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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
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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. Feeding stimulants in semipurified diets for juvenile lake sturgeon (Acipenser fidvescens Rafinesque). Aquaculture Research 27: 953- 957
8 . Moreau, R., Kaushik, S.J. and Dabrowski, K. 1996. Ascorbic acid status as affected by dietary treatment in the Siberian sturgeon (Acipenser baeri Brandt): tissue concentration, mobilisation and L-gulonolactone oxidase activity. Fish Physiology and Biochemistry 15: 431-438
9. Moreau, R. and Dabrowski, K. 1996. The primary localization of ascorbate and its synthesis in the kidneys of acipenserid (Chondrostei) and teleost (Teleostei) fishes. Journal of Comparative Physiology B 166: 178-183
10. Dabrowski, K., Moreau, R , El-Saidy, D. and Ebehng, J. 1996. Ontogenetic sensitivity of channel catfish to ascorbic acid deficiency. Journal of Aquatic Animal Health 8: 22-27
11. Dabrowski, K. and Moreau, R. 1996. Do aU fish need ascorbic acid? Aquaculture Magazine 22(5): 96-98
FIELDS OF STUDY
Major Field: Zoology Research Area: Fish Nutrition
vm TABLE OF CONTENTS
Page
Acknowledgments ...... v Vita...... vii List of Tables ...... xi List of Figures ...... xiü Overview ...... xvüi
Chapters: 1. Introduction ...... L
2. The primary localization of ascorbate and its synthesis in the kidneys of acipenserid (Chondrostei) and teleost (Teleostei) fishes ...... 10
3. Body pool and synthesis of ascorbic acid in adult sea lamprey (Petromyzon marinus): An agnathan fish with gulonolactone oxidase activity...... 17
4. Distribution of L-gulonolactone oxidase among Actinopterygii ...... 22
5. Feeding stimulants in semipurified diets for juvenile lake sturgeon, Acipenser fulvescens Rafinesque ...... 39
6. Renal L-gulonolactone oxidase as affected by dietary ascorbic acid in lake sturgeon (Acipenser fulvescens)...... 45
7. Vitamin C-vitamin E interaction in juvenile lake sturgeon (Acipenser fulvescens R.), a fish able to synthesize ascorbic acid ...... 68
8. Conclusion ...... 94
Appendices
IX Appendix A: Fish acquired ascorbic acid synthesis prior to terrestrial vertebrate emergence ...... 109
Appendix B: Measurement of L-gulonolactone oxidase activity in fish tissues...... 112
List of References ...... 118 LIST OF TABLES
Table Page
1. Characteristics of the fish used in the present study (mean ± SD; n = 3 ) ...... 12
2. AA biosynthesis in the kidney of 300-g adult sea lampreys ...... 20
3. Gulonolactone oxidase activity in the kidney of bowfin (Amia calva), longnose gar (Lepisosteus osseus), and Polyptenis senegalus measured at25°C...... 29
4. Immunologic crossreactivity and specificity of some ascorbic acid synthesizing vertebrates with sturgeon GLO ...... 30
5. Body weight, gonadosomatic index (GSI), hepatosomatic index (HSI), and total ascorbic acid (TAA), dehydroascorbic acid (DHAA) concentrations in selected tissues of bowfin and longnose gar ...... 31
6. Composition of the experimental diets ...... 41
7. Growth and survival of juvenile lake sturgeon of two different sizes fed the experimental diets. Data are shown as mean ± SEM (n = 3); within trials, values of survival or SGR not sharing a common superscript are significantly different (P < 0.05) ...... 42
8. Composition of the basal diet ...... 57
9. Growth rate and hepatopancreas index of lake sturgeon fed graded levels of AA ...... 58
10. Total (TAA), oxidized (DHAA), and reduced (AA) ascorbic acid concentrations in tissues of juvenile lake sturgeon fed graded levels of AA...... 59
11. L-Gulonolactone oxidase (GLO) in posterior kidney, dehydroascorbic acid (DHAA) reductase in posterior kidney and liver, and alkaline phosphatase in blood plasma of juvenile lake sturgeon fed graded levels of AA ...... 60
XI 12. Composition of the basal diet (OC-OE) ...... 81
13. Fatty acid composition of the basal diet (OC-OE) ...... 82
14. Phospholipid fatty acid composition of lake sturgeon dorsal muscle ...... 83
15. Triglyceride fatty acid composition of lake sturgeon dorsal muscle ...... 84
16. Phospholipid fatty acid composition of lake sturgeon liver ...... 85
17. Contribution to the distribution of gulonolactone oxidase (GLO) among extant fishes ...... 103
18. Gulonolactone oxidase (GLO) activities reported in fish and methods used to determine the production of ascorbic acid in vitro...... 105
19. Gulonolactone oxidase (GLO) activity assays using L-gulonolactone as a substrate ...... 116
XU LIST OF nOURES
Figure Paee 1. Gross anatomy of kidneys in the three fish species; the channel catfish (A), the rainbow trout (B) and the white sturgeon (C) ...... II
2. Total ascorbic acid {whole columns) and dehydroascorbic acid {black column) concentrations in the head and trunk kidneys of male channel catfish. Each trunk kidney was first divided into two according to the symmetrical plan of the organ. One semi-trunk kidney per fish was taken for analysis and divided into five portions indicated as 1st to 5th from the anterior extremity to the trunk kidney. Vertical bars represent SD for three animals. Means not sharing a common letter are significantly different at P < 0.01 and 0.05 for TAA and DHA, respectively ...... 13
3. Total ascorbic acid concentrations in the kidney of the rainbow trout fed a diet containing 270.9 mg AA ' kg'*^ {whole columns) or a diet devoid of AA {dotted columns). Kidneys were divided into ten equal portions indicated as 1st to 10th from the anterior extremity of the organ. Vertical bars represent SD for three animals. Means not sharing a common letter are significantly different (P < 0.05) ...... 13
4. Total ascorbic acid {whole columns) and dehydroascorbic acid {black columns) concentrations in the kidney of male white sturgeon. Kidneys were divided into six equal portions indicated as 1st to 6th from the anterior extremity of the organ. Vertical bars represent SD for three animals. Means not sharing a common letter are significantly different at P < 0.01 and 0.05 for TAA and DHA, respectively ...... 14
5. L-Gulonolactone oxidase (GLO) activity {blank columns) and total ascorbic acid (AA) concentration {dashed line) within the kidney of male white sturgeon. Kidneys divided into six equal portions indicated as 1st to 6th from the anterior extremity of the organ. Total ascorbic acid means are from Fig. 4. Vertical bars represent SD for three animals. Means not sharing a common letter are significantly different (P < 0.01)...... 14
xni 6. Hierarchy of living fishes. A + denotes that at least one species in a given order was reported to biosynthesize AA de novo due to the presence of GLO, whereas - denotes the incapability as the result of the loss of the enzyme activity, f R.M. and K.D., unpublished data. (Adapted from ref. 38) ...... 19
7. Total AA (whole columns) and oxidized AA (black columns) concentrations in hver, kidney, and gonads of male {Top) and female {Bottom) sea lampreys trapped in 1995 and 1996 during the upstream migration to the spawning grounds. Mean ± SD of three to four males or females. A * denotes that total AA or oxidized AA concentration in a given tissue was significantly different in 1996 compared to 1995 (P < 0.05). The hepatosomatic indices (liver weight/ body weight, percent) were higher in males [2.6 ± 0.4% (n = 7)] than in females [1.4 ± 0.3% (n = 7)] in 2 subsequent yr. The gonadosomatic index (gonad weight/ body weight, percent) was 1.9 ± 0.5% (n = 5) in males sampled in 1995 and 1996. Fem^e gonadosomatic index was 25.0 ±4.8% (n = 7). The relative weight of kidney was 0.68 ± 0.04% (n = 14) of total body weight irrespective of the year of sampling or the sex ...... 20
8. Total AA distribution in adult sea lampreys trapped during the upstream migration to the spawning grounds. Data collected in 1995 and 1996 were combined for calculations. Proportions are expressed as percentage of total body pool ...... 21
9. Comparison of GLO activities (at 25°C) in the renal microsomes of some vertebrates with an emphasis on fishes. '^Moreau and Dabrowski 1996; "Moreau et al. 1999a; Moreau and Dabrowski 1998; '‘present study...... 32
10. Immunodouble diffusion tests of rabbit IgG to white sturgeon GLO with detergent-solubilized microsomes or crude extracts from various vertebrates. A through L, center well contains protein A-purified rabbit IgG. A-1, white sturgeon posterior kidney microsomes; 2, lake sturgeon posterior kidney microsomes; 3, common carp hepatopancreas crude extract. B-1, white sturgeon posterior kidney microsomes; 2, bowfin posterior kidney microsomes; 3, goldfish hepatopancreas crude extract. C-1, white sturgeon posterior kidney microsomes; 2, longnose gar kidney microsomes; 3, tadpole kidney microsomes. D-1, white sturgeon posterior kidney microsomes; 2, Polypterus kidney microsomes; 3, pig liver crude extract. E>1, lake sturgeon posterior kidney microsomes; 2, bowfin posterior kidney microsomes; 3, common carp kidney crude extract F-1, lake sturgeon posterior kidney microsomes; 2, longnose gar kidney microsomes; 3, goldfish kidney crude extract. G-1, lake sturgeon posterior kidney microsomes; 2, Polypterus kidney microsomes; 3, tadpole kidney microsomes. H I, bowfin posterior kidney microsomes; 2, longnose gar kidney microsomes; 3, pig liver crade extract. I-l, longnose gar kidney microsomes; 2, sea lamprey kidney microsomes; 3, tadpole kidney
XIV microsomes. J 1, bowfin posterior kidney microsomes; 2, sea lamprey kidney microsomes; 3, pig liver crude extract K-1, longnose gar kidney microsomes; 2, Polyptenis kidney microsomes; 3, common carp hepatopancreas crude extract L-1, bowfin posterior kidney microsomes; 2, Polypterus kidney microsomes; 3, goldfish hepatopancreas crude extract; 4, white sturgeon posterior kidney microsomes ...... 33
11. Distribution of gulonolactone oxidase (GLO) in Actinopterygii. Outgroup Petromyzontiformes (the lampreys). A + denotes the presence of GLO, whereas - denotes the absence of the enzyme inTeleostei. Tick mark in the teleost lineage indicates the loss of GLO activity. Cladogram based on Patterson (1973; 1982), Wiley (1976), Carroll (1988), and Nelson (1994). Until the mid-20th century, Lepisosteus was united with Ainia in the Holostei which was thought to represent an intermediate level of actinopterygian evolution between chondrosteans (Acipenseriformes and Polyodontiformes) and teleosts. Patterson Ù973) and Wiley (1976) showed that there are few significant derived characters that are common to Amia and gars. Amia shares many derived features with teleosts that are not evident in Lepisosteus. Wüey (1976) found a number of derived characters that Lepisosteus shares with the neopterygians but not with the living chondrostean orders. The above cladogram indicating that Amiiformes and teleosts are sister groups has been challenged by Olsen and McCune (1991) and Normark etal. (1991). Their studies suggested two alternatives 1) the Seraionotiformes rather than the Amiiformes as the sister-group of teleosts, and 2) the Semionotiformes and the Amiiformes as a monophyletic group that is the sister group of teleosts. The Acipenseriformes include the Acipenseridae (sturgeons) and the Polyodontidae (paddlefishes). It was once thought that the configuration of the mouth and the low degree of ossification of the endoskeleton (jugded to be an ancestral shared character) allied the Acipenseriformes widi sharks. Only with the discovery of Paleozoic bony fish did we realize that the early actinopterygians were heavily ossified and that the loss of ossification in the Acipenseriformes was a secondary specialization. Patterson (1982) places Acipenseriformes closer to the Neopterygii than he does the Polypteriformes. The Polypteriformes are viewed as the sister-group of all other actinopterygians ...... 35
12. Weight gain (%) of juvenile lake sturgeon at the end of the two feeding trials (trial I, 6 w eek; trial II, 4 w eek). Data are shown as mean ± SEM (n = 3). Values not sharing a common letter are significantly different (P < 0.05) ...... 41
13. Effect of dietary AA on GLO activity. Correlations between posterior kidney GLO activity and tissue total AA concentrations in lake sturgeon fed graded levels of AA. Values represent means per tank. One degree of freedom was assigned to each tank. Labels indicate AA supplement expressed as mg/kg diet ...... 61
XV 14. Effect of assay temperature on sturgeon GLO activity. In vitro synthesis of AA was measured at four temperatures in crude extract prepared from lake sturgeon posterior kidney. Data are shown as mean ± SD for 3 replicate measures. was calculated between 15° and 25°C following linear regression ...... 62
15. Effect of L-gulonolatone (S) concentration on sturgeon GLO velocity (V) at 25 °C. In vitro rate of AA synthesis as catalyzed by GLO purified from white sturgeon posterior kidney. (A) Lineweaver-Burk double reciprocal plot. (B) S/V against S plot. Data represent means for 2 replicate measures. Standard deviations lie within the symbols ...... 63
16. Pathways of vitamin E antioxidant action and its regeneration by vitamin C in the cell. MDA, malondialdehyde; DHAA, dehydroascorbic acid reductase; GLO, gulonolactone oxidase ...... 86
17. (A) Total ascorbic acid (whole column) and ascorbic acid (black column) concentrations, and (B) gulonolactone oxidase activity in posterior kidneys of juvenile lake sturgeon fed the four experimental diets. Two diets were devoid of vitamin C and supplemented with either 0 or 200 mg vitamin E/kg dry diet. Two other diets were supplemented with 1250 mg vitamin C&g dry diet and either 0 or 200 mg vitamin E/kg dry diet Data are shown as mean + or - SEM (n = 3 except dietary treatment 1250C-0E for which n = 2). Means not sharing a common letter are significantly different (P < 0.05). Vitamin C supplementation had a significant effect on ascorbate concentrations in the posterior kidney. Vitamin E supplementation and vitamin C-vitamin E interaction had no significant effect on ascorbate concentrations in the posterior kidney. Neither nutrient had any significant effect on GLO activity in the posterior kidney ...... 87
18. (A) Total ascorbic acid (whole column) and ascorbic acid (black column) concentrations, and (B) dehydroascorbic acid reductase activity in livers of lake sturgeon fed the four experimental diets. Two diets were devoid of vitamin C and supplemented with either 0 or 200 rag vitamin E/kg dry diet Two other diets were supplemented with 1250 mg vitamin C/kg dry diet and either 0 or 200 mg vitamin E/kg dry diet Data are shown as mean 4- or - SEM (n = 3 except dietary ureatment 1250C-0E for which n = 2). Means not sharing a common letter are significantly different (P < 0.05). Vitamin C supplementation had a significant effect on ascorbate concentrations in die liver. Vitamin E supplementation and vitamin C-vitamin E interaction had no significant effect on ascorbate concentrations in the liver. Neither nutrient had any significant effect on DKLAA reductase activity in the hver...... 88
19. Correlations between GLO activity in the posterior kidney and total ascorbic acid concentrations in the posterior kidney and the hver of juvenile lake sturgeon. Data are shown as means per tank. One degree of freedom was assigned to each tank ...... 89
XVI 20. (A) a-Tocopherol, and (B) thiobarbituric acid-reactive substances concentrations in livers of lake sturgeon fed the four experimental diets. Two diets were devoid of vitamin C and supplemented with either 0 or 200 mg vitamin E/kg dry diet Two other diets were supplemented with 1250 mg vitamin C/kg dry diet and either 0 or 200 rag vitamin E/kg dry diet Data are shown as mean + SEM (n = 3 except dietary treatment 1250C-0E for which n = 2). Means not sharing a common letter are significantly different (P < 0.05). Vitamin E supplementation and vitamin C-vitamin E interaction had a significant effect on a-tocopherol concentrations in the hver. Neither nutrient had any significant effect on thiobarbituric acid-reactive substances concentrations in the Hver...... 90
21. Distribution of GLO activity among extant fishes through time (Adapted from Nelson 1994). The continuous lines represent the fossil records and the dotted lines represent the inferred phylogeny (Greenwood 1984; Gardiner 1984; Schultze and Wiley 1984; Wüey and Schultze 1984; Carroll 1988; Nelson 1994) ...... 107
22. Phylogeny and tissue location of gulonolactone oxidase (GLO) in vertebrates. The cladogram impHes that the most recent common ancestor to aU vertebrates expressed GLO in the kidney. In the course of vertebrate evolution, the histological site of expression has shifted to the Hver as evidenced by an intermediate state where GLO is expressed in the kidney and Hver concomitantly. Ultimately, GLO was lost at more than one occasion, i.e. in teleost fish, passerin birds, guinea pigs, bats, and primates ...... 108
23. Measurement of GLO activity in teleosts using the DNPH method (524 nm) with or without appropriate correction for interering DNPH- derivatives...... 117
xvu OVERVIEW
My dissertation, is composed of eight chapters and two appendices. Following the introductory chapter (Chapter 1), six self-standing experiments form Chapters 2 through 7. These chapters have been written in the form of scientific articles. Three of them have been published (Chapters 2, 3 and 5), and two are in press (Chapters 6 and 7). Chapter 8 embodies the main findings and their interpretations in a concluding chapter. This dissertation deals with the biosynthesis of AA in extant fishes. Prior to the initiation of this Ph.D. research little was known on the subject. Only ten papers had been published. The knowledge was scarce and fragmented, and some of the data were controversial. As a result a great deal of the data presented in this dissertation are the first of the kind and will likely instigate further research in related fields. The first objective of this dissertation was to study for the first time the distribution of ascorbic acid biosynthesis in a variety of fish taxa including the lamprey (Petromyzon marinus), Polypterus senegalus, the longnose gar (Lepisosteus osseus), and the bowfin (Amia calva). Based on these data, a character tree was generated for present-day fishes. An end was put to the controversy surrounding ascorbic acid (vitamin C) synthesis in teleost fish. The second objective was to investigate the nutritional control of ascorbic acid biosynthesis in fish. Two nutritional experiments were conducted, using the lake sturgeon as a model, in which we looked at the effects of vitamin C and E on the rate of ascorbic acid synthesis. The implications of this work are both fundamental by addressing the evolution of ascorbic acid biosynthesis in vertebrates, and apphed by providing new information on the vitamin C requirement of fish.
xviii CHAPTER 1
INTRODUCTION
L-Ascorbic acid (AA, vitamin C) is naturally present in animals, higher plants, some Fungi (yeast), and some Protista (unicellular algae). It has not been detected in Monera (Bacteria + Archaea). Vitamin C was named ascorbic acid because it prevents and cures scurvy, one of the oldest diseases known to mankind. Ascorbic acid biosynthesis in plants occurs by conversion of D-glucose or D-mannose to L-galactono-1,4-lactone which is directly converted to AA by a mitochondrial enzyme, L-galactono-1,4-lactone dehydrogenase (Oba et al. 1995; Wheeler et al. 1998). This process differs substantially from that of the animals producing AA where D-glucose is oxidized to D-glucuronic acid
(Eisenberg et al. 1959), and L-gulono-1,4-lactone is the most effective precursor of AA. L- Gulono-1,4-lactone oxidase (GLO) has been identified as the enzyme responsible for the oxidation of L-gulono-1,4-lactone to AA, the terminal step in the biosynthesis of AA in animals (Nishikimi et al. 1976; Sato et al. 1976). Whether AA biosynthesis in plants is homologous with the biosynthesis in animals is unknown. Little is known about the functions and metabolism of AA in plants except that it is a precursor of oxalic acid and tartaric acid and may have roles in photosynthesis and transmembrane electron transport (Smirnoff 1996; Foyer 1993; Horemans et al. 1994). AA is a reducing agent acting in the aqueous compartment, both intra- and extracellular, of living organisms by donating one electron to a vast array of oxidized molecules and metals. High concentrations of AA in higher plants (HaUiwell 1982; Dash 1988) and unicellular algae (Shigeoka et al. 1987; Brown and Miller 1992) scavenge oxygen-derived free radicals such as singlet oxygen, superoxide and hydroxyl radical in order to terminate radical chain reaction. This protection against free radical toxicity (Machler et al. 1995) is also believed
to be a key function of AA in animal cell (Bendich et al. 1986; Niki 1991; Chakraborty et al. 1994; Baqa et al. 1994). As a co-factor AA maintains enzyme-bound metal (Mg, Cu, Fe) in the reduced form which sustains enzymatic activity. In contrast to plants, the biosynthesis of AA in animals is part of the glucuronic acid pathway. This pathway is redundant with the hexose monophosphate shunt, in that glucose is channeled into the same pentose pool. The glucuronic acid pathway is important in microsomal drug detoxification mechanisms. By glucuronic acid conjugation, foreign compounds are converted into products which are less toxic, more water-soluble at physiological pH and thereby rapidly excreted in the urine or bile. In mammals, both detoxification pathways and AA biosynthesis are induced by exposure to xenobiotics (Conney et al. 1961; Horio et al. 1983; 1993).
