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Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zed) Road, Arm Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 NOTE TO USERS This reproduction is the best copy avaiiable UMI STUDIES ON ASCORBIC ACID BIOSYNTHESIS IN FISH, EFFECTS OF ANTIOXIDANT VITAMINS ON L-GULONOLACTONE OXIDASE ACTIVITY DISSERTATION Presented in Partial FuUfiUment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Régis Moreau, M.S. ***** The Ohio State University 1999 Approved by Dissertation committee: Konrad Dabrowski, adviser Jerry F. Downhower, co-adviser Patricia G. Parker David L. Stetson Advisers Department of Zoology UMI Number: 9931654 UMI Microform 9931654 Copyright 1999, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTEIACT The inability to biosynthesize ascorbic acid (vitamin C) is rather the exception in vertebrates. Many terrestrial vertebrates can synthesize tlie vitamin owing to the presence of L-gulonolactone oxidase in the liver or the kidney, i.e. the enzyme catalyzing the last step of the ascorbic acid synthetic pathway. However, the primates, the guinea pigs, the bats, and some passerine birds, which all lack the enzyme, are notorious exceptions. L- Gulonolactone oxidase is the only enzyme missing in the ascorbic acid synthetic pathway in vertebrates. Administering chicken L-gulonolactone oxidase to the guinea pig induced the production of ascorbic acid as evidenced by elevated plasma levels and good condition while fed a vitamin C-deficient diet Like the primates, teleost fish, which account for -96% percent of present-day fish biodiversity, have long been thought to be incapable of synthesizing ascorbic acid. Finthermore, this view has been extended imjustifiably to all modem fishes. But the recent discovery of L-gulonolactone oxidase activity in the kidney of the Siberian {Acipenser baen), white (A. transmontanus), lake sturgeon {A. fulvescens), and the paddlefish (Polyodon spathuld) has shed new light on the origin and phylogeny of the character in vertebrates. This dissertation is organized around two major objectives, (1) to investigate the distribution of ascorbic acid biosynthesis in extant fishes, and (2) to study the nutritional control of the synthetic pathway in a fish. We asked (i) what is the organ localization of L- gulonolactone oxidase in fish, (ii) when was the origin of ascorbic acid biosynthesis in fishes, (iii) what is the character tree for extant fishes, (iv) do some teleost fish synthesize u ascorbic acid, and (v) can dietary vitamin C or E affect the activity of L-gulonolactone oxidase. After réévaluation and extension of earlier work, we provided evidence that, while the teleosts studied lack the enzyme L-gulonolactone oxidase, and thus require a dietary source of vitamin C, all non-teleost fish investigated thus far possess the enzyme in the kidney. The present work supported the view that kidneys are the site of ascorbic acid synthesis in poïkilothermie vertebrates such as the fish, amphibians, and reptiles. L- Gulonolactone oxidase activity was not uniform in the kidneys of sturgeon. The major site ofascorbic acid synthesis was in the posterior kidney iP < 0.01). In contrast, ascorbic acid concentration was significantly higher (P < 0.01) in the head kidney. An active transport of ascorbic acid to the cortical and chromaffin tissues may provide a suitable explanation for the concentration gradient The finding of L-gulonolactone oxidase activity in the kidney of the sea lamprey {Petromyzon marinus), the most phylogenetically ancient group of fishes ever studied for ascorbic acid biosynthesis, suggested that the function may have originated in the ancestor of present-day fishes as early as 590-500 miUion years ago in the Cambrian period, and later was past on to terrestrial vertebrates. The daily rate of ascorbic acid synthesis in a fish was estimated based on kinetics data obtained with L-gulonolactone oxidase purified from white sturgeon (A. transmontanus) posterior kidneys. The projections showed that a 1-kg sturgeon could synthesize 3 mg ascorbic acid per day, and a 1-kg sea lamprey, 4 mg at 15°C. These rates were approximately 7 times slower than in the rat per unit weight, and suggested to correlate with basal metabolic rate. Unlike in mice, feeding lake sturgeon {A. fulvescens) with high levels of vitamin C did not slow-down