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Hill’s Pet Nutrition, Incorporated, Topeka, KS
3To whom correspondence should be addressed. E-mail: karen_wedekind{at}hillspet.com.
KEY WORDS: • antioxidants • dogs • oxidative stress • vitamin E • vitamin C
EXPANDED ABSTRACT
A variety of bioactive compounds in foods contribute functionality to feedstuffs. In particular, antioxidants (AOX) play an important role in reducing the risk of free-radical–related oxidative damage associated with aging and degenerative diseases. Fruits and vegetables are high in flavonoids, carotenoids, vitamins and minerals, and other phytochemical components that have been shown to have high AOX properties. Epidemiologic evidence (1,2) as well as a number of prospective trials (3,4) have shown that diets rich in fruits and vegetables and/or vitamins and minerals are associated with delayed aging and reduced risk of cancer, coronary heart disease (CHD) and other degenerative diseases. It is clear from the literature that AOX biomolecules in combination are more effective than single sources of antioxidant ingredients (2,5–7). Thus, it was our objective to evaluate various combinations and levels of antioxidants to determine which combination yields the greatest efficacy in dog foods.
The levels of antioxidants used in our study were based on kinetic studies in humans. Traber et al. (8) determined that 150 IU 
-tocopherol/d were necessary to reach steady-state conditions. Similarly, a number of other reviews (9, 10) suggest this level of vitamin E to be protective. A pharmokinetics study (11) determined that 200 mg vitamin C/d was necessary to reach plasma saturation as well as steady-state plasma concentrations. Recommendations by the National Cancer Institute, the American Heart Association and the U.S. Department of Agriculture suggest a minimum of five servings of fruits and vegetables daily, which provide by calculation approximately 200 mg vitamin C/d (5). Thus, the canine metabolic equivalent of 150 mg/d vitamin E and 200 mg/d vitamin C formed the basis for our minimum level of antioxidant inclusion along with 1 mg/kg ß-carotene. Different levels of this antioxidant combination as well as addition of fruits and vegetables were evaluated in our study.
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MATERIALS AND METHODS |
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Dogs were blocked by age and sex and allotted to a randomized complete block design with eight dogs/treatment. Treatments included: 1) a control (ctrl) formula; 2) ctrl + 1x inclusion of AOX vitamins consisting of vitamin E provided as -tocopheryl acetate (Roche Vitamins, Parsippany, NJ), vitamin C provided as ascorbyl-2-monophosphate (Stay-C; Roche Vitamins,) and ß-carotene (Roche Vitamins,) added topically; 3) ctrl + 2x inclusion of AOX vitamins; 4) ctrl + 2x inclusion of AOX vitamins + fruits and vegetables consisting of tomato pomace, grape pomace, dried carrots, spinach and citrus pulp, each at 1% inclusion; and 5) ctrl + fruits and vegetables only (same inclusion as in treatment 4). Analyzed concentrations of vitamins E, C and ß-carotene for these formulations are shown in Table 1.
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Measures of antioxidant status included serum alpha tocopherol, ascorbate, and ORAC. Serum -tocopherol was analyzed by high-performance liquid chromatography with UV detection after methanol/hexane extraction (12). Serum vitamin C was measured using a fluorometric assay (13). An automated ORAC assay was used for measuring serum ORAC according to the procedures of Cao et al. (14) and was performed by a commercial laboratory, and appropriate controls were used in the measure of these samples (Genox, Baltimore, MD). It is not known whether the ORAC method has been validated for the dog, but there is no reason to believe that there would be interspecies specificity given the nature of this assay.
The study was evaluated as a factorial design using the general linear models (GLM) procedure of SAS (15) with a model appropriate for a randomized complete block design. Dogs were blocked by sex and age, such that within each block, each treatment had a similar sex and age distribution. There was a tendency (P < 0.10) for animals to have differing initial ORAC values; thus postmeans were adjusted to a common value of the covariate. Two covariates were used in the model, adjusting for both initial value and average intake. Data are presented as initial and as the change between end and beginning measurements or difference (i.e., wk 4 - time 0). Preplanned orthogonal contrasts included linear and quadratic effects for treatments 1–3, main effects of AOX (treatments 3 and 4 vs. treatments 1 and 5), main effects of fruits and vegetables (treatments 4 and 5 vs. treatments 1 and 3) and the interaction between AOX and fruits and vegetables (treatments 3 and 5 vs. treatments 1 and 4). Probability values of P < 0.05 between treatments were considered significant.
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RESULTS |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Serum C concentrations were likewise higher than values in other published literature (20). Despite the fact that dogs in the Marshall study were given a 1 g sample of vitamin C as a supplement (this approximates 3400 mg/kg diet), compared to only 224 and 341 mg/kg diet in our study, serum C concentrations in the Marshall study ranged from 28.4 to 39.8 µmol/L relative to our range of 3 to 143 µmol/L. These differences in serum C concentrations could be attributable to methodological differences (i.e., laboratory procedure used, timing of blood collection, lack of acidification of samples, etc.). The serum C changes observed in our study were difficult to explain. For example, we did not see a dose effect for serum vitamin C with increasing dietary C. Other published literature (21), however, has shown that vitamin C fed in the Stay-C form is bioavailable and well utilized by the dog. Surprisingly, we saw higher serum C as a result of feeding fruits and vegetables. This higher serum C for dogs fed the fruit and vegetable treatments cannot be explained by the vitamin C contributed by these ingredients. It is possible that the higher serum C observed in dogs fed the fruits and vegetables results from other antioxidants or some other factor (i.e., flavonoids and/or carotenoids, etc.) that caused a regeneration of vitamin C not seen in the serum of dogs fed diets not containing fruits and vegetables. A number of studies have shown that antioxidants in combination are more effective (5,6). The fact that many of the antioxidants are linked in cascades or interrelated processes resulting in a recirculation of certain metabolites may offer some explanation for the higher serum C observed for dogs fed the fruit- and vegetable-containing treatments.