L-Ascorbic acid is an essential molecule in the overall health of animals, including bone formation (Peterkofsky 1991; Phillips et al. 1992; Gosiewska et al. 1994), growth (McLaren et al. 1947; Halver et al. 1969; Wilson and Poe 1973; Lim and Lovell 1978), and fertility (Blom and Dabrowski 1995; Ciereszko and Dabrowski 1995; Luck et al. 1995). The functions of AA have been reviewed frequently and wül not be discussed in detail here. In short, AA participates in a variety of biological processes such as collagen, steroid, and neurotransmitter biosyntheses, immune response, metal ion transport, and cholesterol metabolism. The most widely accepted function of AA is that of a water-soluble free radical chain-breaker. Throughout the hody AA acts as a reducing agent by donating one hydrogen to a broad range of oxidized biomolecules and trace elements including metal co-enzyme. In turn, AA becomes oxidized. Oxidized AA in the form of ascorbyl radical or dehydroascorbic acid can be regenerated in vivo chemically by reduced glutathione (GSH) or enzymatically by GSH- or NAD(P)H-dependent reductase (Rose 1989; Choi and Rose 1989; Meister 1994; Winkler et al. 1994). Ultimately dehydroascorbic acid is metabolized into compounds (Niemela 1987) that lack antioxidant property. Endogenous (biosynthesis) and/or exogenous (diet) supply of AA contribute to maintaining AA body pool. The synthesis of an essential antioxidant, such as vitamin C, might only evolve when adequate supplies are unavailable in the diet When vitamin C becomes available through numerous generations, then specific organisms may lose their ability to synthesize AA (Bimey et al. 1976) with no significant consequence on survivorship as long as vitamin C intake meets the nutritional requirement Alternatively, these organisms may specifically decrease the catabolic rate of AA and/or acquire more efficient means of its utilization (active transport). After prolonged vitamin C deprivation, however, scurvy might develop, ultimately causing death as early as three weeks in the guinea pig (Barnes et al. 1973) and in six weeks in the channel catfish, Ictulurus punctatus (Dabrowski et al. 1996). If nutritional criteria are invoked to understand the phylogenetic distribution of AA synthetic capacity in mammals, then natural diets do not provide the clue (Jukes and King 1975; Bimey et al. 1980). Indeed, consuming large quantities of plant food rich in AA has allowed the ancestor of the guinea pigs to survive the loss of AA synthesis, whereas other herbivorous species such as goats and rabbits (Sato et al. 1976) have retained a synthetic capability. In other words, high dietary content of AA does not indicate whether GLO activity has been lost, and inferences drawn from examining natural diets may be misleading. The current knowledge about AA biosynthesis in animals is fragmented. Most research work both in biochemistry and evolution has been conducted in vertebrates, and in mammals in particular. It is assumed that all amphibians and reptiles have the ability to synthesize AA (Nishhdmi and Udenftiend 1976; Chatteijee 1973; Dabrowski 1990), whereas the birds and mammals are diversified regarding the presence of GLO activity (Roy and Guha 1958; Chaudhuri and Chatterjee 1969; del Rio 1997). Vertebrates that cannot synthesize AA are devoid of GLO activity, and GLO is the only enzyme of the AA synthetic pathway missing in scurvy-prone vertebrates (Sato et al. 1976). Vertebrates that synthesize AA only express the enzyme in the liver and/or the kidney. Its tissue localization has shifted from the kidney in lower vertebrates to the liver in higher vertebrates. The enzyme is associated with the subcellular microsomal fraction, likely to be the endoplasmic reticulum. GLO is a 400-kD flavoprotein in the chicken (Kiuchi et al. 1982). The amphibian GLO has different antigenic determinants than the mammalian enzymes. Detergent-solubüized microsomal preparations from the kidneys of bullfrogs do precipitate with antisera directed against either rat or goat GLO, whereas liver extracts from ten mammals crossreact with these antibodies (Nishikimi and Udenfriend 1976). These antisera did not react with tissue extracts from scurvy-prone mammals, i.e. guinea pigs, monkeys or man. Guinea pigs and humans lack the mRNA for GLO, but the genome of these mammals contains a pseudogene with DNA sequence related to this enzyme
(Nishikimi et al. 1988; 1989). The origin of AA biosynthesis in animals is unclear. Twenty years ago the common view was that amphibians first acquired AA biosynthesis (origin of LLssamphibia which includes frogs and salamanders 360 million years ago, Kumar and Hedges 1998). But research in crustaceans suggest that the trait may have originated in invertebrates (Wallace et al. 1985). Two studies on lobster (Homanis americanus) conducted by the same group suggested that marine decapods can synthesize AA (Kean et al. 1985; Desjardins et al. 1985). But this view has been criticized because insects are unable to synthesize AA (Gupta et al. 1972; Kramer and Seib 1982), and in the case of crayfish {Astacus leptodactylus), a freshwater decapod, no GLO activity was detected in the hepatopancreas (Dabrowski 1990d). In penaeid shrimp, also, vitamin C deficiency referred to as "black death" disease has been demonstrated (Margarelli et al. 1979; Shigueno and Itoh 1988). Today there is stül insufficient data to establish firmly the origin of AA biosynthesis in invertebrates. This is because the distribution of AA-synthesizing capability in extant animals is only partially known. Too few taxa have acmally been studied for the presence of the trait. Prior to the onset of this dissertation, findings in our laboratory as well as elsewhere have challenged earlier conclusions (Chatterjee 1973; Nandi et al. 1997, see Appendix A) denying the presence of AA biosynthesis in fishes. Studies by Chatteijee’s group had been conducted solely in teleosts. However, four non-teleost species, i.e. the Australian lungfish (Neoceratodus forsteri), the Siberian sturgeon (Acipenser baerf), the white sturgeon (A. transmontanus), the lake sturgeon (A. fulvescens), and the paddlefish iPolyodon spathiild) were found to have the capacity to synthesize AA as they possess GLO activity in the kidney (Dykhuizen et al. 1980; Moreau 1992; Dabrowski 1994). Based on these data we hypothesized that while the teleosts have lost the ability to synthesize AA, some non-teleosts species have retained it. This view, however, contradicts reports claiming that some teleosts including the common carp {Cyprinus carpio) and the goldfish {Carassius auratus) can synthesize AA (Yamamoto et al. 1978; Sato et al. 1978; Soliman et al. 1985; Thomas et al. 1985). We believe that the latter reports are erroneous due to the use of inappropriate analytical methods. Beyond the controversy surrounding the incapability of teleosts to synthesize AA which is addressed in this dissertation, these recent findings pose questions regarding the peculiar nature of AA biosynthesis presence and disappearance in fish evolution as well as its origin in vertebrates. This dissertation is organized around two major objectives, (1) to investigate the distribution of AA biosynthesis in fishes, and (2) to study the nutritional control of AA biosynthesis in a fish. Each major objective consists of several questions. The first objective is addressed in Chapters 2. 3 and 4. We asked (i) what is the organ localization of AA synthesis in fish, (ii) how far back can we trace the origin of AA biosynthesis in fishes, and (iii) what is the character tree for AA biosynthesis in extant fishes.
The first objective required the preliminary study of the localization of L- gulonolactone oxidase, the enzyme responsible for the last step of AA synthesis pathway, in the organs of a fish. We used the white sturgeon, i.e. a non-teleost fish species able to synthesize AA, as a model. Based on previously published data in vertebrates, two organs, the hver and the kidney, were studied in the white sturgeon. For comparison, two teleost species, the rainbow trout (Oncorhynchus mykiss) and the channel catfish (Ictalunis punctatus), were included in the study. The results of this study are presented in Chapter 2. They indicated that in sturgeon, the pathway for AA synthesis is located in the kidney, particularly in the posterior kidney. In 1995, Touhata et al. reported the presence of GLO activity in a shark (Mustelus manazo) and a ray (JDasyatis akajei), and in the Japanese lamprey (Lampetra japonica). These data have added to the growing literature on the AA synthesis in non-teleosts. The discovery that a lamprey (a jawless fish with an ancient lineage) can synthesize AA was significant from an evolutionary viewpoint as it positioned the origin of AA synthesis pathway further in the past, possibly as early as the origin of lampreys in the Cambrian. However, the level of GLO activity reported by Touhata et al. was very low in comparison to our measurements in sturgeon kidney, thus questionable. Chapter 3 describes the study we have conducted with the sea lamprey (JPetromyzon marinus) collected from the Lake Erie tributaries in 1995 and 1996. In this study, we also examined for the first time the presence of GLO activity in the bowfin {Amia calva). The results confirmed ± at the lampreys are able to synthesize AA and added the bowfin to the list of fish species with GLO activity.
Prior to the initiation of this dissertation, there was no phylogenetic scheme of GLO distribution in fishes. This is partially due to the paucity of data, often rendered ambiguous by the use of unreliable techniques. Due to the rarity of some fish species, such as the coelacanth, and the technical and logistic difficulties associated with sampling fish specimens living in very different habitats both in fresh- and salt-water under high and low
latitudes, it was hardly possible to survey all orders of living fishes. Therefore, in Chapter 4, we have focused our efforts on the Actinopterygians, which account for 42 out of the 57 orders of modem fishes. The controversy surrounding the incapability of teleosts to synthesize AA was also addressed by including in the survey the common carp (C. carpio) and the goldfish (C. auratus), two teleosts for which the presence of GLO activity in the hepatopancreas and/or the kidney had been claimed. The survey also included the bowfin (A. calva), the longnose gar (Lepisosteus osseus) both Neopterygii, and Polypterus senegalus (Chondrostei). The presence of GLO was analyzed both by in vitro activity assay (see Appendix B) and immunoreactivity using anti-sturgeon GLO antibodies. This work led to the conclusion that all modem Actinopterygians can synthesize AA except the teleosts. The second objective of this dissertation presented in Chapters 5. 6 and % deals with the nutritional control of AA synthesis in fish, in particular, the effects of two antioxidant vitamins, ascorbic acid and vitamin E, on the level of GLO activity in the lake sturgeon (A. fulvescens). The estabhshment that some non-teleost species synthesize a significant amount of AA has obvious implications to aquaculture in defining the vitamin requirement of teleost species. Nearly all fish species raised for economical purposes, such as the salmonids, ictalurids, cichlids, and cyprinids, belong to the teleosts, thus require a dietary source of vitamin C. It is well known that their requirements may vary depending upon the biological stage, or the envirorunental and rearing conditions. One could argue that these modulators of vitamin C requirement in teleosts are also active in non-teleosts able to synthesize AA, such as the sturgeon. Under controlled conditions, a sturgeon’s response to an increased need for vitamin C could be an increased synthesis by the kidney, which should be translated into increased GLO activity. Among all possible modulators, nutritional ones have been investigated in this study. Prior to conducting nutritional trials, a semi-purified diet had to he developed for our fish model, i.e. the lake sturgeon, the sturgeon representative for aquaculture in the Great Lakes region. Preliminary attempts to feed lake sturgeon with casein-based diets were unsuccessful due presumably to low diet palatability. Strategies were implemented to overcome poor voluntary feed intake. Early training with mixed diets (artemia nauphi- compounded diets) offered to larvae did not improve later acceptance of a casein-based diet by juveniles. The decision was made to modify the casein diet in order to improve its palatabüity. Chapter 5 describes this experiment. Five combinations of various feeding stimulants (betaine, krill meal, fish protein concentrate) were incorporated to the basal casein diet and fed to lake sturgeon of two body sizes (2 and 200 g). Irrespective of the body size, the incorporation of 2% betaine and 6% fish protein concentrate improved growth rate and survival of juvenile lake sturgeon significantly. The growth rate was comparable with those obtained with Siberian sturgeon of similar sizes indicating that our modified casein-based diet was satisfactory to conduct further studies on the nutritional requirements of the lake sturgeon. Whether AA synthesis in fish is affected by dietary manipulation is unknown. A cost-efficient model dictates that mechanisms of regulation operate at the lowest cost possible. For an organism synthesizing AA in which vitamin C supplies to the body pool come firom the diet and the endogenous synthesis, it imphes that one or both flows of vitamin C are regulated so that excessive accumulation and depletion of AA in the tissues do not occur. Since the protection against oxygen-driven toxicity is a major function of vitamin C, nutritional factors that either increase or decrease the animal’s overall protection against oxygen toxicity are most Likely to impact the rate of AA biosynthesis in vivo. With
8 the knowledge that GLO activity in mice was lowered by increased levels of dietary AA, an emphasis was on determining whether similar control existed in the sturgeon. This study is reported in Chapter 6. Along with vitamin C, vitamin E, uric acid, cysteine, and reduced glutathione are potent chain-breaking antioxidants in vivo. The availability of any of these antioxidants alone or combined could influence the rate of AA biosynthesis in vivo. In Chapter 7. we evaluated the effects of dietary vitamin E on the tissue concentrations of AA, the protection against lipid peroxidation, the AA synthesis and enzymatic regeneration in sturgeon. Vitamin E was chosen as the best possible candidate since it interacts perhaps synergistically with vitamin C in scavenging free radicals in vivo. Chapter 8 concludes the dissertation by collecting the most significant data from the previous chapters. In the light of the new data, earlier findings and claims have been critically reviewed. An attempt was made to suggest further studies in the fields of evolution and nutrition. The presence of GLO in species having ancient lineages calls for building a new character tree among animals. For that purpose the presence of GLO activity in the kidney of the sea lamprey {Petromyzon marinus) (Moreau and Dabrowski 1998) and the Australian lungfish (Neoceratodus forsteri) (Dykhuizen et al. 1980) is phylogenetically significant as it anchors the character tree at the origin of vertebrates and suggests its radiation throughout the animal kingdom. Ultimately, the trait was lost on several occasions independently such as in the teleosts, birds and mammals (del Rio 1997; Bimey et al. 1980). The reasons for such a disappearance are discussed. CHAPTER!
