma -tocopherol was higher after antioxidant supplementation compared to placebo administration (P = 0.04) but was not altered by the SET (Fig. 1B).
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Pulmonary antioxidant status and lipid peroxidation
The median percentage of BALF recovery on the placebo diet was 55% (range, 40–75%) and was not different after antioxidant supplementation or the SET. BALF ascorbic acid concentration did not increase significantly after antioxidant supplementation either before or after the SET (12.8; range, 0.3–33.9 and 13.5; range, 0.0–17.0 µmol/L, respectively) compared with placebo administration (5.5; range, 0.0–11.6 and 9.7; range, 1.5–14.2 µmol/L, respectively). BALF dehydroascorbate (2.9; range, 0.2–6.0 µmol/L), ascorbic acid redox ratio values (0.34; range, 0.02–1.00), GSH (0.9; range, 0.4–1.5 µmol/L), glutathione redox ratio values (0.06; range, 0.03–0.12), uric acid (10; range, 5–25 nmol/L), malondialdehyde (29; range, 17–33 nmol/L), and BALF-pellet ascorbic acid concentrations (57; range, 13–88 nmol/mg of protein) at rest on the placebo diet were not altered after antioxidant supplementation or the SET (P > 0.05). A trend toward a decrease in BALF GSSG concentration (P = 0.06) was observed after antioxidant supplementation (28; range, 8–103 nmol/L) compared to the placebo (70; range, 25–140 nmol/L). BALF ascorbic acid redox ratio was not related to glutathione redox ratio. However, there was a small correlation between BALF GSH and BALF GSSG concentrations after controlling for the effect of repeated measures in individual horses (ß-coefficient = 6.76; P = 0.03).
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DISCUSSION |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In the present study, exercise failed to induce either pulmonary or systemic oxidative stress. A similar level of exercise was previously demonstrated to produce a marked increase in plasma uric acid concentration in RAO-affected horses that was reduced after antioxidant supplementation (7). Furthermore, BALF uric acid concentration was also increased after exercise (7). This difference may reflect the higher level of remission of the RAO-affected horses in the present study based on BALF cytology.
We previously demonstrated that the BALF ascorbic acid redox ratio is correlated with the BALF glutathione redox ratio in healthy control horses (1) and when controls are compared with RAO-affected horses that have chronic airway inflammation (3). In the present study, however, there was no relationship between these two variables. Interestingly, BALF GSH correlated with BALF GSSG concentration in RAO-affected horses. An increase in GSSG content with a simultaneous equivalent increase in GSH concentration will prevent or reduce any increase in glutathione redox ratio. An increased oxidant load, indicated by the increase in GSSG, may have led to an increase in BALF GSH via the redox-sensitive transcriptional upregulation of mRNA for 
-glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis (12).
In conclusion, oral antioxidant supplementation significantly elevated plasma ascorbic acid and -tocopherol concentrations in RAO-affected horses. Interestingly, the concentration of GSH correlated with the concentration of GSSG in BALF from RAO-affected horses, which may reflect a protective compensatory mechanism to an oxidant load.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This study was supported by WALTHAM Centre for Pet Nutrition, Leicestershire, UK.
4 Abbreviations used: BAL, bronchoalveolar lavage; BALF, bronchoalveolar lavage fluid; GSH, reduced glutathione; GSSG, oxidized glutathione; RAO, recurrent airway obstruction; SET, standard exercise test; VO2max, maximum oxygen uptake.
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LITERATURE CITED |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Deaton, C. M., Marlin, D. J., Smith, N. C., Roberts, C. A., Harris, P. A., Kelly, F. J. & Schroter, R. C. (2002) Antioxidant supplementation and pulmonary function at rest and exercise. Equine Vet. J. Suppl. 34: 58–65.
2. Art, T., Kirschvink, N., Smith, N. & Lekeux, P. (1999) Indices of oxidative stress in blood and pulmonary epithelium lining fluid in horses suffering from recurrent airway obstruction. Equine Vet. J. 31: 397–401.[Medline]
3. Deaton, C. M., Marlin, D. J., Smith, N. C., Harris, P. A., Roberts, C. A., Schroter, R. C. & Kelly, F. J. (2004) Pulmonary epithelial lining fluid and plasma ascorbic acid concentrations in horses affected by recurrent airway obstruction. Am. J. Vet. Res. 65: 80–87.[Medline]
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6. Schunemann, H. J., Freudenheim, J. L. & Grant, B. J. (2001) Epidemiologic evidence linking antioxidant vitamins to pulmonary function and airway obstruction. Epidemiol. Rev. 23: 248–267.
7. Kirschvink, N., Fievez, L., Bougnet, V., Art, T., Degand, G., Smith, N., Marlin, D., Roberts, C., Harris, P. & Lekeux, P. (2002) Effect of nutritional antioxidant supplementation on systemic and pulmonary antioxidant status, airway inflammation and lung function in heaves-affected horses. Equine Vet. J. 34: 705–712.[Medline]
8. Liau, L. S., Lee, B. L., New, A. L. & Ong, C. N. (1993) Determination of plasma ascorbic acid by high-performance liquid chromatography with ultraviolet and electrochemical detection. J. Chromatogr. 612: 63–70.[Medline]
9. Kelly, F. J., Rodgers, W., Handel, J., Smith, S. & Hall, M. A. (1990) Time course of vitamin E repletion in the premature infant. Br. J. Nutr. 63: 631–638.[Medline]
10. Smith, N. C., Dunnett, M. & Mills, P. C. (1995) Simultaneous quantitation of oxidised and reduced glutathione in equine biological fluids by reversed-phase high-performance liquid chromatography using electrochemical detection. J. Chromatogr. B Biomed. Appl. 673: 35–41.[Medline]
11. Young, I. S. & Trimble, E. R. (1991) Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection. Ann. Clin. Biochem. 28: 504–508.
12. Rahman, I. & MacNee, W. (1999) Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am. J. Physiol. 277: L1067–L1088.
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) or a placebo (•). Statistical analysis was performed using Friedman analysis of variance and Wilcoxon signed-rank tests. *P < 0.05, antioxidant treatment significantly different from placebo; #P = 0.04, exercise time point significantly different from rest.