the rate of synthesis in the kidney. On the contrary, the activity of the enzyme L-gulonolactone oxidase was stimulated by dietary vitamin C. The deprivation of both vitamin C and E did not affect significantly ( P > 0.05) m L-gulonolactone oxidase activity. These observations indicated that neither vitamin exerted a negative feedback on the rate of ascorbic acid synthesis in the juvenile of lake sturgeon. The importance of synthesizing ascorbic acid in non-teleost fishes and the reasons for the loss of the function in teleosts are discussed. IV ACKNOWLEDGMENTS I wish to express my gratitude to ray adviser. Dr. Konrad Dabrowski, for accepting rae as a graduate student, for providing intellectual stimulation and financial support Many thanks for your belief in ray abilities not only to establish a research program but also to overcome all the difficulties therein. I admire your enthusiasm, dedication, and professionalism. Special thanks to my M.S. advisor. Dr. Sadasivam Kaushik, in France, for recommending me to Dr. Dabrowski, encouraging me to grow professionally, and supporting my efforts to conduct research. I wish to thank Dr. Jerry Downhower for serving as my co-advisor, for sharing his wisdom, expertise, and spirit of academic excellence. This educational experience with you has been a privilege. I acknowledge and appreciate the effort and attention of my Dissertation Committee Members, Dr. Patricia Parker and Dr. David Stetson, for critical review of my writings. Thank you to Dr. Paul Sato and Dr. Diane McClure for providing their expertise, valuable guidance, and assistance in achieving my goals. I appreciate your patience and clarity in helping me understand the heart of the matter. Sincere appreciation is extended to my colleagues and friends who provided assistance and encouragement throughout these years. This research could have never been accomplished without the personnel of the Aquaculture Program at the Piketon Research and Extension Center. I am grateful for the assistance received from the personnel of the School of Natural Resources, and the Department of Zoology. I am also thankful to the School of Natural Resources for providing financial support when research funding was lacking. Last but never far from my mind and heart despite the miles, my family. To my parents whose support in my educational and professional goals has been life-long. Had it not been for your confidence in me this would not have been possible. I am grateful. VI VTTA December 19, 1968 ......................Bom - Dreux, France 1989..............................................B.S. Biology, University of Tours, France 199 1..............................................M.S. Oceanography, University of Western Brittany, Brest, France 199 2..............................................Degree of Advanced Studies in Biology and Agronomy, University of Rennes 1, France 1995-present ..................................Graduate Research Associate, The Ohio State University PUBLICATIONS 1. Moreau, R , Dabrowski, K. and Sato, P.H. 1999. Renal L-gulono-1,4-lactone oxidase as affected by dietary ascorbic acid in lake sturgeon {Acipenserfulvescens). Aquaculture (in press) 2. Kolkovski, S., Czesny, S., Yackey, C., Moreau, R., Chila, F., Mahan, D. and Dabrowski, K. 1999. The effect of vitamin C and E in (n-3) highly unsaturated fatty acids enriched artemia nauplii on growth, survival, and stress resistance of fresh water walleye larvae. Aquaculture Nutrition (in press) 3. Moreau, R , Dabrowski, K., Czesny, S. and Cihla, F. 1999. Vitamin C-vitamin E interaction in juvenile lake sturgeon {Acipenser fiilvescens R.), a fish able to synthesize ascorbic acid. Journal of Applied Ichthyology (in press) 4. Moreau, R. and Dabrowski, K. 1998. Fish acquired ascorbic acid synthesis prior to terrestrial vertebrate emergence. Free Radical Biology and Medicine 25: 989-990 vu 5. Moreau, R. and Dabrowski, K. 1998. Body pool and synthesis of ascorbic acid in adult sea lamprey (Petromyzon marinus): An agnathan fish with gulonolactone oxidase activity. Proceedings of the National Academy of Sciences of the USA 95: 10279-10282 6. Moreau, R , Cuzon, G. and Gabaudan, J. 1998. Efficacy of silicone-coated ascorbic acid and ascorbyl-2-polyphosphate to fast-growing tiger shrimp (JPenaeus monodon). Aquaculture Nutrition 4: 23-29 7. Moreau, R. and Dabrowski, K. 1996.