THE PRIMARY LOCALIZATION OF ASCORBATE AND ITS SYNTHESIS IN THE KIDNEYS OF ACIPENSERID {CHONDROSTEI) AND TELEOST {TELEOSTEI) FISHES
10 J Ounp Ptuxu'l H il'Wfti IMi; I 's is.î Sprtnticr-Vcrhii: I'Wfi
OR KilN \l PAIM R
R. Moreau * K. Dabrowski The primary localization of ascorbate and its synthesis in the kidneys of acipenserid {Chondrostei) and teleost {Teieostei) fishes
Accepted: 29 January 1996
Abstract The kidneys of the rainbow trout Oncorliyn- chus mykiss. the channel catfish laalurus ptmctiitus Introduction (both Teieostei). and the white sturgeon, Acipenser transmantamis [Chondrostei] displayed similar profiles Ascorbic acid (AA) is found in particularly high con of ascorbate distribution irrespective of the capability centrations in many endocrine tissues; adrenal gland, of synthesizing ascorbic acid. The head kidney was pituitary, pancreas, gonads and thyroid in comparison found to be the richest in ascorbate. whereas the trunk with liver, muscle, intestine and skin. The high concen kidney showed significantly lower ascorbate levels in tration of this molecule in adrenal gland of mammals is all three species. The head kidney richness in ascorbate well established (Levine and Pollard 1983; Diliberto et was correlated with the localization of the cortical and al. 1983). In fishes there is no discrete adrenal gland. chromaffin tissues known to accumulate ascorbate in • The cortical tissue and chromaffin tissue are associated some fish and mammals. Based on ascorbate concen with a specific location in kidney. The kidney of teleosts tration. it was possible to distinguish the head from the is composed of several tissues with different functions. trunk kidney in salmonids and sturgeons which have In salmonids. the kidney is anteriorly dominated by an antero-posterior-fused kidney. The absence of L- hematopoietic.cortical and chromaffin tissues, whereas gulonolactone oxidase activity in the kidneys of the posteriorly it includes nephrons embedded in blood- channel catfish and the rainbow trout was asserted forming tissues (Hendricks 1983). It has been shown in biochemically. We also confirmed that the ascorbic some salmonids that the anterior kidney has a higher acid-synthesizing enzyme exists in white sturgeon kid ascorbate concentration than intermediate and poste ney. and found that the enzyme distribution was in rior kidneys due to the location of cortical tissue versely correlated with ascorbate concentrations. An (Fontaine and Haley 1954). This was confirmed in active transport of ascorbate might exist in the head other teleost species (Wilson and Poe 1973) and was the kidney of both acipenserids and the teleosts in order to reason why several authors chose the anterior kidney maintain this vitamin at high concentrations. This re as a physiological indicator of ascorbate status (Lim port suggests a link between ascorbate concentration and Lovell 1978: Lovell and Lim 1978: Agrawal and and its physiological functions in kidneys of lower Mahajan 1980: Lanno el al. 1985:- Lovell and El- vertebrates. Naggar 1989: White et al. 1993: Li and Robinson 1994). However, there has been no systematic study which has Key words Vitamin C • Gulonolactone oxidase • examined the distribution of ascorbate along the head Adrenal gland • Scurvy-prone fish • Sturgeon and trunk kidneys in fish and that elucidates the ab sence or presence of AA synthesizing capability. .Abbreviations AA ascorbic acid • T.-l.-l total ascorbic Given the variety of tissues and functions of kidney acid • DHA dehydroascorbic acid • DC IP dichloro- in fish, we attempted to map the distribution of ascor- indophenol • EOT A ethylcncdiamineieira-acetic batc in this organ and. based on its concentration, acid - GLO L-gulonolactonc oxidase generate new ideas for its functional importance parti cularly in the syntheses of neuropeptides, such as norepinephrine (Levine and Morita 1985: Levine et al. R. .Moreau • K. Dahrou 11 R. Moreau and K. Dabrouski: A&corbaïc in lîsh kidneys Dabrowski 1994). with teleost fish matiilesting signs a maximal absorbance of A;«i “ 14.98 per I mmol-1" *-cm "L of scurvy when dietary vitamin C source is lacking GLO activity was expressed as pg AA form ed-h"‘ -g* * tissue. With (Lim and Lovell 1978: Mahajan and Agrawal 1979: the intent of improving the assay for further research, we investi gated the effect of freezing and thawing on GLO activity. Using the Dabrowski et al. 1990). above method, we assayed fresh and frozen crude extracts prepared from the kidneys of 2-year.old male white sturgeon. In addition to the direct assay, frozen kidneys from the three Gsh species were analy^ for GLO activity using the colorimetric ascorbate deter Matotfals and methods mination (Dabrowski 1990). In short, 200 pi of enzyme preparation was incubated at 25’C in 650 pi of phosphate buffer (pH 7.4), 50 pi The ascorbate distribution in fish kidneys was investigated in two of 50mmol-l"' glutathione and lOOpl of KXImmol 1"' L- Teieostei, the rainbow trout {Oncorhynchus mykiss) and the channel gulonolactone.The sub-sample was taken at time 0 and the reaction catfish Uctalurus punctatus), and one member of Chondrostei, the was stopped after 2 h by transferring 4(X) pi of the mixture to 4(X) pi white sturgeon (Acipenser transmontanus). Rainbow trout, channel of 5% (w/v) trichloroacetic acid protein-precipitating solution con catfish and white sturgeon were obtained from The Ohio State taining 250mmol-l"' of HCIO* and 0.08% (w/v) EDTA. University's aquaculture facility, Piketon Research and Extension Results are shown as mean ± SEM except where indicated. Data Station, Piketon, Ohio. USA. Channel catfish were reared in pond, •were compared by one-way ANOVA followed by Fisher's multiple and rainbow trout and white sturgeon in fiberglass tanks. Males of range tesL 4-year.old channel catfish (23S0-26S0 g) and 2-year.old white stur geon {503-639 g) were fed commercial diets (Table I). Three-year-^jld male rainbow trout (976-1773 g) were sampled from two experi mental groups fed diets containing either a high supplementation of Results AA (270.9 mg AA-kg"*) or a diet devoid of AA for 12 months (Ciereszko and Dabrowski 1995). The highest AA concentrations tn the three fish species At the sampling time, fish were stunned by a blow on the head and studied were associated with the anterior part of the the spinal cord was sectioned. For the channel catfish and the rainbow trout. AA concentrations and AA-synthesizing enzyme kidney. The head kidney of the channel catfish, which is activity were analyzed in kidneys of different individuals. Each anatomically separated from the trunk kidney (Fig. 1), channel catfish trunk kidney was first divided into two sections had a significantly (P < 0.01} higher concentration of according to the symmetrical plan of the organ (Fig. 1). One semi total AA (reduced and oxidized forms combined; TAA) trunk kidney was taken per Ksh for AA measurements and divided (109.3 ± 9.4 ng-g“ ‘; mean ± SD; n - 3) than any into five portions. The whole head kidney was analyzed as one portion. "The kidneys of rainbow trout fed with experimental diets other portion of the trunk kidney (80.4 ± 1.9 pg-g"*; were divided into ten equal portions for the purpose of total AA n = 5). No significant (P = 0.46) difference was found measurement only. Rainbow trout used for enzyme analysis were between the portions of the trunk kidney (Fig. 2). In the taken from a batch of Gsh fed a commercial feed. The white sturgeon rainbow trout and the white sturgeon, for which head kidneys were separated into two along the symmetrical axis (Fig. 1). Each half was allotted to either ascorbate measurements or enzy and trunk kidneys are fused, TAA concentrations were matic assay. The halves were then divided into six equal portions. significantly (P < 0.05 and P < 0.01, respectively) high TAA and DHA concentrations were measured by the 2,4- er in the anterior portions (Figs- 3 and 4). In the rain dinitrophenylhydrazine method in kidney samples held at — 82 "C bow trout fed a diet lacking vitamin C, the highest TAA prior to analysis (Dabrowski and Hinterleitner 1989). Modification concentrations.were found within the first three por of the original method included incubation with DCIP reduced to 20 min. tions (28.8 ± 1.9 pg- g“ n = 3) compared to the other GLO activity was determined by direct spectrophotometric assay portions (15.6 ± 0.8 ng*g~‘: n = 7). In the white stur in crude extract (Dabrowski 1990). Fresh renal samples of the geon, TAA was higher in the first two portions three fish species were homogenized (1.2-1.6 g of tissue in 6 ml (121.4 ± 3.1 ng-g“ ‘; n=2) compared to the others of 50 mmol-1"' phosphate buffer, pH 7.4. containing 1 mmol-1"' (87.1 ± 1.6 ng-g“ ‘;n = 4). Based on ascorbate concen EDTA and 0.2% sodium deoxycholate) in a spin homogenizer (Omni SOOO) at 4°C. The homogenate was then centrifuged for trations, two subunits were differentiated in the kidneys 15 min at 15000 p at 4'C. The measurements were performed at of the rainbow trout and the white sturgeon. The divi 25 °C on the supematanL In these assay conditions, AA showed sion can be placed approximately after the first third of Tabic 1 Charactcrislics of the Gsh used in the present study Species Sampling date Dietary status Fish body Relative kidney (mean ± SD: n = 3) weight (g) weight (%)“ Rainbow trout April No ascorbic acid 1132 + 192 1.14 + 0.04 270.9 mg AA -kg"'* 1364 + 363 1.26 ±0.13 Channel caiGsh October Commercial feed 2467 + 161 0.73 +0.1 (002 + 0.04)' White sturgeon June Commerdal feed 561 + 7 0 0.55 ± 0.06 "Ascorbic add (AA) in the form of ascorbyl-2.monophosphate (Phospitan C. Showa Dcnko K. K.. New York) “Relative kidney weight (%) = (kidney weight x 100)/(Gsh body weight) ‘ Value in parentheses represents the relative head kidney weight (“/•) 12 R- Moreau and K. Dabrowski: Ascorbaicin fish kidneys headb'dney yW aJSüüüj bead kidney aiuerior kidney trunk kidney - ^ / posterior kidney / \ Fig. lA-C Gross anatomy of kidneys in the three fish species; the channel catfish (A) , the rainbow trout (B) and the white sturgeon (C) T3 150- 50- Ist 2nd 3rd 4th 5th £lh 7th 8th 9th 10th Portions of kidney Fig. 3 Total ascorbic acid concentrations in the kidney of the rain bow trout fed a diet containing 270.9 mg AA k g "' (who/e columns) or a diet devoid of AA (dolled columns). Kidneys were divided into ten equal portions indicated as 1st to 10th from the anterior extrem 1st 2nd 3rd 4th Sih ity of the organ. Vertical bars represent SD for three animals. Means Head not sharing a common letter are significantly different (P < 0.05) kidney Portions of trunk kidney. Fig. 2 Total ascorbic acid (wAofr rolumnsl and dehydroascorbic acid (black columns) concentrations in the head and trunk kidneys of male the kidney from the anterior extremity. In comparing channel catfish. Each trunk kidney was first divided into two accord the kidney anatomy of the channel catfish, the anterior ing to the symmetrical plan of the organ. One semi-trunk kidney per subunit of the rainbow trout and white sturgeon kidney fish was taken for analysis and divided into five portions indicated as may be considered as the head kidney, and the poste 1st to 5th from the anterior extremity of the trunk kidney. V'ernco/ bars represent SD for three animals. Means not sharing a common rior subunit as the trunk kidney (Fig. I). letter are significantly difiercnt al P < 0.01 and 0.05 for TAA and The DHA distribution showed a similar pattern as DHA. respectively TAA although larger variations were observed ( + SD) 13 R. Moreau and K. Dabrowski: Ascorbate in lïsh kidneys Freezing and storage of the crude enzyme prepara 150- tion at — 82 ’C for I day had no significant (P = 0.60) effect on G LO activity (246.3±56.8 pg AA-h~i-g~‘; -T 125 eo n = 3 versus 241.0 +49.1 pg AA-h~‘-g~i in fresh éo crude extract kept on ice for up to 10 min; n — 3). 3 100 Moreover, the enzymatic activity did not seem to be O affected by two thawings within 2 days (236.0 + 45.0 pg 75 AA-h“ ‘ -g" 1; rt = 2). Using both enzymatic assays (sec I Materials and methods), GLO activity was found to be 50 missing in the kidneys (head and trunk kidney) of the rainbow trout and the channel catfish. In contrast, the 25 enzyme activity was found in the kidneys of the white sturgeon. Using the colorimetric ascorbate determina- 0-1- . tion, the GLO activity level in the trunk kidney was found to be as high as 244 pg AA formed h"^ g"i tissue, similar to the results obtained using the direct Portions of kidney spectrophotometric assay. Furthermore, GLO activity was significantly (P < 0.01) higher in the posterior kid Fig. 4 Total ascorbic acid (whole columns) and dehydroascorbic acid (black columns) concentrations in the kidney of male white sturgeon. ney than in the head kidney, and inversely correlated Kidneys were divided into six equal portions indicated as 1st to 6th with ascorbate concentration (Fig. 5). frqm the anterior extremity of the organ. Vertical bars represent SD Tor three animals. Means not sharing a common letter ate signiR- cantly diflerent at P < 0.01 and 0.05 for TAA and D H A respectively Difcusaion The results presented here corroborate the observa (Figs. 2,4). The head kidneys of the channel catfish as tions on renal AA distribution in salmonids by Fon anatomirâlly defined; and of the white sturgeon as taine and Hatey (1954). O ur data confirm that the head physiologically identified, were significantly (P < 0.05) kidney had the highest AA concentration of the whole the renal portions richest in DHA. The DHA con organ in teleosts, and extended that finding to one centrations represented 95.4 ± 0.8% (n = 6) and representative of acipenserids. As ascertained in eel and 74.6 ± 2.8% (n = 6) of TAA in the channel catfish and cat sharks by the latter authors, who measured AA in the white sturgeon, kidneys, respectively. isolated cortical tissue, the high AA concentrations can For the rainbow trout group fed the diet containing be associated with the localization of cortical and 270.9 mg AA-kg" \ TAA reached a high concentration chromaffin tissues in the head kidney of the fish species (164.7 ± 3.3 p g -g " ‘; n — 10) in both head and trunk we studied. This structure appeared to be homologous kidney portions but no significant (P = 0.97) differ to the mammalian adrenal cortex and adrenal medulla ences were found between any portions (Fig. 3). (Grizzle and Rogers 1976; Barannikova et al. 1978; Fig. 5 L-Culonolactone oxidase 130 (GLO) activity (blank columns) □ GLO and total ascorbic acid (AA) concentration (dashed line) within the kidney of male white sturgeon. Kidneys were divided into six equal portions indicated as 1st to 6th from the anterior -110 extremity of the organ. Total ascorbic acid means are from Fig. 4. Vertical bar represents SD for three animals. Means not sharing a common letter are signiRcantly diflerent (P <0.01) Portions of kidney 14 R. Moreau and K. Dabrowski: Ascorbate in fish kidneys Jônsson « al. 1983). AA concentration in adrenal cor make the head kidney a relevant criterion of AA status tex is known to be one of the highest among mam in both fish groups. In addition, the determination of malian tissues beside corpus luteum (Luck and Zhao TAA concentrations was proposed to be a reliable way 1993), adrenal medulla, pituitary (Sheldrick and Flint to distinguish the head from the trunk kidney which 1989) and pancreas (Zhou and Thorn 1991). There are could be expanded to any fish species having antero- at least two explanations for AA abundance in mam posterior-fused kidneys. malian adrenal gland; either AA is synthesized in the Although, acipenserids possess an advantage over tissue, or it is accumulated against a concentration teleosts in their ability to produce A A, they would gradient (Levine and Morita 1985). First, the existence conserve the active transport of that molecule in of an AA transport system was demonstrated in freshly the head kidney. Likewise, adrenal gland of rats, a isolated bovine adrenal cortical cells (Finn and Johns species able to synthesize AA, contains an AA concen 1980) and in bovine adrenomedullary chromaffin cells tration of more than 300-fold higher than blood (Levine and Pollard 1983; Diliberto et al. 1983). Sec- ' (Levine and Morita'1985). Maintaining a high AA ond, the adrenal gland of mammals does not synthesize level in cortical and chromaffin tissues implies a physio AA (Grollman and Lehninger 1957; Chatteijee et al. logical importance of the compound, such as its 1958). Similarly, the channel catfish (Wilson 1973) and involvement in the syntheses of neuropeptides and the rainbow trout (Soliman et al. 1985) are unable to corticosteroids (Wedemeyer 1969). Thus, the results synthesize AA in kidney. Therefore, the active transport we obtained with the species of Chondrostei and of AA may provide a suitable explanation of the high Teieostei, suggested that endocrine functions are inti AA concentration in the head kidney of fish species mately correlated with AA concentration among lower prone to scurvy. vertebrates. Previous work indicated that some mammals can synthesize AA in kidney (Bimey et al. 1980). Similarly, Acknovrtedgancnts We thank Dr. M. Wat! for critical reading of the acipenserid fishes have GLO in kidney but not in liver manuscript and J. Ebeling for fish maintenance and sampling. (Moreau 1992; Dabrowski 1994). The present study Tbanlcs are also due to Dr. A. Ciereszko for providing raint>ow trout showed, for the first time, that the AA-synthesizing samples from his experimenU This study was supported by USDA OICD, US-French Cooperation Program (project no. 58-319R-0- enzyme distribution was not uniform along the kidney 026). and Piketon Research and Extension Station. Piketon, Ohio, and the major site of synthesis was found in the poste USA. Salaries were panly provided by state and federal funds rior half. Consequently, the separation of both sites of allocated to the Ohio Agriculture Research and Development AA synthesis and AA storage in kidney is in agreement Center, The Ohio State University. Manuscript No. 46/95. • with the hypothesis of an AA transport from the poste rior site of synthesis to the cortical and chromaffin tissues in the head kidney. Reference* AA