Based on the findings from this study, these data would suggest that the 2x inclusion of AOX was efficacious in improving antioxidant status based on higher serum vitamin E and ORAC observed in dogs fed the 2x AOX concentrations. The combination of 2x AOX + fruits and vegetables did not increase serum vitamin E or ORAC but was instead numerically decreased relative to the 2x AOX treatment only; thus there was not a synergistic effect, as expected. Serum vitamin C concentrations were, however, increased in dogs fed the fruit and vegetable addition.
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FOOTNOTES |
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2 Supported by Hill’s Pet Nutrition, Inc., Topeka, KS.
4 Abbreviations used: AOX, antioxidants; ctrl, control; 8OHdG, 8-hydroxy-2'-deoxyguanosine; ORAC, oxygen radical absorbance capacity.
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LITERATURE CITED |
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TOPMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Block, G. (1992) The data support a role for antioxidants in reducing cancer risk. Nutr. Rev. 50:207-213.[Medline]
2. Langseth, L. (1995) Oxidants, Antioxidants and Disease Prevention 1995 International Life Sciences Institute (ILSI Europe) Brussels, Belgium .
3. Rimm, E. B., Stampfer, M. J., Ascherio, A., Giovannucci, E., Colditz, G. A. & Willett, W. C. (1993) Vitamin E consumption and the risk of coronary disease in men. N. Engl. J. Med. 328:1450-1456.
4. Stampfer, M. J., Hennekens, C. J., Manson, J. E., Colditz, G. A., Rosner, B. & Willett, W. C. (1993) Vitamin E consumption and the risk of coronary disease in women. N. Engl. J. Med. 328:1444-1449.
5. Lachance, P. A. (1997) Micronutrients in Cancer Prevention. ACS Symposium Series no. 1994 1997:49-64 American Chemical Society Washington, DC .
6. Sayhoun, N. R. (1997) Vitamin C: what do we know and how much do we need?. Nutrition 13:835-836.[Medline]
7. Schorah, C. (1995) Micronutrients, antioxidants and risk of cancer. Bibl. Nutr. Dieta 52:92-107.
8. Traber, M. G., Ramakrishnan, R. & Kayden, H. J. (1994) Human plasma vitamin E kinetics demonstrate rapid recycling of plasma RRR--tocopherol. Proc. Natl. Acad. Sci. U.S.A. 91:10005.
9. Charleux, J. (1996) Beta-carotene, vitamin C and vitamin E: the protective micronutrients. Nutr. Rev. 54:S109-S114.[Medline]
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12. Driskell, W. J., Neese, J. W., Bryant, C. C. & Bashor, M. M. (1982) Measurement of vitamin E in human serum by high-performance liquid chromatography. J. Chromatogr. 231:439-444.[Medline]
13. Vuilleumier, J. P. & Keck, E. (1989) Fluorometric assay of vitamin C in biological materials using a centrifugal analyser with fluorescence attachment. J. Micronutr. Anal. 5:25-34.
14. Cao, G., Verdon, C. P., Wu, A.H.B., Wang, H. & Prior, R. L. (1995) Automated oxygen radical absorbance capacity assay using the COBAS FARA II. Clin. Chem. 41:1738-1744.[Abstract]
15. SAS (1989) 4th ed. SAS User’s Guide. Statistics, version 6 2 SAS Institute Inc. Cary, NC .
16. Harper, E. J. & Alblas, J. (1999) Supplementation of dietary tocopherol increases plasma values outside the normal ranges. FASEB J 13:A564.
17. Biourge, V., Grandjean, D., Sergheraert, R. & Driss, F. (1999) Vitamin E supplementation in sled dogs 1999:727 Proc. of the 17th ACVIM Chicago, IL (abstract).
18. Baskin, C. R., Hinchcliff, K. W., DiSilvestro, R. A., Reinhart, G. A., Hayek, M. G., Chew, B. P., Burr, J. R. & Swenson, R. A. (2000) Effects of dietary antioxidant supplementation on oxidative damage and resistance to oxidative damage during prolonged exercise in sled dogs. Am. J. Vet. Res. 61:886-891.[Medline]
19. McCall, M. R. & Frei, B. (1999) Can antioxidant vitamins materially reduce oxidative damage in humans?. Free Radic. Biol. Med. 26:1034-1053.[Medline]
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21. Schulze, J., Broz, J. & Ludwig, B. (1993) Efficacy of L-ascorbate-2-polyphosphate as a source of ascorbic acid in dogs. Int. J. Vitam. Nutr. Res. 63:63-64.[Medline]
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