concentration in the head kidney of the rainbow trout fed with experimental diets reflected the dietary Agrawal NK. Mahajan CL (1980) Comparative tissue ascorbic acid vitamin C level. Our data agreed with those for the studies in fishes. J Fish Biol 17:135-141 Barannikova I A, Vasilyeva VeV, Trenkler IV, Tscpelovan PG rainbow trout (Lanno et al. 1985), the Atlantic salmon (1978) The interrenal gland in the life of diadromous sturgeons (White et al. 1993) and the channel catfish (Lovell and (family Acipenseridae). J Ichthyol 18:633-647 El-Naggar 1989; Li and Robinson 1994). However, Bimey EC. Jenness R. Hume ID (1980) Evolution of an enzyme results obtained with the channel catfish fed a scorbu- system: ascorbic acid biosynthesis in monotremes and marsu togenic diet in which head kidney AA level remained pials. Evolution 34:230-239 Chatieijee IB. Ghosh JJ. Ghosh NC. Guha BÇ (1958) Effect of consistently high (Lim and Lovell 1978; Lovell and Lim cyanide on the biosynthesis of ascorbic acid by an enzyme prep 1978), contradict previous findings and our present aration from goat liver microsomes. Biochem J 70: 50^515 data. Nevertheless, the last-mentioned authors re Ciereszko A. Dabrowski K (1995) Sperm quality and ascorbic ported variations in their results that could not be acid concentration in rainbow trout semen arc affected by dietary vitamin C: an across-season study. Biol Reprod 52: explained. Therefore, we believe that below dietary 982-988 saturation threshold, AA concentration in the head Dabrowski K (1990) Gulonolactone oxidase is missing in teleost kidney is higher than that in the trunk kidney. In the fish - the direct spectrophotometric assay. Biol Chem Hoppe- case of the rainbow trout fed a diet highly supple Seyler 371: 207-214 mented with vitamin C, AA reached a similar level in Dabrowski K (1994) Primitive actinopterigian fishes can synthesize ascorbic acid. Expcrientia 50: 745-748 both head and trunk kidneys. As observed in mammals Dabrowski K. Hinterleitner S ( 1989) Simultaneous analysis of ascor (Finn and Johns 1980; Levine and Pollard 1983), the bic acid, dehydroascorbic acid and ascorbic sulphate in biolo AA transport appeared to be concentration dependent gical materials. Analyst 114: 83-87 in rainbow trout kidney, and saturable at the high Dabrowski K. El-Fiky N. Kock G. Frigg M. Wieser W (1990) dietary A A levels (Ciereszko and Dabrowski 1995). Requirement and utilization of ascorbic acid and ascorbic sulfate in juvenile rainbow trout. Aquaculture 9 1: 317-337 Similarities in AA distribution within kidneys of Diliberto El Jr. Heckman GD. Daniels AJ (1983) Characterization acipenserid and teleost fish fed diets containing AA of ascorbic acid transport by adrcnamcdullary chromaffin cells. levels either close to or lower than the requirement J Biol Chcm 258: 12886-12894 15 R. Moreau and K. Dabrou>ki: A.scorbaic in li»h kidneys Finn FM. Johns PA (I9S0I Ascorbic acid transport by isolated Lovell RT. Lim C 11978) Vitamin C in pond diets for channel catfish bovine adrenal cortical cells Endocrinology 106: SI 1-817 Trans Am Fish Soc 107: 321-325 Fontaine M. Hatey J 11954) Teneur en acide ascorbique de llnter- Luck MR. Zhao Y 11993) Identification and measurement of col rênal des poissons Isclactens et têlêostêcns). Bulletin de l'Institut lagen in the bovine corpus luteum and its relationship with Océanographique 51: 1-7 ascorbic acid and tissue development. J Reprod Fertil 99- Grizzle J.M. Rogers WA 11976) Endocrine system - Excretory 647-652 system. In: Rouse RO (ed.) Anatomy and histology of the channel Mahajan CL, Agrawal NK 11979) Vitamin C deficiency in Channa catfish. Agricultural Experiment Station. Auburn University. punctatus Blocft. J Fish Biol 15: 613-622 Alabama, pp 29-39 Moreau R ( 1992) Stockage tissulaire et mobilisation de Taeide ascor Grollman A P. Lehninger AL (1957) Enzymatic synthesis orL-ascor- bique - Evaluation des symptômes de carence en vitamine C chez bic acid in different animal species. Arch Biochem Biophys 69: Testurgcon Siberian (Acipenser baeri). Diplôme d'Etudes Ap 458-^67 profondies en Biologie et Agronomie, Université de Rennes 1, Hendricks JD (1983) Urinary system. In: Yasutake WT. Wales JH France (eds) Mieroscopic anatomy of salmonids: an atlas. United States Sheldrick EL, Flint APF (1989) Post-translational processing of Department of the Interior, Fish and Wildlife Service. Resource oxytocinneurophysin prohormone in the ovine corpus luteum: Publication 150. Washington. D.C, pp 97-103 activity of pcptidyl glycine x-amidating mono-oxygenase and Jenkins JS (1962) The effect of ascorbic acid on adrenal steroid ' .' concentrations of its factor, ascorbic acid. J Endocrinol 122: synthesis in vitro. Endocrinology 70: 267-271 313-322 Jônsson A-C. Wahlqvist L Hansson T (1983) Effects of hypophysec- Soliman AK.Jauncey K, Roberts RJ (1985) Qualitative and quantit lomy and cortisol on the catecholamine biosynthesis and cat ative identification of L-gulonolactone oxidase activity in some echolamine content in chromafHn tissue from rainbow trout. teleosts. Aquacult Fish Manag 16: 249-256 Salmo gairdneri. Gen Comp Endocrinol 51: 278-285 Wedemeyer G (1969) Stress-induced ascorbic acid depletion and Lanno RP. SlingerSJ. Hilton JW (1985) Effect of ascorbic acid on cortisol production in two salmonid fishes. Comp Biochem dietary copper toxicity in rainbow trout (Salmo gairdneri Richar Physiol 29: 1247-1251 dson). Aquaculture 49: 269-287 White A, Fletcher TC, Secombes CJ, Houlihan DF (1993) The effect Levine M. Morita fC (1985) Ascorbic acid in endocrine systems. In: of different dietary levels of vitamins C and E on their tissue levels Aurbach GD. McCormick DB (eds) Vitamins and Hormones 42; in the Atlantic salmon, Salmo solar L. In: Kaushik SJ. Luquet 1-64 P (eds) Fish nutrition in practice. INRA, Paris, Biarritz France, Levine M. Morita K. Pollard HE (1985) Enhancement of June 24-27 1991. pp 203-207 norepinephrine biosynthesis by ascorbic acid in cultured bovine Wilson RP (1973) Absence of ascorbic acid synthesis in channel chromafhn cells. J Biol Chem 260: 12942-12947 catfish. Ictalurus punctatus, and blue catfish, Ictalurusfructatus. Levine M. Pollard HB (1983) Hydrocortisone inhibition of ascorbic Comp Biochem Physiol 46B: 635-638 acid transport by chromaffin cells. FEBS Lett 158: 134-138 Wilson RP. Poe WE (1973) Impaired collagen formation in the Li MH, Robinson EH (1994) Effect of dietary vitamin C on tissue scorbutic channel catfish. J N utr 103: 1359-1364 vitamin C concentration in channel eatfish. Ictalurus punctatus. Zhou A, Thom NA (1991) High ascorbic acid content in the rat and clearance rate at two temperatures - a preliminary investiga endocrine pancreas. Diabetologia 34: 839-842 tion. J Appl Aquacult 4: 59-71 Lim C. Lovell RT (1978) Pathology of the vitamin C deficiency syndrome in channel catfish (Ictalurus punctatus). J Nutr 108: Communicated by H. Langer 1137-1146 Lovell RT, El-Naggar GO (1989) Vitamin C activity for L-ascorbic acid, L-ascorbyl-2-suIfate, and L-ascorbyl-2-phosphate Mg for channel catfish. In: Proc "Third Int Symp on Feeding and Nutri tion in Fish. Toba, Japan, Aug 28-Sept I. pp 159-165 16 CHAPTERS BODY POOL AND SYNTHESIS OF ASCORBIC ACID IN ADULT SEA LAMPREY {PETROMYZON MARINUS): AN AGNATHAN FISH WITH GULONOLACTONE OXIDASE ACTIVITY 17 P utc- A kuU. T if L S.-i Vol. 05. pp. uc7')-iu:.s:. a u c u m Phv\«i*U»^y Body pool and synthesis of ascorbic acid in adult sea lamprey (JPetromyzon marinus'): An agnathan fish with gulonolactone oxidase activity R ÉG IS M o r e a u a n d K o n r a d D a b r o w s k i * Sch(N3l o f N atural Rcsnurcea. O hio State U niveraity. Colum bus. O H 43210 Contmunicotcrf by John £, Hûlvcr. L'niventty o f Washington, Seattle. WA. -Vfuy 29. 799S Ireceived fo r eeviesv April 17. 19981 ABSTRACT Although many vertebrates can synthesize with dietary A A levels in rainbow trout [Oncorhynchus mykiss) ascorbic acitl (vitamin C), it is still unclear from the evolu females (15). The sea lamprey {Petromyzon marinus, Agnatha) tionary perspective when the ability to synthesize the vitamin represents an excellent model for reproductive physiologists first appeared in the animal kingdom and how frequently the because maturing sea lamprey allocate essential nutrients into trail has been lost. We report here ascorbic acid biosynthesis the gonads while fasting, reach sexual maturity, and die shortly ability in sea lamprey [Petromyzon marinus) which represent after spawning. the most ancient vertebrate lineage examined thus far for Adult landlocked sea lamprey enter rivers and streams presence of gulonolactone oxidase, the enzyme catalyzing the tributary to the Great Lakes and migrate to the spawning terminal step In biosynthesis of vitamin C. This finding grounds where reproduction occurs in the spring and summer. supports the view that the ancestors o f living vertebrates were When migration starts, sea lamprey stop feeding and begin to not scurvy prone and that the loss of gulonolactone oxidase m ature. The cessation of feeding is associated with progressive activity subsequently occurred several times in vertebrate atrophy of the intestine (16). Gonadal development during this phylogeny. Adult sea lamprey allocate significant amounts of period depends on fat and protein reserves mainly in muscles ascorbic acid to the gonads to guaranty high-quality gametes. and skin. Tissue stores of ascorbate were maintained by de novo syn This investigation was conducted (i) to examine whether sea thesis (I J —U mg of ascorbic acid/3Q0-g sea lamprey per day lamprey, a phylogenetically ancient vertebrate, can synthesize at 15*Q while sea lamprey fast during spawning migration. AA de novo and (») to quantify AA allocation to specific We estimate that the in vivo daily renewal rate of ascorbate is tissues during reproduction. Kinetics data of fish GLO were 4-5% of the whole-body aScorbate pool based on measurement used to project a daily rate of AA synthesis in upstream- of its biosynthesis and concentration in the whole animal. migrating sea lamprey. Since the discovery of its function in scurvy-prone humans and MATERIALS AND METHODS guinea pigs in 1932 (I), L-ascorbic acid (AA . vitamin Q has attracted research in the areas of nutrition, aging, cancer Adult sea lamprey used in this investigation originated from ethiology (2). U V-B skin damage (3), and hormonal regulation Lake Erie. Upstream-migrating sea lamprey were trapped in (4). Less attention has been given to the evolution of AA May-June 1995 arid 1996 at (Zattaraugus Creek. Springville, synthesis in vertebrates. It is unclear when the ability to NY (water temperature; I3-IS*C). Sea lamprey weighed 289 zz synthesize AA first appeared in the animal kingdom and how 40 g (n = 6) in 1995 and 340 z 41 g (n = 8) in 1996. Female frequently the trait has been lost (S—7). We previously de sea lamprey were 5% heavier than males in both years. scribed gulonolactone oxidase (GLO. EC I.IJ.8) activity in GLO activity was measured in the kidneys and livers of each Chondrostei fishes (sturgeon and paddlefish) (8-10), the male (three in 1995 and four in 1996) and each fem ale (three enzyme responsible for the oxidation of L-gulonolactone to in 1995 and four in 1996) sea lamprey using the direct AA (11). The general view is that all animals lacking GLO spectrophotometric assay (17). Kidneys and livers were re activity require an exogenous source of AA (12). whereas it is moved. frozen in liquid nitrogen, and stored at —81*C until psum ed that animals producing AA do not need an AA supply analyzed. Tissue samples were homogenized in 0.25 M sucrose in their diet to achieve optimal growth and accomplish suc (1 g of tissue in 5 ml of sucrose) at low speed for two periods cessful reproduction. of 10 sec using a spin homogenizer Omni 5(XX) (Omni Inter AA is an essential molecule in the overall health of humans national. Waterbury. CT). Homogenates were centrifuged at 15.0(K) X g for 15 min at 4'C. Pellets were discarded and and animals, including growth, bone development, immune supernatants were centrifuged at lOO.fXX) x g for 60 m in at 4*C. system, and fertility. The roles of AA in reproduction arc to The pellets (or microsomes) were resuspendcd with 1 ml of 20 participate in the biosynthesis of collagen, to take part in the mM Tris-acetate (pH 8.0) containing 10 mM KCl. 1 mM biosynthesis of steroid and peptide hormones, and to prevent EDTA. and 0.2% (wl'voI) sodium deoxycholate using a glass the oxidation of biomolecules. Specific transport systems for rod and a spin homogenizer (Omni 5(100). The suspensions dehydroascorbic acid (oxidized vitamin C) have been identi were then centrifuged at 14.(X)0 x g for 35 min at 4*C to fied in oocytes (13) in which AA accumulates against a solubilize the microsomes and release the enzyme into solu concentration gradient, indicating a specific requirement of tion. The top fraction was assayed at 25°C for GLO as the vitamin C in the gametes. In scurvy-prone teleost fishes, sperm microsomal fraction of the tissue preparation. Protein content concentration and motility were reduced by dietary AA defi of the microsomal fraction was determined with the Bradford ciency (14). Fecundity and embryo survival both increased procedure (IS) using commercial Coomassie protein assay reagent (Pierce). BSA was used as the standard. GLO activity The publication costs of this article were ttefraycd in part bv page charge payment. This article must thcrefiirc tie hereby market! —aji erttsement" in accnrJancc with IS U-S.C- 51734 solely to indicate this fact. Abbreviations: GLO. L-gulono-I.4-Iaciunc oxidase (EC 1.1.3 S): A.A. ascorbic acid. C luuR by The National Academy of Sciences UU2'-lt424.‘ns.'V51i»;ru_rS2lKl tl “ To whom reprint requests should be addressed e-mail: dabrowski. PN’a S is as-ailabic online al ww-w pnas ore Itiiosu cdu 18 Phvsiolücv: Moreau and Dabrowski Proc. Sari. .-\cad. Sci. 9S (1998) was expressed as micrograms of AA formed per milligrams of nized in 59c (wt.'vol) trichloroacetic acid solution containing 250 microsomal protein per hour at 25°C. The rate of AA biosj-n- mM HCIO, and 0.089c (wtvol) EDTA using a spin homogenizer chesis m vivo per sea lamprey per day was calculated at 15’C (Omni 5000) and centrifuged at 29,OCX) x g for 30 min at 4’C following the procedure used previously (19). The in viiro Supernatants were stored at -81°C until assayed. Michaelis constant {K„) of white sturgeon GLO for L- Total A.A concentration in the whole body minus liver, kidney, gulonolactone (0.92 mM; R.M.. K.D.. and P. H. Sato, submit and gonads was determined using HPLC with an electrochenucai ted for publication) was used for calculation. Since the con detection (HPLC-EC) procedure from three males and three centration of L-gulonolactone in our enzyme assay (10 mM) females sampled in 1996. The HPLC system comprised a 25-cm x was 10-fold higher than K„, Michaelis-Menten kinetics were 4.6-mm, 5-fun particle size, PLRP-S column (Polymer Labora applied with the assumption that the measured velocities were tories, Amherst, MA), an amperometric detector LC-4C (Bio- close to the estimates of The concentration of L- analytical Systctns, West Lafayette, IN), and a cross-flow cell gulonolactone (S) in renal cells of sea lamprey was assumed to equipped with a glass carbon electrode and an AgfAgO reference be no greater than 0.05 mM (19). A theoretical observed rate electrode. The applied potential was -t-0.575 V (oxidative mode). (ko) at 2S°C was calculated for male and female sea lamprey The mobile phase consisted of 20 mM NaH:PO*-HiO. 0.06% (wtNol) metaphosphoric acid, and 0.4% (vol/vol) acctonitrUe (pH by substituting into the equation ki = k'niaj-S/ffm + S. The 3.0) delivered at a flow rate of 0.4 ml/mitL Samples were values were then multiplied by 6 (since 1 g of sea lamprey homogenized (1 g of tissue in 10 ml) in cold 0.1% (wt/vol) kidney contains approximately 6 mg of microsomal protein). ' metaphosphoric add containing 0.1 mM EDTA and 1 mM followed by times 24 (to convert to I day), then times 2 (weight thiourea (pH 22), and centrifuged at 29,000 x g for 30 min at 4'C. in grams of the kidneys in a 300-g male or female sea lamprey), The supernatants were kept at -81'Cunta assayed. One milliliter and divided by 1.73 (Qio betw een 15 and 25*C as determined of supernatant was incubated with 1 ml of 1% (wt/vol) dl - for sea lamprey GLO) to express the rate of synthesis at 15'C homocysteine and 8 tnl of 50 raM sodium phosphate buffer (pH (water temperature at Cattaraugus Creek). 72) containing 0.1 mM EDTA and 1 mM thiourea for 30 min at Total and oxidized AA concentrations in liver, kidney, and rtxjm temperature. The reaction was stopped by transferring 0 J testis of each male (three in 1995 and four in 1996) and in liver, ml o f incubate into 0.5 ml of cold 3.5% (wt/vol) metaphosphoric kidney, and eggs of each female (three in 1995 and four in 1996) add. The samples were filtered through a 0.45-fun nylon mem were determined using the 2,4-dinitrophenylhydrazinc colorimet brane prior to injection. ric method (20). The sum of reduced and oxidized forms of AA Statistical analysis was performed using the unpaired Stu represents the total AA concentratioiL Tissues were homoge dent's r test. The accepted level of significance was 0.05. SupertJassg Order —^orders ofTcleojtci (W-) « B ag Ci M^Semionotifonn&s (•} ^Adtnopteiygu Aapensciiformcs (+) * ^rCboodroctei PolypceriTonnes (-) ^Lepidosireniform es ('•') ^ Djpnot -----=— Gomihoaommi# — Ceratodomiformes (+) ^ C—Sarcopicfygu - -*"Coelacanlhimorpha «^Coelacanihironnes Baamobnmchu ■ -orders of Eutelachii (•►) ^ i^-Chondrichthyes ~GHolocephalx — -Chimaeriformes Cephalaspidomorphi. “ PcifomyzDniiforrncs (■►) • Agnatha . Myxini "Myxiniformcs F tc.6 Hierarchy of living fishes. A ■*' denotes that at least one species in a given order v,*as reported to biosynihc^izc AA dc novo due to the presence of GLO. whereas — denotes the incapahiliiy as the result of the loss of the enzyme activity, t. R.M. and K.D.. unpublished data. (Adapted from ref. ,'K). 19 Phvsiology: Morciiu anJ Dubru^ski Pruc. Stitl. Acad. Sci. USA 95 (1993) RESULTS AND DISCUSSION synihcsis,-whole-body pool, percent), approximately 4 - 5 % of Lamprey (Agnatha. Petromyzontiformcs) belong to the most the whole-body AA pool is renewed daily via biosynthesis in phylogenetically ancient group of fishes (origin of Petromy- 300-g sea lamprey at 15"C irrespective of the sex. For com zontiformes 420 million yr before the present: ref. 21) ever parison. the whole-body pool o f A A in 270- to 355-g adult male studieti regarding AA biosynthesis (Fig. 6)- GLO activity was rats amounted to 24-43 mg of A A and approximately 5-g mg found in the kidney of sea lamprey and was associated with the of A A was synthesized per day (37). Thus, the turnover rate of microsomal fraction. The levels o f enzyme activity (126-159 the body pool per day in the rat ranged from 19 to 21%. fig A A/mg microsomal protein per h at 25*C) were not AA allocation was influenced by sea lamprey sex. In males, significantly different (P > 0.05) irrespective o f the sex o r the tissue total AA concentrations did not differ significantly year of sampling (Table 2). No enzyme activity was detected between 1995 and 1996 (Fig. 7)- The oxidized AA concentra in the liver. GLO activity is reported in the kidney of land tions in the liver and testis were significantly (P < 0.05) lower locked sea lamprey (P. marinus), corroborating previous data in males sampled in 1996 as compared with those sampled in of trace activity in Lampetra japonica (29). The present study 1995. Large variations am ong individuals were observed in the supports the notion that kidneys arc the site of AA biosynthesis testes. Most of the A A found in the liver, kidney, and testis was in poïkilothermie vertebrates such as amphibians and reptiles in the oxidized state. Female sea lamprey sampled in 1995 (31, 32), Dipnoi (29, 30), and Chondrostei fishes (8-10) and differed significantly (P < 0.05) from those sampled in 1996 extends this view to a representative of Jawless vertebrates (Agnatha). Since there is no instance of re-acquisition of GLO 300 activity once lost, present evidence suggests that the common ancestor of Agnatha and Gnaihostomata, that existed in the Males Cambrian period (590-500 million yr before present), pos sessed GLO from which living aquatic and terrestrial verte brates evolved.' Independent episodes of evolution led to the loss of GLO activity (due to missense mutations in the ctxiing region of the gene; refs. 33 and 34) several times in vertebrate phylogeny, such as in teleost fishes (17), passeriform birds (6), bats (7), guinea pigs (5, 33), and primates (5, 34). Parasitic sea lamprey rely on endogenous^ produced AA to maintain the AA pool in their reproductive organs. Because blood plasma of fish fed upon by lamprey has a low concen tration of AA (1-5 PS /ml) p5), dietary AA is likely to be a marginal source compared with A A produced enzymatically in the kidneys. When examined in relation to terrestrial mam mals, the calculated rate of AA synthesis in the 300-g sea lamprey (1.2-U mg AA/300-g sea lamprey per day at 15’Q was found to be intermediate to that of the 350-g rat (5.4 mg AA/liver per day at 3TC) and that of the 25-g mouse (03 mg AA/liver per day at 37*C) (19). Also, upstream-migrating sea lamprey do not feed and depend exclusively on endogenously produced vitamin C to compensate for metabolized AA. Thus, the synthesis rate can be viewed as a reliable estimate of the recommended daily allowance for AA maintenance in migrat ing lamprey. In vivo studies using 220- to 347-g male rats and i.p. "C-labeled AA administration as the sole exogenous supply of vitamin C showed that the amount of A A synthesized daily (2.6 mg AA/IQO g body weight) was about the sam e as the amount required each day in the diet of guinea pigs of similar body weight to maintain tissue saturation (36). Assuming that migrating sea lamprey are in a steady state in respect to AA synthesis and degradation and consequently the body pool of AA remains constant during the upstream migration, the synthesis rate corresponds to the turnover rate, i.e., the amount of body A A renewed per day. Based on our calculations (daily KidneyLiver Table 2 AA biosynthesis in the kidney of 300-g adult or testis sea lampreys Fto. Total AA (whole columns) and oxidized AA (black Y ear M ale Fem ale 7 columns) concentrations in liver, kidney, and gonads of male (Top) Gulonolactone oxidase activity and female (Bottom ) sea lampreys trapped in 1995 and 1996 during the (pg AA' mg microsomal upstream migration to the spawning grounds. Mean = SD of three to protein h 1995 126 = 17. 159 r 62 four males or females. An • denotes that total AA or oxidized AA at2 5 * Q 1996 142 = 17 150 a 36 coneentration in a given tissue was significantly different in 1996 Rate of AA biosvnthesis compared with 1995 (P < 0.05). The hepatosomatie indices (liver weighL'body weight, percent) were higher in males [2.6 = 0.496 (n =■ (mg AA/sea lampreyday at 15*0 1.2 1.3 — 7)] than in females [1.4 ~ 0.3% (n = 7)] in 2 subsequent yr. The Mean r SD of three to four males or females. Gulonolactone gonadosomatic index (gonad weight/body weight, percent) was 1.9 = oxidase activity was not signilicantly different (P > 0.05) txtwcen 05% (n “ 5) in males sampled in 1995 and 1996. Female gonadoso sexes in 1995 or in 1996. Likewise, the enzym e activity was not matic index was 25.0 a 4.8% (n * 7). The relative weight ofkidney was significantly different among males or among females in 1995 eom- 0.68 - 0.04% (n "V 14) of total body weight irrespective of the year of pared to 1996. sampling or the sex. 20 Physiology; Moreau and Dahro«.ski PrtK.-. \'atl. .-hue/. Sci. L5.4 VJ (IWS) with respect tolivcr AA concentrations. Significant tiifferences and stresses the importance of AA in gonadogcnesis of all were also found in kidney total A.A and egg oxidized AA vertebrates irrespective of the ability to biosynthesize vitamin C. concentrations in female sea lamprey. In the livers of females, total A.A concentrations were three times higher than in males. Wc thank Dr. A Ciereszko for assistance with sea lamprey sampling Liver, kidney, a.id gonad represented a small portion (a.7%) and Drs. F. W. H. Beamish and J. H. Youson for critical reading of the of the total AA in males, whereas the same three tissues draft. This study was partly funded by the Great Lakes Fishery accounted for 43 of the total A A in females (Fig. 8). The Commission (Ann Arbor. MI) and the Ohio Sea Grant College Program. Salaries were partly provided by state and federal funds disparity was related to a much higher gonadosomatic index in appropriated to the Ohio Agriculture Research and Development females and a concomitantly lower concentration of total AA Center (Wooster. OH). in testes (79J-104.1 p.g/g) than in eggs (130-133.2 Whole-body total AA was not significantly different between 1. Svirbely. J. L. & Szent-Gvdrgyi. A (1932) .Varune (London) 129, females and males: 28.8 i 1.2 vs. 26.7 r 1.6 mg AA/300-g sea 576-577. lamprey, respectively. The greater content of A A in eggs than 2. Weber. P.. Bendieh. A Sc. Sehalaeh. W. (1996) Int. J. Vitam. Nutr. in testes was balanced by the lower AA concentration in the Res. 66. 19-30. . 3. Kobayashi. S.. Takehana. M.. Itoh. S. Se Ogata. E. (1996) remainder of the females (once liver, kidney, and gonads were Photoehem. Photobiol. 64, 224-228. removed): 16.4 — 1.8 mg A A/individual compared with male 4. Wells, W. W„ Dou, C.-Z., Dybas, L. N., Jung, C -H - tCalbach. bodies; 25.2 z: 1.7 mg AA/individual. Total A A concentrations H. L & Xu. D. P. (1995) Proe. Natl. Acad. Set. USA 92, in muscle tissue and notochord averaged 40 jig/g in both male 11869-11873. and female sea lamprey. Total AA concentrations in sea 5. Bums, J. J. (1957) Nature (London) 180, 553. lamprey skin were the highest among the tissues analyzed and 6. Chaudhuri. C .R .& C hatterjee, 1. B. ( 1969) Science 164,435-436. differed markedly between sexes: 360 fig/g in females versus 7. Bimey, E. C - Jenness, R. Sc Ayaz, K .M . ( 1976) Nature (London) 260, 626-628. 460 ftg/g in m ala. Further study is required to explain the role 8. Moreau, R. Sc Dabrowski, K. (1996) I. Comp. Physiol. B 166, of the skin as à storage site of vitamin C, possibly in osmo 178-183. regulation and body defenses. 9. Dabrowski. D. (1994) Expcrientia SO, 745-748. Upstream-migrating sea lamprey studied in 1995 and 1996 10. Moreau, R., Kaushik. S. J. Sc Dabrowski. K. (1996) Fish Physiol. displayed similar AA concentrations in their tissues, indicating Biochem. 15, 431-438. that males and females had consistent AA status in both years 11. Hassan, M. Sc Lehninger, A L (1956) J. BioL Chem. 223, 123-128. while approaching the spawning grounds. Male and female sea 12. Sato, P., Nishikimi, M. Sc UdenfrientL S. (1976) Biochem. Bio lamprey also showed consistency in whole-body A.A content phys. Res. Commun. 71, 293-299. and rate of AA biosynthesis. However, AA was differently 13. Vera. J. C , Rivas, C. L Fisehbarg, J. & Golde, D. W. (1993) distributed within the botfy depending on the sex. AA depo Ntttttre (London) 364, 7 9 -5 2 sition in eggs exceeded that of any other organs, representing 14. Ciereszko, A Sc Dabrowski, K. (1995) Biol. Reprod. 5 2 982-988. 38.6% of the total pool df AA in female sea lampreys. In 15. Blom, J. H. & Dabrowski. K. (1995) BioL Reprod. 52, 1073-1080. contrast, testis AA amounted to 2.2% of total body pool in 16. Vladykov, V .D .& Muketji,G.N.(1961)J. AtJt.Res. BoardCan. 18, 1125-1143. males. Male and female sea lam prey showed comparable levels 17. Dabrowski, K. (1990) Biol. Chem. Hoppe-SeylerSTl, 207-214. of GLO activity, indicating that discrepancies regarding the 18. Bradford. M. (1976) Anal. Biochem. 72 248-254. tissue A A allocation between sexes were the result of distinct 19. Rueker, R- B- Dubick. M. A & Mouritsen, J. (1980) A m . J. Clin. patterns of distribution within tissues rather than different Num. 33, 961-964. rates of AA biosynthesis. It also suggests that AA deposited 20. Dabrowski, K. Sc Hinterleitner, S. (1989) Analyst 114, 83-87. into the gonads was transferred from reserve tissues such as the 21. Goodman,'M- Miyamoto, M. M. Sc Czelusniak, J. (1987) in muscle and the skin, transported via the blood, and accumu Molecules and Morphology in Evolutiort: Conflict or Compromise?. eds. Patterson, C. (Cambridge Univ. Press, Cambridge, U JC), pp. lated against a concentration gradient into the gametes re 141-176. quiring specific transporters (13). 2 2 Chatterjee, I. B. (1973) S e t Cub. 39, 210-212 The process of A A accumulation into sea lamprey eggs is in 23. Wilson, R. P. (1973) Comp. Biochem. Physiol. 46B, 635-638. agreement with previous studies conducted in scurvy-prone 24. Yamamoto, Y- Sato, M. Sc Ikeda, S. (1978) Bull. Jpn. Soc. ScL teleost fishes (14, 15). This results in high concentrations of AA Ftsh. 44, 775-779. 25. Sato, M., Yoshinaka, R„ Yamamoto, Y. & Ikeda, S. (1978) Bull. MALEFEMALE Jpn. Soc. Sci. Fish. 44, 1151-1156. 26. Soliman, A K- Jauneey, K. Sc Roberts, R. J. (1985) Atjuacult. Fish. Manag. 16, 249-256. 27. Thomas. P - Bally. M. B. Sc Neff. J. M. (1935) J. Fish Biol. 27, 47-57. 28. Dabrowski. K. (1991) Fish Physiol. Biochem. 9, 215—221. Muscle, skia, Muscle, skto. 29. Touhata. K- Toyohara. H.. Mitani. T., Kinoshita. M - Satou, M. viscera, skeleton viscera, skeleton Sc Sakaguehi. M. (1995) Ftsh. Sci. 61, 729-730. 2.7% 30. Dykhuizen. D. E.. Harrison. K. M. & Richardson. B. J. (1980) 94,3% D.8% Expcrientia 36. 945-946. 0.8% 31. Roy. R. N. &. Guha. B. C. (1958) Nature (London) 182,319-320. 3 2 Chatterjee. 1. B. (1973) Science 182, 1271-1272 33. Nishikimi. M- Kawai. T. Sc Yagi. K. (1992) /. Biol. Chem. 267, 21967-21972. □ Liver 34. Nishikimi. M - Fukuvama. R.. Minoshima. S.. Shimizu. N. & Y agi. K. (1994)2 Biol. Chem. 269, 13685-13688. 35. Dabrowski. K.. Lackner. R. i Doblander. C. ( 1990) Can. J. Fish O Testis Aqttat. Sci. 47, 1518-1525. 36. Bums, J. J_ Mosbach. E. H. Sc Schulenbcrg. S. (1954) J. Biol. F ig . 8 Total AA distribution in adult sea lampreys trapped during Chem. 207, 679-687. the upstream migration to the spawning grountls. Data collected in 37. Curtin. C . O . Sc King. C. G. ( 1955) /. Biol. Chem. 216. 539-548. 1995 and 1996 were com bined for calculations. Proportions arc 38. Nelson. ]S. (1994) Fishes o f the World (Wiley, New York), 3rd expressed as percentage of total body pool. Ed. 21 CHAPTER 4 DISTRIBUTION OF L-GULONOLACTONE OXIDASE AMONG ACTINOFTERYGn INTRODUCTION It has now been demonstrated beyond doubt that some modem fish can synthesize ascorbic acid (AA, vitamin C) de novo due to the presence of the enzyme L-gulonolactone oxidase (GLO) in the kidney. The origin of AA biosynthesis capability in vertebrates is currently postulated to have occurred some time in the Cambrian period (500-590 million years ago) in the common ancestor of modem lampreys and jawed fishes (Moreau and Dabrowski 1998a; b). However, the character distribution in extant fishes is not fuUy elucidated. The study of AA biosynthesis in fishes has gained increasing attention over the past few years. Besides data generated from our laboratory (Dabrowski 1994; Moreau et al. 1996; Moreau and Dabrowski 1996; 1998a), two contributions (Touhata et al. 1995; Mæland and Waagb0 1998) have added to the rediscovery and réévaluation of AA biosynthesis in fish. Present data indicate that while teleosts are unable to synthesize AA, a variety of non-teleost fish such as lamprey, shark, ray, lungfish (Dykhuizen et al. 1980), paddlefish, and sturgeon can produce AA in the kidney. Since overstated and wrong generalization has been made in the past on the basis of observations reported by a single 22 laboratory, the confirmation of already existing but sparse data is most needed. Bowfin, gar and Polypterus have already been studied for the presence of GLO activity. Touhata et al. (1995) found no activity in the gar (Lepisosteus occulatus) and in Polypterus senegalus. Although we reported the qualitative presence of GLO in bowfin (Amia calva) from a few individuals, no quantitative measurement had been made (Moreau and Dabrowski 1998a). On the basis of these studies and considering fish phylogeny, one would have to suppose that GLO activity was lost on three separate occasions in Actinopterygii, i.e. in the Polypterus, the gars and the teleosts. We reevaluate the situation here. The objectives of the present research were 1) to quantify GLO activity in the kidney, liver and spleen of three representatives of Actinopterygii (bowfin, gar, and Polypterus), 2) to evaluate the immunologic reactivity of Actinopterygii tissues with antibodies directed against white sturgeon GLO, and 3) to measure the concentrations of AA in the tissues of wild bowfin (A. calva) and longnose gar (L. osseus). MATERIALS AND METHODS Fish The capability to synthesize AA was examined in the following Actinopterygians; common carp (Cyprinus carpio, Teieostei, Neopterygii), goldfish (Carassius auratus, Teieostei, Neopterygii), bowfin (Amia calva, Amüformes, Neopterygii), longnose gar (Lepisosteus osseus, Semionotiformes, Neopterygii), and Polypterus senegalus (Polypteriformes, Chondrostei). Common carp were obtained from the Piketon Research and Extension Center (Ohio). Goldfish and Polypterus were obtained from a local fish dealer. Bowfin originated from the Lake Erie (Sandusky, OH) and longnose gar from the Ohio River. White sturgeon 23 {Acipenser transmontanus) and lake sturgeon {A. fulvescens) were used as positive control for GLO activity, and sea lamprey {Petromyzon marinus) as outgroup for rooting the character tree. Gulonolactone oxidase activity Gulonolactone oxidase (GLO, EC 1.1.3.8) activity was quantified by direct spectrophotometric assay (Dabrowski 1990) on crude extracts and microsomes prepared from frozen (-80°C) kidney, Hver, hepatopancreas, and spleen. For crude extracts, tissue samples were homogenized (1 g tissue in 4-5 ml buffer) in cold 50 mM Na phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.2% Na deoxycholate in a spin homogenizer (Omni 5000) at 4°C, and centrifuged at 15,000 g for 15 min at 4°C. To prepare microsomes, a tissue sample was homogenized in cold 0.25 M sucrose (1 g tissue in 4 ml) and centrifuged at 10,000 g for 15 min at 4°C. The supernatant was then spun at 100,000 g for 60 min at 4°C to produce the microsomes. The pellet was resuspended with 1 ml of 20 mM Tris-acetate (pH 8.0) containing 10 mM KCl, 1 mM EDTA, and 0.2% Na deoxycholate using a glass rod and a spin homogenizer (Omni 5000), kept on ice until assayed for GLO activity at 25°C. The assay mix contained 10 mM L-gulonolactone, 2.5 mM reduced glutathione in 50 mM Na phosphate buffer (pH 7.4). A blank containing D- gulonolactone in place of L-gulonolactone was assayed simultaneously. While common carp, goldfish, bowfin and gar were analyzed individually, tissues from 11 specimens of Polypterus were pooled for GLO activity assay. Antibodies Antibody directed against purified white sturgeon GLO (Moreau et aL 1999a) was raised in the New Zealand white rabbit using standard procedure in compHance with the 24 guidelines of the Institutional Laboratory Animal Care and Use Committee of The Ohio State University. Three injections were given intradermaUy at 3-week intervals. The animals were bled 10 days after the last injection and the resulting sera were tested for antibody to white sturgeon tissue preparations of kidney and liver by immunodouble diffusion (Ouchterlony 1968). Antisera IgG were purified by affinity chromatography using protein A-Agarose, and used for crossreactivity test with vertebrate GLO by immunodouble diffusion. Immunodouble diffusion Immunodiffusion plates were made of 1% agarose containing 0.05% Na azide (Pierce, Rockford, EL), stored at 4°C soaked in 20 mM Tris-acetate (pH 8.0) containing 10 mM KCl, 1 mM EDTA and 0.2% Na-deoxycholate. Twenty pi (70-120 fig protein) of tissue preparations, antisera or purified IgG were loaded onto the plates and diffused overnight at room temperature. Immunoprecipitins were revealed by GLO activity staining. GLO activity stain cocktail consisted of 50 mM Na phosphate buffer (pH 7.5) containing 1 mM EDTA, 0.1% phenazine raethosulfate, 0.1% nitroblue tetrazoUum, and 20 mM L- gulonolactone. A single immunoprecipitin staining positive for GLO activity was found when either crude extract or microsomes prepared from white sturgeon kidney were tested against either antisera or purified IgG. When D-gulonolactone in place of L-isomer was employed in GLO activity stain cocktail no precipitation line was observed. Crude tissue extracts or microsomes (prepared as above) from the kidneys, the livers and the spleens of common carp, goldfish, bowfin, longnose gar, lake sturgeon, Polypterus and sea lamprey, plus tadpole kidney, and pig liver were tested for material antigenically related to white sturgeon GLO. First, crude extracts were tested for material that crossreact with antibodies directed against white sturgeon GLO. When crossreactivity was demonstrated. 25 immunoreactants prepared firom microsomes were further tested for antigen relatedness by multiple paired comparisons. Microsomes prepared from white sturgeon kidney were used as a positive control. In the comparative test, the patterns formed by the extensions of the precipitation lines and their eventual interactions were considered. Ouchterlony (1968) described three major type reactions where each reaction is associated to a precipitation pattern; (i) a reaction of antigen identity indicated by a complete fusion of precipitation Lines forming a continuous arc, (ii) a reaction of non-identity resulting from an absence of deviation of lines where they intersect implying that the two lines have unrelated antigen- antibody compounds, and (üi) a reaction of partial identity illustrated by interference of the precipitation lines with partial deviation or not, and a spur projecting from where the two lines meet Ascorbic acid analysis Tissues from wild bowfin and longnose gar were examined for ascorbic acid concentration. Blood plasma, liver and spleen total (TAA) and oxidized (DHAA) ascorbic acid were analyzed using the 2,4-dinitrophenylhydrazine colorimetric method (Roe and Kuether 1943) modified as in Dabrowski and Hinterleitner (1989). RESULTS Gulonolactone oxidase activity No GLO activity was detected in the tissues (kidney, hepatopancreas and spleen) of common carp and goldfish from either crude extracts, 100,000g pellet (microsomes) or 100,000g supemates. However, bowfin, longnose gar and Polypterus demonstrated GLO activity in the kidney but not in the liver or spleen. For the three species, the enzyme activity was associated with renal microsomes. On average, the activities amounted to 0.7 ± 0.2, 1.5 ± 0.3, and 0.4 pmol AA/mg protein ' h in the bowfin, the longnose gar and 26 Polypterus, respectively (Table 3). Fish body weight or sex did not correlate with GLO activity. The enzyme activities found in bowfin and Polypterus were comparable to those previously reported in sturgeon and sea lamprey. However, the longnose gar specimens presented an activity twice as high (Fig. 9). Immunologic crossreactivity and antigen relatedness Kidney, liver, and spleen preparations from common carp, goldfish, Polypterus, sea lamprey, tadpole, and pig, and liver and spleen preparations from white sturgeori, lake sturgeon, bowfin, longnose gar, and Polypterus did not show material antigenically related to white sturgeon GLO. However, kidney preparations from lake sturgeon, bowfin, and longnose gar contained material that crossreacted with antibodies directed against white sturgeon GLO (Table 4). Antigenic relatedness was further studied by paired comparisons. Lake and white sturgeon antigens were found to be identical, as did the antigens from bowfin and longnose gar. The latter species showed partial identity with either lake or white sturgeon antigen (Fig. 10). Ascorbic acid content in wild bowfin and longnose gar tissues Ascorbate concentrations (TAA and DHAA) in the blood plasma, the liver and the spleen are shown in Table 5. In the tissues of bowfin, ascorbate concentrations were not linearly correlated with either sex, body weight, GSI or HSI. Oxidized ascorbate was not correlated with total ascorbate. Blood plasma TAA concentration was less in bowfin (0.7 to 2.7 [ig/ml) than in longnose gar (8.8 to 9.1 [ig/ml). In contrast, hver TAA concentrations were higher in bowfin (142.1 to 198.7 (J-g/g) than in longnose gar (84.8 to 109.4 jig/g). In the hvers of longnose gar, TAA was almost entirely in the oxidized form. Bowfin and longnose gar had comparable TAA concentrations in the spleen (170 ± 50 |ig/g) where most of ascorbate was in the oxidized state. 27 DISCUSSION The present data contradict the report of Touhata et al. (1995) that GLO is absent from gar and Polypterus kidney. All non-teleost Actinopterygians under study herein possess GLO activity, thus are capable of synthesizing AA. Our data refute earlier reports that the common carp (C. carpio) and the goldfish (C. auratus) possess GLO (Yamamoto et al. 1978; Sato et al. 1978; Soliman et al. 1985; Thomas et al. 1985). As a consequence the present study supports the claim that present-day teleosts lack GLO (Fig. 11). In order to complete the survey of the presence of GLO in fishes, representatives of Mixiniformes, Chimaeriforraes and the coelacanth {Latimeria chalumnae), the sole living species of Coelacanthiformes, are yet to be analyzed. Among the non-teleost species shown to have GLO activity, sea lamprey and Polypterus GLOs were not antigenetically related to sturgeon GLO. Hundreds of million years of evolution in the GLO protein since the divergence between sea lamprey and sturgeon (-420 million years ago, Goodman et al. 1987), likewise since the divergence between Polypterus and sturgeon (-380 million years ago), have led to antigenic dissimilarity. The GLO proteins from the bowfin and the gars, on one hand, and from the lake and white sturgeons, on the other hand, which have diverged since -150 and -80 million years ago (Carroll 1988; Birstein and DeSalle 1998), respectively, have multiple antigenic determinants in common. Partial identity between either the bowfin or the gars and the sturgeons (the latter originated 175-200 million years ago, Birstein and DeSalle 1998) illustrates an intermediate situation where within each paired comparison both species share at least one determmanL 28 Species Sex Body Gulonolactone oxidase activity weight (g) (|imoi AA/mg microsomal protein • h) Bowfin (Amia calva) F 984 0.5 F 1328 1.0 F 1411 0.7 F 2883 0.9 F 3346 0.6 F 3886 0.5 M 1321 0.7 M 1432 0.8 M 1472 0.8 M 1890 0.6 Longnose gar (Lepisosteus osseus) F 870 1.1 M 730 1.6 I 340 1.8 Polypterus senegalusj I 17±4 0.4 F = female, M = male, I = immature. No gulonolactone oxidase activity was found in the liver and the spleen of the three fish species, f Pool of tissues from 11 individuals. Table 3: Gulonolactone oxidase activity in the kideny of bowfin (Amia calva), longnose gar (Lepisosteus osseus) and Polypterus senegalus measured at 25°C 29 White Lake Bowfin Gar Polypterus Sea Tadpole Pig sturgeon sturgeon lamprey Crossreactivity with antibodies directed against white sturgeon GLO Yes Yes Yes Yes No No No No Antigen specifieity (assuming crossreactivity) W. sturgeon - id part id part id n/a n/a n/a n/a L. sturgeon - - part id part id n/a n/a n/a n/a Bowfin id n/a n/a n/a n/a Gar - n/a n/a n/a n/a U) o id = identical part id = partial identity n/a = not applicable Table 4; Immunologic crossreactivity and specificity of some ascorbic acid-synthcsi/ing vertebrates with sturgeon GLO Sex Body GSI HSI Blood Blood Liver Liver Spleen Spleen weight (%) (%) TAA DHAA TAA DHAA TAA DHAA (g) (pg/mL) (% of TAA) (fig/g) (% of TA A) (ng/g) (% of TAA) Bowfin (Amia calva) F 984 1,0 1.2 2.1 33 180.2 30 189,3 100 F U28 0,5 1,3 2.1 27 198.7 54 189.8 83 F 1411 0.3 1.8 l.l 34 148,6 63 153.3 98 F 2883 9,8 1,2 1.5 59 142,1 76 158.2 98 F 3346 8,3 1,3 l.l 36 162,0 65 170.8 91 F 3886 8,7 1,6 0.7 36 177,7 44 176.4 86 M 1321 2,0 1,5 2.5 61 180,5 59 221,9 100 M 1432 2,2 1.7 2,1 78 185,9 56 170,6 95 M 1472 2,4 1.4 1.4 35 196,3 59 163.8 100 w M 1890 2.1 1.3 2,7 80 156,7 51 127,8 100 Longnose gar (Lepisosteus osseus) F 870 4,2 2.0 8.8 29 93,4 94 184,9 100 M 730 3,5 2.4 8.9 17 84,8 100 170,6 100 1 340 - 2.8 9,1 26 109,4 100 176,7 100 F = female, M = male, I = immature, Table 5: Body weight, gonadosomatic index (GSI), hepatosomatic index (HSI) and total ascorbic acid (TAA), dehydroascorbic acid (DHAA) concentrations in selected tissues of bowfin mid longnose gm\ Tadpole Polypterus 4 Longnose gar^ Bow6n^ Sea lamprey^ Lake sturgeon" White sturgeon ^ 0 0.5 1.5 GLO activity (pmol AA mg-i microsomal protein h -i) Fig. 9: Comparison of GLO activities (at 25°C) in the renal microsomes of some vertebrates with an emphasis on fishes. ‘Moreau and Dabrowski 1996; "Moreau et al. 1999a; ^Moreau and Dabrowski 1998; ‘‘present study. 32 Fig. 10: Immunodouble diffusion tests of rabbit IgG to white sturgeon GLO with detergent-solubüized microsomes or crude extracts from various vertebrates. A through L, center weU contains protein A-purified rabbit IgG. A-1, white sturgeon posterior kidney microsomes; 2, lake sturgeon posterior kidney microsomes; 3, common carp hepatopancreas crude extract. B-I, white sturgeon posterior kidney microsomes; 2, bowfin posterior kidney microsomes; 3, goldfish hepatopancreas crude extract. C-1, white sturgeon posterior kidney microsomes; 2, longnose gar kidney microsomes; 3, tadpole kidney microsomes. D 1, white sturgeon posterior kidney microsomes; 2, Polypterus kidney microsomes; 3, pig liver crude extract. E-1, lake sturgeon posterior kidney microsomes; 2, bowfin posterior kidney microsomes; 3, common carp kidney crude extract. F-1, lake sturgeon posterior kidney microsomes; 2, longnose gar kidney microsomes; 3, goldfish kidney crude extract. G-1, lake sturgeon posterior kidney microsomes; 2, Polypterus kidney microsomes; 3, tadpole kidney microsomes. H I, bowfin posterior kidney microsomes; 2, longnose gar kidney microsomes; 3, pig liver crude extract. I-l, longnose gar kidney microsomes; 2, sea lamprey kidney microsomes; 3, tadpole kidney microsomes. J-1, bowfin posterior kidney microsomes; 2, sea lamprey kidney microsomes; 3, pig liver crude extract. K-1, longnose gar kidney nficrosomes; 2, Polypterus kidney microsomes; 3, common carp hepatopancreas cmde extract. L-1, bowfin posterior kidney microsomes; 2, Polypterus kidney microsomes; 3, goldfish hepatopancreas crude extract; 4, white sturgeon posterior kidney microsomes. 33 K Fig. 10 34 Fig. 11: Distribution of gulonolactone oxidase (GLO) in Actinopterygii. Outgroup PetromyzontLformes (the lampreys). A + denotes the presence of GLO, whereas - denotes the absence of the enzyme in Teleostei. Tick mark in the teleost Uneage indicates the loss of GLO activity. Cladogram based on Patterson (1973; 1982), Wiley (1976), Carroll (1988), and Nelson (1994). Until the mid-20th century, Lepisosteus was united with Amia in the Holostei which was thought to represent an intermediate level of actinopterygian evolution between chondrosteans (Acipenseriformes and Polyodontiforraes) and teleosts. Patterson (1973) and Wiley (1976) showed that there are few significant derived characters that are common to Amia and gars. Amia shares many derived features with teleosts that are not evident in Lepisosteus. Whey (1976) foimd a number of derived characters that Lepisosteus shares with the neopterygians but not with the living chondrostean orders. The above cladogram indicating that Amiiformes and teleosts are sister groups has been challenged by Olsen and McCune (1991), and Normark et al. (1991). Their studies suggested two alternatives 1) the Semionotiformes rather than the Amiiformes as ± e sister- group of teleosts, and 2) the Semionotiformes and the Amiiformes as a monophyletic group that is the sister group of teleosts. 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Examination of the qualitative ability of some cold water marine teleosts to synthesise ascorbic acid. Comp. Biochem. Physiol. A 121: 249-255 Moreau, R. and Dabrowski, K. 1996a. The primary localization of ascorbate and its synthesis in the kidneys of acipenserid {Chondrostei) and teleost (Teleostei) fishes. J. Comp. Physiol. B 166: 178-183 Moreau, R., Kaushik, S.J. and Dabrowski, K. 1996. Ascorbic acid status as affected by dietary treatment in the Siberian sturgeon (Acipenser baeri Brandt): tissue concentration, mobilisation and L-gulonolactone oxidase activity. Fish Physiol. Biochem. 15: 431-438 Moreau, R. and Dabrowski, K. 1998a. Body pool and synthesis of ascorbic acid in adult sea lamprey (Petromyzon marinus): An agnathan fish with gulonolactone oxidase activity. Proc. Natl. Acad. Sci. USA 95:10279-10282 37 Moreau, R. and Dabrowski, K. 1998b. Fish acquired ascorbic acid synthesis prior to terrestrial vertebrate emergence. Free Radio. Biol. Med. 25:989-990 Moreau, R., Dabrowski, K. and Sato, P.H. 1999a. Renal L-gulono-1,4-lactone oxidase as affected by dietary ascorbic acid in lake sturgeon (Acipenser fulvescens R). Aquaculture (in press) Nelson, J.S. 1994. Fishes of the World. 3rd ed., John Wiley & Sons, Inc., New York, 600 p. Normark, B.B., McCune, A.R. and Harrison, R.G. 1991. Phylogenetic relationships of Neopterygian fishes inferred from mitochondrial DNA sequences. Mol. Biol. Evol. 8:819-834 Olsen, P.E., McCune, A.R. 1991. Morphology of the Semionotus elegans species gruop from the early Jurassic part of the Newark Supergroup of Eastern North America with comments on the family Semionotidae (Neopterygii). J. Vert. Paleontol. 11: 269-292. Ouchterlony Ô., 1968. Handbook of Immunodiffusion and Immunoelectrophoresis. Ann Arbor Science Publishers, Inc., Ann Arbor, Ml, 215 p. Patterson, C. 1973. Interrrelationships of holosteans. J. Linn. Soc. (Zool.) Suppl.l, 53: 233-305 Patterson, C. 1982. Morphology and interrelationships of primitive actinopterygian fishes. Am. Zool. 22: 241-259 Roe, J.H. and Kuether, C.A. 1943. The determination of ascorbic acid in whole blood and urine through the 2,4-dinitrophenylhydrazine derivative of dehydroascorbic acid. J. Biol. Chem. 147: 399-407 Touhata, K., Toyohara, H., Mitani, T., Kinoshita, M., Satou, M. and Sakaguchi, M. 1995. Distribution of L-gulono-1,4-lactone oxidase among fishes. Fisheries Sci. 61:729-730 Wiley, E.O. 1976. The phylogeny and biogeography of fossil and recent gars (Actinopterygii: Lepisosteidae). Misc. Pub. Univ. Kansas Mus. Nat. Hist. 64: 1- 111 38 CHAPTERS FEEDING STIMULANTS IN SEMIPURIFIED DIETS FOR JUVENILE LAKE STURGEON, ACIPENSER FULVESCENS RAFINESQUE 39 Aquaculture Research. 1996. 27. 953-937 Short Communication Feeding stimulants in semipurified diets for juvenile lake sturgeon, Acipenser fulvescens Rafinesque* R Moreau & K Dabrowski School of Natural Resources. The Ohio State University. Columbus. Ohio, USA Cacreipon d m rr; Or K onisl Dabrowski. School of Natural R aoorca. The Ohio Stale tJobrasUy. 2021 CoOqr Rood. Onlnmhita. OH 43210. USA Furthermore. In-depth nutritional studies require The lake sturgeon..Acipenser ^vescens Raflnesque. the use of purified diets that give growth rates populations present In the Great Lakes and the comparable to those of lake sturgeon when fed lakes of southern Canada, although protected live food. The present work evaluates growth and (Johnson 1987). are still considered to be vulnerable survival of lake sturgeon juveniles of two body (lUCN 1988) or threatened (Williams. Johnson. sixes when fed with casein-based semipurlfied diets Hendrickson. Contreras-Balderas. Williams. supplemented with dlfierent compotinds as feeding Navarro-Mendoza. McAllister & Deacon 1989). stimulants. Conservation and restoration strategies Involve Three compounds. Le. betalne. krill and fish artificial propagation and rearing oC juveniles (or protein concentrate, were tested for their palatabtlity later release into the wlltL in diets of juvenile lake sturgeon. The composition Under culture conditions, information is available of the experimental-diets Is given in Thble6. Diets on nutrient requirements and feeding rates of white were formulated to contain at least 40% crude sturgeon. A. tixmsmonumus Richardson, and Siberian protein and 12% crude (at as recommended for sturgeon. A. baeri Brandt. Earlier studies have white sturgeon Juveniles (Hung 1991). A basal diet demonstrated that these species can be raised on containing 2% betalne was used as a control (diet live food (Monaco. Buddington & Doroshov 1981). B). In addition to 2% betalne. either krill or fish dry fish meal-based diets (Buddington & Doroshov protein concentrate was added at either 3% (diets 1984; Dabrowski. Kaushik & Faucoimeau 1985) BK and BF. respectively) or 6% (diets B ^ and BF2. and semipurified diets (Hung 1991; Kaushik. Brique respectively) levels, with substitution made of dextrin & Blanc 1994). In contrast, the literature on lake sturgeon feeding and nutrition under culture to avoid changes in the essential amino add conditions Is limited. In our experience, juvenile lake composition as provided with the basal formula. sturgeon has shown poor food consumption and Krill was obtained (rom Dr R. Hardy. Marine Fisheries thus poor growth on a casein-based semipurlfied diet. Service. Seattle. WA. USA Fish protein concentrate This study was Initiated because disadvantages was a commercial product (CPSP 90) (rom related to live food procurement (cost and labour) Sopropêche SA. (Boulogne-sur-mer. France). One have Increased Interest in compound diet among way analysis of variance was performed to Infer sturgeon culturlsts. In practice, commercial starter the effect of the dietary treatment on the growth feeds yield poor growth and low survival when fed performance. Chi^ test and arcsin transformation to lake sturgeon in comparison with white sturgeon. (Sokal & Rohlf 1969) were used to assess survival. Dilferences were considered significant at the 0.05 ’Presented at the 24ch Fish Feed and Nutrition level. Workshop. 19-21 October 1995. The Ohio State Unlversiqr. Columbus. Ohio. USA. Five thousand newly hatched lake sturgeon larvae O 1996 BliekwdI Science Ltd. 40 Feeding sUmuIanu Tor juvenile lake sturgeon R Morrau & K Dabrmnki Aquaculture Research. 1996. 27. 953-957 Table 6 Composition of the experimental diets Trill I 160- IngmdtonU (%) DIeU 140- 120- 8 BK BK2 BF BF2 100- 80- Casein (vttamin-rree)' 36.0 36.0 36.0 36.0 36.0 60- Com starch* 14.0 14.0 14.0 14.0 14.0 40- Dextrin (80% water-sokjbie}* 13.0 10.0 7.0 10.0 7.0 & Bataine (anhydrous)* ZO ZO ZO ZO ZO 20- Kr% - 3.0 6.0 -— I 0- Ash protein concentrate^ - - - 3.0 6X) 40- 1 Trill n Egg white* 5.0 5.0 5.0 5.0 5.0 » Gelatin* 4.0 4.0 4.0 4.0 4.0 30- Cod Over oa* 4.0 4J0 4.0 4.0 4.0 Soybean ladthin* 4.0 4J0 4.0 4J0 4.0 20- Comoa* ZO ZO ZO ZO ZO Lard* ZO ZO ZO ZO ZO Vltamtn mixture^ 4.0 4.0 4.0 4.0 4.0 10- Bemhart TomareA salt mixture* 3.0 3.0 3.0 3.0 ZO Choline chloride (99%)* ZO ZO ZO ZO ZO 0- Carboxymethyi ceOuiose ZO ZO ZO ZO ZO sodHjm salt* -10- CeOutoae (AlphaceO* Z99 Z99 Z99 Z99 Z99 BK BKZ BF BF2 Vitamin C^ 0.01 o m 0.01 0.01 0.01 Dietuy cceatment 'ICN Blomedkals. Inc.. Costa Mesa. CA. Flgnre12Wdght gain (%) of Juvenile lake sturgeon at the ^A.B. Staley Mfg Co.. Decatur, 0 . end of the two feeding trials (trial i. 6 weeks; trial n. 4 ^Slgma Chemical Co.. St Louis. MO. weeks). Data are shown as mean m SEM (n - 3). Values Concentrate of Bsh soluble proteia (CPSP 90: crude not sharing a common letter are slgnlflcantly different (P protein. 82-84% WW: crude Upld. 9-13% WW). < 0.05). Soproptebe S.A.. Boulogne-sur-mer. Prance. *Roche Perfocuiance Premix. composition per g of the vitamin mixture: vitamin A. 2645.50 lU; vitamin Dj. 220.46 ID: vitamin E. 44.09 lU: vitamin B;i. 13 pg: rlboSavln. 13.23 mg: nladn. 61.73 mg: n-pantotbenlc Water temperature was 19.2 ± 0.6'C throughout add. 22.05 mg: menadione. 1.32 mg; loUc add. 1.76 mg: the experiment The five diets were cold-pelleted Into pytldoxlne. 4.42 mg; thiamin. 7.95 mg: o-blotln. 0.31 mg. 2.5-nun-dlameter size. Greeze-dried to 10% humidity, Cbospltan C (ascocbyl phosphate Mg). Showa Denko KJC. crushed Into 0.5-0.8-mm particles and stored at New York. 4°C until needed. Each diet was randomly allotted to three tanks. Fish were fed at the daily feeding rate of 3.3—3.5% (of dry food per wet body weight] were received Gmm Wild Rose Hatchery (Wisconsin for 43 days. The daily rations were given In six Department of Natural Resources. OSA) and meals. Fish were weighed alter one month and at transferred to the Piketon Research and Extension the end of the trial (6 weeks). 36 h after the last meal. Center (Ohio. USA). Larvae were Initially fed with Sturgeon fed diet B showed the poorest growth naupUl of brine shrimp. Artemla frandscana, and rate (1.2 i 0.5% day"*) as well as the lowest reared on sturgeon starter diet (courtesy of Dr R. survival (35.6%) alter 6 weeks flhble 7). Sturgeons Barrows. Fish Technology Center. Bozeman. on diet B were lethargic. Supplementation with M ontana. USA) to the body weight of 1-5 g. At the either krill or fish protein concentrate Improved onset of the nutritional experiment (trial I). 225 weight gain and survival (60-80%) significantly Juveniles of body weights ranging fimm 1.4 to 4.2 (Flg.12). Increase of either krill or fish protein g were divided Into 15 circular tanks (volume 40 1) concentrate levels (rom 3% to 6% was of some equipped with a central outlet protected by mesh benefit to growth but did not significantly influence screen. The average weight of Esh per tank was the survival. adjusted to 2.20 Z 0.05 g (mean z: SD; n = 15). During trial H. one-year-old juveniles of lake C 1996 Blackwell Sdence LuL A/juaadatrt Baeardi. 27. 9S3-957 41 Aquaculture Research. 1996. 27. 95J-957 Feeding stimulants lor juvenile laite sturgeon R Moreau & K Dabrowikl Tiible 7 Growth and jurvival of juvenile lake sturgeon of two dlHereot shes led the experimental diets. Data are shown as mean r SEM (n » 3): within trials, values of survival or SCR not sharing a common superscript are significantly dînèrent (P < 0.05) Dietary traetment Initial body Rnal tiody Survival (%) SQR* walghtlS) walflMlg) day-1) Trial c B 2.2 e 0.1 3.5 = 0.8 35.6' 15 = 05® BK 2.2 e 0.1 4.6 = 0.4 77.6® 25 = 0.4 “ BK2 22 = 0.1 5.1 = 0.3 77.6® 3 5 = 05* BF 22 = 0.1 4.6 = 0.6 80.0® 24 = 0.6“ BF2 22 = 0.1 55 = 05 60.0“ 21 = 05 • Trial»: a 210.0 = 27 2105 = 122 100 05=05®* BK 205.4 = 1.7 1985 = 3.7 100 -0.1 = 0.1 * BK2 205.9 = 0.9 221.4 = 65 100 05 = 0.1 ® BF 204.5 = 20 249.4 = 5.7 100 0.7 = 0.1 • BF2 205.6 = 11 2565 = 4.6 100 0.8 = 0.1 * 'Specific growth rate — {[In (final body weight) — In (initial body weight)] X 100} / (duration of trial). sturgeon (strain Grom WblT River. Wisconsin. USA), formula Improved growth rate and survival of of body weight ranging Gom 180 to 260 g. were Juvenile lake sturgeon significantly. The growth rate, divided Into 15 eqierlmental groups of eight fish. 0.8% day"', reported here with 180-26O-g lake Each group was In a 4004 circular tank supplied sturgeon given diet BF2 compared lavourably with with oxygen-saturated water. The Initial average the growth rates of 1% day"' and 0.6-0.7% day"' weight of fish per tank was equal to 206.3 ± 3.9 g obtained with 90-g Siberian sturgeon led fish meal- (mean ± SD; n •> 15). Water temperature w as a t based diet at 17.5°C (Kaushik. tuquet. Blanc & Paba 19.6 ± 0.9°C throughout the ezperimenL The five 1989) and 160-g Siberian sturgeon cultured on a diets were cold-pelleted bito 2.5-mm-dlameter size casein-based diet at 18'C (Kaushik et aL 1994). alter addition of 30% water, sealed In plastic bags respectively. Both the lake sturgeon and the Siberian and stored at —15*C until needed. Each diet was sturgeon Inhabit the coldest geographical areas of randomly allotted to three tanks. Dietary rations the Northern Hemisphere and would have close were weighed and thawed dally. Fish were fed at a phylogenetic relationships (ArQruklln 1995). dally feeding rate of 1.1% (on dry weight basis) for Therefore, comparisons between these two species 4 weeks. The dally rations were given In three In terms of growth rate appear relevanL A growth meals. Fish were weighed on the 29th day of the rate of 0.8% day"' as obtained In the present work trial 36 h alter the last meal with one-year-old lake sturgeon fed a casein-based, Fish survived 100% Gi all groups. The fish protein semipurlfied diet Is satisfactory to conduct further concentrate supplementation at either 3% or 6% studies on the nutritional requirements of the generated significantly higher growth rates (0.7- species. 0.8% day"'. Thble 7) than In other treatments. It Is unlikely that the beneficial ellect of the fish Rations of diets BF and BF2 were consumed within protein concentrate on sturgeon growth Is the result a few minutes and sturgeons were active during of higher levels of essential amino acids and/or forty feeding In comparison with fish fed other diets. Diet adds In the supplemented diets compared with the BK was not consumed by the one-year-old lake control diet. The control diet contained sturgeon, which exhibited weight loss. Diet BK2 approximately 45% crude protein, which Is 5% gave an Intermediate growth rate. The addition of higher than the recommended level for optimal 2% betalne to the casein-based formula (diet B) growth of white sturgeon Juvenile (Moore. Hung & resulted In no growth. No pathological signs were Medrano 1988). On the contrary, the soluble observed In any group. peptides, particularly the short polypeptide chains Irrespective of the body size, the Incorporation of of low molecular weight (technical document no. 6% fish protein concentrate Into the basal diet 1. Sopropiche SA.) contained In the fish protein O 1996 Bladcwdl Sdence Lid. AtpjacuUiUt Reaardi. 27. 953-957 42 Feeding itknulants Tor Juvenile lake Muigeon R Moreau & K Dabrowski Aquaculture Research. 1996. 27. 953-937 concentrate, probably stimulated the consumption Abstracts. World Marlculture Society. Annual Meeting. of rood by acting upon the olfactory and/or gustatory Washington. DC receptors located on Bsh body and In the mouth. Buddington R.K. & Doroshov SX (1984) Feeding trials with Sturgeon led fish protein concentrate supplemented hatchery produced white sturgeon Juveniles [Adpenzer Cnmsmontanus). Aquaculture 36,237-243. diets were active and searched for food alter the first Carr WXS. (1978) Chemoreceptlon In the shrimp. portion was given. The composition as well as Foluenianetexpiigia; the role of amiiu adds and betalne In the concentration of peptides In the fish protein dldtatlon of a feeding response by extracts. Comparative concentrate could have played an Important role In BlodiemlstryandPhystoloeyBlA, 127-131. chemoattractlon mechanism. It could also explain Carr WXS- Blumenthal ICM. & Netherton J.C (1977) the limited ellect of betalne alone and the variable Chemoreceptlon tar the pigfish. Orthcpristls chrysopienat effects of the betalne—krill combination. At the contribution of amino acids and betalne to stimulation equivalent level of supplementation, krill and fish of feeding behavior by various extracts. Comparative protein concentrate gave similar Improvements of Blodtemlstrg and Physiology S8A. 69-73. growth with 2-g Juveniles but not with one-year- Dabrowski IC,XausfalkS.J.&FaucooneaaB. (1983) Rearing old Juveniles. This suggests changes In of sturgeon (Ac(penaer&asrt Brandt) Iaivie.LFeedlng trial Aquocultura 4 7 , 183-19X attractant—stimulant preferences In relatlott to fish cm C (1989) Diet design: aquaculture. Feed International age (Mackle. Adron & Grant 1980; Shparkovsldy. KK2). 12-14. Pavlov & Chlnarina 1983). It Is worth noting that Goh t & Tunura T. (1980a) Olfactory and gustatory Incorporation of 2% betalne alone was Inefiectlve In responses to amino adds In two marine teleosts — red sea making lake sturgeon leed on the casein-based bream and muBet. Comparative Biochemistry and semipurlfied diet. In contrast with previous worit on Physiology 6 5 C 217-224. the white sturgeon (Patterson. Lellls & Barrows Goh Y. & Thmura t . (1990b) Effect of «minn adds on the 1994) as well as with many fish (Carr. Blumenthal feeding behaviour In red tea bream Comparative & Netherton 1977; Goh & Thmura 1980a. 1980b: Biochemistry and Physiology 66C 225-229. Gill 1989) and crustaceans (Carr 1978). Finally, the Hung S SdJ. (1991) Nutrition and feeding of hatchery- produced Juvenile' white. sturgeon (Aefpenser present study demonstrates that. In lake sturgeon Iranstrumtansa): an overview. Itu ProceeeBngs o f the First feeding, the combination of betalne and fish protein International Symposium an the Sturgeon, ŒMAGSEF. Oct, concentrate Is an alternative to tublfac and white 3-6. 1989. Bordeaux, France (ed. by P. WUUot), worm extracts as previously reported (Blnkowsld & pp. 63-77. Meyers 1983). International Union Ibr Conservation of Nature (1988) 1988 lUCN R e d -list o f Threatened Animals. The lUCN Conservatloii Mbnlttxing Centre, Cambridge, England. Johnson JX. (1987) Protected Fishes of the United States and AcknowI*dgm**xtm Canatfa(ed. by BJ?,McAleer). American Fisheries Sodety, Bethesda. MD. The authors th an k M r S. FIfer, Wisconsin Kaushik S.J» tuqiiet D- Blanc D. & Paba A. (1989) Studies Department of Natural Resources, Wild Rose on the nutrition of Siberian sturgeon, Adpenser baeri. L Hatchery, WI, and Dr R. Barrows, Bozeman, MT. Utilization of digestible carbohydrates by sturgeon. We also thank Mr J. Ebellng for technical support Aguaculture 797-107. 6 , during the growing trials. Support for this project Kaushik S.J- Brique J. & Blanc D. (1994) Apparent amino was obtained from the US Department of Agriculture, add availability and plasma feee amino add levds In OICD, project 58-319R -0-026, and DSDA Grant Siberian sturgeon [Acipenser baeri). Comparative 92-37203-7949. a n d partial funding to OARDC Biochemistry and Physiology 107A, 433-438. manuscript no. 1/96. Madde AM - Atbon J.W. & Grant P.T (1980) Chemical nature of feeding stimulants for the Juvenile Dover sole. Solettsolett(L.).Joum alofFldi Biology 16, 701-708. Monaco G-Buddington RJC & Doroshov SX ( 19 81 ) Growth R«for«ncas of white sturgeon (Acipenser transmontanus) under hatchery conditions. Journal o f World Marlculture Sodety Aityuklln EN. (1993) Contribution on biogeography and 12(1). 113-121. relationships within the genus Acipmstr. Sturgeon Moore B.J„ Hung S.S.O. & Medrano JF. (1988) Protein Quanerly 3(2). 6-8. requirement of hatchery-produced Juvenile white Blnkowsb F.P. & Meyers S.P. (1983) Culture and feeding sturgeon [Acipenser transmontanus). Aquaculture 71, response of the lake sturgeon. Acipenser fulvescens. Itu 235-245. O 1996 Blackwell Science Ltd. Aquaadture Research. 27.953-957 43 Aquaculture Research. 1996. 27. 953—957 Feeding stimulants for Juvenile lake sturgeon R Mortau & K DabmvsU Patterson T_ Lellls W.A. & Barrows F.T. (1994) Starter diets ■ Sokal R.R. & Rohlf F.f. (1969) Biom etry - The P rlndpla and Ibr the Snake River white sturgeon (Acipenser Practice of Statistics In Biological Research. p%i. 386-287. transmontanus). In: Book of Abstracts. World Aquaculture W.H. Freeman and Company. San Francisco. '94. Jan. 14-18.1994. New Orleans. LA. USA. pp. 225. Williams J £ . Johnson J.&. Hendrickson D.A~ Contreras- Shpatkovskly LA_ Pavlov (J). & Chlnarina AD. (1983) Balderas S« Williams JD_ Navarro-Mendoa &L. Behavior of young hatchery-reared Atlantic Salma solar. McAllister Oon E. & Deacon J.E. (1989) Fishes of North (SalnuMldae) Influenced by amino adds, foum ai o f America endangered, threatened, or of special concern: hhthyology 23(4). 140-147. 1989. Fisheries 14.2-20. C 1996 BlickweU Science Ltd. Aguaeulture Research. 27. 953-957 44 CHAPTER 6 RENAL L-GULONOLACTONE OXIDASE ACTIVITY AS AFFECTED BY DIETARY ASCORBIC ACID IN LAKE STURGEON (ACIPENSER FULVESCENS) INTRODUCTION L-Ascorbic acid (AA, vitamin C) is synthesized in animals from either D-glucose or D-galactose as part of the glucuronic acid pathway (Hassan and Lehninger 1956). Branching from L-gulonic acid, the biosynthetic pathway of AA comprises three consecutive steps: first, the enzymatic lactonization of L-gulonic acid catalyzed by L- gulonolactone hydrolase (EC 3.1.1.18) (Stubbs and Haufrect 1968), second, the oxidation of L-gulonolactone catalyzed by L-gulonolactone oxidase (GLO, EC 1.1.3.8) and third, the spontaneous isomerization of 2-keto-L-gulonolactone leading to AA (Chatterjee et al. 1960). The general view is that animals lacking GLO are unable to synthesize AA and thus depend upon a dietary source of the vitamin (Sato et al. 1976). In guinea pigs and human, the enzyme deficiency is the result of missense mutations within the gene for GLO (Kawai et al. 1992; Nisltikimi et al. 1992; 1994). The inability to biosynthesize AA is rather the exception in the animal kingdom. Many of higher vertebrates can synthesize the molecule due to the presence of a functional 45 GLO associated with the microsomal fraction in either the liver or the kidney or in both organs. In mammals, however, the primates, the guinea pigs (Bums 1957) and the bats (Bimey et al. 1976) cannot produce AA de novo. Sixteen species of passeriform birds were found to be incapable of producing AA (Chaudhuri and Chatteqee 1969). Among the lower vertebrates analyzed to date, reptiles, amphibians (Roy and Guha 1958; Chatteijee 1973), and fishes retaining numerous ancestral characters, such as lamprey, shark, ray, lungfish and sturgeon (Dykhuizen et al. 1980; Dabrowski 1994; Touhata et al. 1995; Moreau et al. 1996; Moreau and Dabrowski 1998; Maeland and Waagbd 1998), have been shown to have GLO in the kidney, whereas teleost fish lack GLO activity (Dabrowski 1990). The mechanisms by which fish GLO activity is regulated are unknown. In six- month-old white sturgeon (Acipenser transmontanus) fed a diet devoid of AA, tissue total AA concentrations were not decreased while survival and growth rates were comparable to those of AA-supplemented groups (Dabrowski 1994). This observation suggested that white sturgeon GLO produced adequate amounts of AA to meet the fish needs at a stage of rapid growth while the dietary source was withheld. However, the question of whether enhanced dietary AA intake exerts a negative control on fish GLO is of great interest since such a feedback seems to operate in rabbit (Jenness et al. 1978) and mice (Tsao and Young 1989). The present study was specifically designed to address this question. The experiment followed the finding that white sturgeon GLO activity is significantly higher in the posterior than anterior kidney (Moreau and Dabrowski 1996a). To avoid non-controlled sources of AA, the casein-based semipurified diet was given to sturgeon. Also, AA dietary levels were increased up to 1250 mg/ kg, that is more than 10 times the recommended dietary vitamin C level in fish (NRG 1993). In addition to quantifying AA concentrations in selected tissues and GLO activity in the kidney, the activities of two enzymes involved in AA metabohsm, i.e. dehydroascorbic acid reductase (Bode et al. 1993) and alkaline 46 phosphatase (Matusiewicz and Dabrowski 1996), were measured in lake sturgeon {Acipenser fulvescens) fed graded levels of AA. Kinetic characteristics of sturgeon GLO were used to project a daily rate of AA synthesis in this fish. MATEEÜALS AND METHODS Feeding trial We used two-year-old juveniles of lake sturgeon, strain from the Wolf River (Wisconsin, USA), a species native of the Great Lakes region. Prior to the trial, sturgeon were fed a semipurified diet devoid of AA for 9 days. Experimental fish (initial body weight 253± 89 g, mean ± SD) were tagged and sorted into 12 experimental groups of 8 fish. Each group was assigned to a 400-1 circular tank supplied with oxygen-saturated water. Water temperature was 19.8° ± 0.4°C throughout the experiment. AH animal procedures and handling were conducted with care in comphance with the guidelines of the Institutional Laboratory Aitimal Care and Use Committee, The Ohio State University. The basal diet was formulated according to previous work (Moreau and Dabrowski, 1996b), and is shown in Table 8 . Four experimental diets were prepared by adding either 0, 50, 250 or 1250 mg AA in the form of ascorbyl-2-monophosphate Mg (Phospitan C, Showa Denko K. K., New York) per kg of the basal formula with substitution being made for cellulose. Ascorbyl-2-monophosphate Mg was chosen because of its stability (Dabrowski et al. 1994) and bioavaüabüity to fish (El Naggar and Lovell 1991; Matusiewicz and Dabrowski 1995). The whole amount of feed required for the experimental period was prepared at one time. Diets contained 30% water, were sealed in plastic bags, and frozen at -15°C until needed. Three groups of fish were randomly given one of the test diets for a period of 38 days. Daily, the feed ration was weighed and thawed. Fish were fed 7 days a week, three times a day. The feeding rate was adjusted at 1.1-1.3% (on dry weight basis) of fish body weight per day. 47 Sampling An intermediate weighing was carried out on day 32. On day 39, two fish per tank, among those which had showed the best weight gain on day 32, were sampled. Tanks were sampled in the same order as the last meal was given the day before ( 10-min interval time between 2 tanks). Fish were anesthetized on ice and blood was drawn at the caudal vein region. Fish were stunned by a blow on the head and the spinal cord was sectioned. Liver and kidney were removed. The kidneys were divided into the anterior kidney and the posterior kidney as previously described (Moreau and Dabrowski 1996a). Anterior kidneys were analyzed for ascorbate concentrations. Posterior kidneys were further divided into halves according to the symetrical axis of the organ. Each half was designated to either ascorbate concentration measurements or enzyme activity assays. Tissues were immediately frozen in liquid nitrogen and stored at -STC until analysis. Blood samples were centrifuged at 7,000 X g for 5 min and plasma was stored at -81°C until assayed. Ascorbic acid analysis Total AA (TAA, sum of reduced and oxidized AA), and oxidized AA (DHAA) in liver, kidney and blood plasma were assayed using the 2,4-dinitrophenylhydrazine (DNPH) colorimetric method (Roe and Kuether 1943) modified as in Dabrowski and Hinterleitner (1989). Hepatic and renal tissues were homogenized in 5% trichloroacetic acid (TCA) solution containing 250 mM HCIO 4 and 0.08% ethylenediaminetetra-acetic acid (EDTA) using an Omni 5000 homogenizer (Omni International, Inc., Waterbury, CT), and centrifuged at 29,000 x g for 30 min at 4°C. Plasma extracts were prepared similarly with the exception that the TCA concentration of protein precipitating solution was increased to 10% and added to blood plasma with a 1:1 (v:v) ratio. 48 L-Gulono-1,4-lactone oxidase activity Gulonolactone oxidase activity was determined by quantifying AA produced in crude enzyme preparation using the DNPH colorimetric method (Dabrowski 1990). Posterior kidneys were homogenized (1 g of tissue in 4 ml of 50 mM phosphate buffer, pH 7.4, containing 1 mM EDTA and 0.2% sodium deoxycholate) in a spin homogenizer (Omni 5000) at 4°C. The homogenates were centrifuged for 15 min at 15,000 x g at 4°C. Four hundred |il of supernatant were then incubated with 100 p.1 of 50 mM reduced glutathione, 1300 fil of 50 mM phosphate buffer (pH 7.4) and 200 |il of 100 mM L-gulono-1,4-lactone at 25°C. Three hundred-pl aliquots were withdrawn at 0, 10, 15, 20, 30 and 50 min, mixed with 300 |il of 5% cold TCA containing 250 mM HCIO 4 and 0.08% EDTA and stored at -81°C until analysis the following day. After thawing, the incubates were centrifuged for 30 min at 14,000 x g at 4°C and TAA was assayed in the supernatant (Dabrowski and Khnterleimer, 1989). The concentration of TAA formed versus time was plotted and the initial velocity was calculated from the linear portion of the curve. GLO activity was expressed as nmol AA formed/mg soluble protein - h. This procedure was used to compare GLO activities in anterior and posterior kidneys, and to study the effect of various assay temperatures (0°, 15°, 25° and 35°C) on GLO activity in the posterior kidney of lake sturgeon fed a commercial trout diet Purifîcation of sturgeon gulonolactone oxidase Gulonolactone oxidase was partially purified from white sturgeon posterior kidney following the procedure described for chicken kidney (Kiuchi et al. 1982). Purification steps included differential centrifugation, solubilization with Brij 35, DEAE Sephadex A-50 ion exchange chromatography, hydroxyapatite chromatography, and ultrafiltration. 49 Dehydroascorbic acid (DHAA) reductase (EC 1.8.5.1) Tissue samples were sliced and rinsed twice with cold 1.15% KCl containing 10 mM EDTA (pH 7.4). One g of tissue was homogenized for 30 sec at a moderate speed using a spin homogenizer (Omni 5000) in 3 ml of cold 0.1 M Tris-HCl (pH 7.5) containing ImM EDTA. The homogenate was then centrifuged at 20,000 x g for 15 min at 4°C. Aliquots of 250 pii were dialyzed for 1 h at 4°C against 0.1 M Tris-HCl (pH 7.5) containing ImM EDTA using an oscillating motion microdialyzer (OMS 101, National Labnet Company) and cellulose membranes of 12,000-molecular weight cut-off. The enzymatic formation of AA was measured based on the change of absorbance at 265 nm (Stahl et al. 1983). The reaction mixture contained 890 |J.l of 50 mM phosphate buffer (pH 6.7) supplemented with 0.147 mM EDTA, 50 fil of 50 mM reduced glutathione, and 10 pi of dialyzed enzyme preparation. The reaction was initiated by adding 50 pi of 25 mM DHAA (Aldrich Chemical Company, Inc., Milwaukee, WI). A blank was carried out simultaneously without enzyme preparation in order to determine the non-enzymatic reduction of DHAA. The increase in absorbance at 265 nm was recorded at 25 °C for 5 min. We used the molar extinction coefficient of AA at 265 nm, i.e. 14,700 l/(mol • cm), for calculation. DHAA reductase activity was expressed as nmol of AA formed/mg of soluble protein in the dialyzed enzyme preparation • h. Alkaline phosphatase activity Total alkaline phosphatase in blood plasma was determined by monitoring spectrophotometrically at 405 nm the release of nitrophenol for 10 min at 25°C (Matusiewicz and Dabrowski 1996). Thawed blood plasma was centrifuged at 14,000 x g for 30 min at 4°C prior to an assay. Fifty-pl aliquots of supernatant were incubated with 50 950 (il of 100 mM diethanolamine/HCl buffer (pH 9.8) containing 5 mM p- nitrophenylphosphate and 5 mM MgCl 2. The molar extinction coefficient for nitrophenol at 405 nm was 14,767 I/(mol ' cm). Total alkaline phosphatase activity was expressed as (imol nitrophenol released/1 of blood plasma • min. Protein determination Protein content of enzyme preparations was measured by the Bradford procedure (Bradford 1976) using commercial Coomassie protein assay reagent (Pierce Chemical Company, Rockford, IL). Bovine serum albumin was used as the standard. Statistical analysis The effects of dietary treatment on AA concentrations and enzyme activities were analyzed using nonparametric BCruskal-WaUis test followed by Dunn’s multiple comparison procedure (Dunn 1964). Correlations between tissue TAA concentrations and posterior kidney GLO activity were assessed by linear regression using the StatView 512+ (Brainpower, Inc., Calabasas, CA) package for Macintosh. One degree of freedom was assigned to each tank. Differences of GLO activity between lake sturgeon anterior and posterior kidneys were assessed by paired Student’s f-test. The accepted level of significance was 0.05. RJESULTS Growth and survival At the end of the feeding experiment, survival was 100% in all groups. No pathologies were observed among fish. Sturgeon body weight approximately doubled. Specific growth rate and hepatosomatic indices are shown in Table 9. 51 AA concentrations in sturgeon tissues Total AA concentrations were approximately double in head kidney than in liver, and 8-15 fold higher in liver than in blood plasma. Sturgeon fed 1250 mg AA/kg diet had in aU selected tissues TAA concentrations significantly higher (P < 0.05) than sturgeon fed either 0 or 50 mg AA/kg diet (Table 10). A supplement of 1250 rag AA/kg diet significantly increased (P < 0.05) TAA concentrations in the liver, posterior kidney and blood plasma as compared to a supplement of 250 mg AA/kg diet. Sturgeon fed 250 mg AA/kg diet had posterior kidney and blood plasma TAA concentrations significantly higher (P < 0.05) than sturgeon fed either 0 or 50 mg AA/kg diet. No statistical differences were found in tissue TAA concentrations between fish fed 0 and 50 mg AA/kg diet. TAA was found predominantly in reduced state in the posterior kidney (50.5-65.8% of TAA) and blood plasma (52.3-74.4% of TAA). Oxidized AA (DHAA) was predominant in the hver and head kidney (> 68 % of TAA). Enzymes related to AA metabolism At the end of the feeding trial, posterior kidney GLO activity varied from 45.1 to 74.6 nmol (or 7.9 to 13.1 |ig) AA/mg soluble protein • h at 25°C among fish fed the experimental diets. The enzyme activity was significantly (P < 0.05) lower by 28%-40% in the group fed 50 mg AA/kg diet as compared to the groups fed 0, 250 or 1250 mg AA/kg diet (Table 11). GLO activity was significantly correlated with posterior kidney (P < 0.01), head kidney (P < 0.02), and blood plasma (P = 0.031) TAA concentrations (Fig. 13). No statistical differences were found in DHAA reductase activities between fish fed 0 and 1250 mg AA/kg diet (Table 11). DHAA reductase activity was 70% higher in hver 52 than in posterior kidney. The analysis of the total alkaline phosphatase activity in blood plasma showed no significant difference between dietary treatment (Table 11). Sturgeon GLO characteristics Gulonolactone oxidase activity was significantly higher (P < 0.05) in the posterior kidney than in the anterior kidney of lake sturgeon (62.7 ± 19.9 versus 11.4 ± 2.3 nmol AA/mg soluble protein • h; n = 4 fish). Posterior kidney GLO velocity increased linearly (r- = 0.998, P < 0.001, n = 3 assays at each temperature from a pool of tissue) with assay temperature over a 0-28“C range. Further increases in the reaction temperature had little effect on the activity. Qio between 15° and 25°C was 1.65 (Fig. 14). This coefficient was used to calculate the in vivo synthetic rate in lake sturgeon. Affinity constant (K^) at 25°C of purified sturgeon GLO for L-gulonoIactone was calculated by averaging values derived from Lineweaver-Burk double reciprocal plot and S/V against S plot, so that = (0.66 + 1.18)/2 = 0.92 mM (Fig. 15). DISCUSSION AA concentrations in sturgeon kidney Total AA concentrations in the head kidney were consistently higher than in the posterior kidney which confirmed our earlier observation (Moreau and Dabrowski 1996a). This distribution most likely reflects the biochemical specificities of the sturgeon kidney among which the anterior kidney is a site of AA accumulation, whereas the posterior kidney is the primary location for its synthesis. Higher proportions of oxidized AA in the tissues of fish unable to synthesize AA has been used as an indicator of vitamin C deficiency (Dabrowski 1990b). MetaboHcally, it signifies that AA is oxidized faster than it is regenerated. However, it would be speculative to infer that the proportion of reduced AA 53 is higher in the posterior kidney of sturgeon solely because AA is mainly synthesized in this region. The head kidney could possibly demonstrate a faster rate of AA oxidation. Effect of dietary AA on GLO activity The results showed that lake sturgeon GLO activity was inconsistently affected by graded dietary levels of AA. Supplementing lake sturgeon diet with 0, 250 or 1250 rag AA/kg did not significantly affect GLO activity despite significant increases in tissue TAA concentrations. A significant -40% decrease in GLO activity was found, however, between sturgeon fed 50 and sturgeon fed either 250 or 1250 rag AA/kg diet. This observation is yet to be explained. Additional levels of supplemental AA between 0 and 250 mg/kg would be needed to confirm this finding. The fact that sturgeon fed 250 and 1250 rag AA/kg diet stored dietary AA in their tissues while maintaining enzymatic synthesis of AA at a rate as high as that of sturgeon fed 0 mg AA/kg diet implies that dietary AA did not exert a negative feedback over GLO activity. Positive significant correlations between posterior kidney GLO activity and TAA concentrations in the kidney and blood plasma also supported the absence of a negative feedback exerted by tissue or circulating AA. The stronger correlation found in the posterior kidney (r^ = 0.584, Fig. 13) than in any other tissues indicated that in situ synthesis contributed to further increase posterior kidney TAA concentrations. The present results differed from those of previous studies in rabbit (Jenness et al. 1978) and mice (Tsao and Young 1989) in which a significant decline of GLO activity was found in the livers of animals fed diets supplemented with large amounts of AA. Because the oxidation of L-gulonolactone to AA is accompanied by the production of hydrogen peroxide (Kiuchi et al. 1982) and the equimolar consumption of reduced glutathione (Banhegyi et al. 1996), lacking a negative feedback mechanism on GLO activity 54 while AA is abundant in the diet does not seem most cost-efficient. Reduced glutathione would directly scavenge hydrogen peroxide enzymatically by the action of glutathione peroxidase, and nonenzymatic reaction is also possible. Thus AA appears to be synthesized at the expense of cellular antioxidant defense system. Rate of AA synthesis in vivo We applied to sturgeon the mathematical method used previously to quantify the biosynthetic rate of AA in the liver of mammals (Rucker et al. 1980) and in the kidney of sea lamprey (Moreau and Dabrowski 1998). The model uses GLO maximum velocity (Vmax) expressed per mg microsomal protein. Since L-gulonolactone concentration (10 mM) in our GLO activity assay was 10 times Km (0.92 mM) we assumed that the initial velocities we calculated were near Vmax. Assuming that the concentration of L- gulonolactone (S) in renal cells, as in hepatic ceUs, is less than 0.05 mM (Rucker et al. 1980), and knowing that 100 mg of fish kidney soluble protein yields approximately 8 mg of microsomal protein, Vmax ranged from 568 to 931 nmol AA/mg kidney microsomal protein • h at 25°C. The theoretical rate (V q) was calculated by substituting in the equation Vo =Vmax S/Km + S which averaged 39.4 nmol AA/mg kidney microsomal protein ■ h at 25°C. The rate was then multiplied by 6 (since 1 g of fish kidney contains approximately 6 mg of microsomal protein), followed by times 24 (to convert to one day), then times 5 (weight in gram of the kidneys in a 1-kg lake sturgeon), and divided by 1.65 (Qio)- The theoretical net synthesis in lake sturgeon amounted to 17 pimol (or 3 mg) AA/kg wet weight • day at 15°C. This estimate of the daily synthesis of AA in sturgeon (3 mg AA) compares favorably to vitamin C requirement for teleost fish. For instance, a 1-kg rainbow trout held at 15°C and fed at 1% of its body weight per day a diet supplemented with 100 mg AA/kg 55 (NRC 1993) will ingest 1 mg AA daily. From this comparison, it can be argued that first, the vitamin C requirement of trout and sturgeon is in the same order of magnitude, and second, that juvenile sturgeon possess adequate synthetic capabilities to meet their vitamin C need and therefore do not require an exogenous supply of vitamin C. Since environmental factors (Wise et al. 1988), stress (Lovell and Lim 1978) and pathologies (Wahli et al. 1995) can increase significantly the vitamin C requirement of teleost fish, it is reasonable to think that these conditions wül also increase sturgeon vitamin C needs. We hypothesize that sturgeon can respond to increased nutritional needs by synthesizing more AA. Practically, the measurement of GLO activity and the calculation of AA synthesis rate in sturgeon held under specific experimental conditions could be employed as a biochemical indicator to identify these factors and predict AA requirement in teleost fish. 56 Ingredient Concentration (g/kg dry diet) Casein (vitamin-free) ^ 360 Dextrin (80% water-soluble) ^ 150 Com starch ^ 100 Fish protein concentrate ^ 50 Egg white ^ 50 Gelatin ^ 40 Cod hver oü * 40 Soybean lecithin ^ 40 Com oil ^ 30 Vitamin mixture 40 Bemhart TomareUi salt mixture ^ 30 Choline chloride (99%) ' 20 Carboxymethyl cellulose (sodium salt) ^ 20 L-Methionine ^ 5 Cellulose (Alphacel) ^ 25 * ICN Biomedicals, Inc.. Costa Mesa. CA. - A.E. Staley MFG. CO., Decatur, IL. ^ CPSP 90, Sopropêche S.A., Boulogne-sur-raer, France. Roche Performance Premix composition per g of the vitamin mixture : vitamin A (retinyl acetate), 2645.50 lU; vitamin D3, 220.46 lU; vitamin E, 44.09 lU; vitamin B 12, 13 p.g; riboflavin, 13.23 mg; niacin, 61.73 mg; d-pantothenic acid, 22.05 mg; menadione, 1.32 mg; folic acid, 1.76 mg; pyridoxine, 4.42 mg; thiamin, 7.95 mg; d-biotin, 0.31 mg. ^ Degussa Corporation, Chemicals division, Ridgefield Park, NJ. Table 8 : Composition of the basal diet 57 AA Initial fish body Final fish body s g r ' H s r supplement weight weight (% BW ■ day) (%) (mg/kg diet) (g) (g) 0 320.2 ± 40.3 582.3 ± 74.2 1.6 ±0.1 3.0 ±0.1 50 361.2 ± 54.1 639.2 ± 94.5 1.5 ±0.1 2.9 ± 0.2 250 343.7 ± 52.8 603.0 ±75.1 1.5 ± 0.1 3.0 ±0.1 1250 276.8 ±21.4 526.5 ± 43.0 1.7 ±0.1 3.0 ±0.1 'specific growth rate = 100 x (In final body weight - In initial body weight) / (duration in days). 'Hepatosomatic index = 100 x (liver weight) / (body weight). Data are presented as mean ± SEM for 3 replicate tanks. Table 9: Growth rate and hepatosomatic index of lake sturgeon fed graded levels of AA 58 Tissue AA supplement (mg/kg diet) 0 50 250 1250 Liver TAA (nmol/g) 409.6 ±28.8' 393.9 ± 28. 1' 435.2 ± 26.6' 585.8 ± 8.2' DHAA (nmol/g) 316.3 ±23.7“' 286.2 ± 17.6' 324.1 ± 22.5“' 400.2 ± 14.3“ AA(% of TAA)' 23.7 ± 0.4 27.9 ± 4 26.4 ± 1.5 32.4 ± 2.8 Head kidney TAA (nmol/g) 719.5 ± 30. l' 718.0 ± 26.5' 896.1 ± 100. 1"' 1056.2 ± 18.4“ DHAA (nmol/g) 672.2 ± 38.1 660.9 ±41.4 655.4 ± 39.8 786.3 ± 80.7 AA (% of TAA) 7.7 ± 4.4 9.2 ± 2.5 26.7 ± 4.6 26.5 ± 6.8 Posterior kidney LA TAA (nmol/g) 527.9 ± 17.o' 503.7 ± 6.3' 761.3 ± 31.6' 940.9 ± 32.7 “ DHAA (nmol/g) 219.0 ± 22.9' 252.0 ± 17 . 1“' 261.2 ± 12. 1“' 326.1 ± 15.7 “ AA (% of TAA) 59.1 ±3.4 50.5 ± 2.9 65.8 ± 1.1 65.2 ± 3.0 Blood plasma TAA (pM) 25.4 ± 3.7 ' 23.5 ± 3. 1' 41.2 ± 2.2' 68.4 ± 3.6“ DHAA (pM) 1 1 .6 ± 1.9 10.4 ± 1.7 13.5 ± 2.7 17.6 ± 1.2 A A (% of TAA) 52.3 ±11.8 56.4 ± 1.4 67.8 ± 5.6 74.4 ± 2.2 AA Wcis calculated by subtracting DHAA from TAA. Data are shown as means ± SEM for 3 replicate tanks. Values not sbming a common letter within rows are significantly different (P < 0.05). Table 10: Total (TAA), oxidized (DHAA), and reduced (AA) ascorbic acid concenualions in tissues of juvenile lake sturgeon fed graded levels of AA ' '5...... AA supplement G LO' DHAA reductase Alkaline phosphatase^ (mg/kg diet) Posterior kidney Posterior kidney Liver Blood plasma 0 62.8+ 2.4"* 232.8 ± 19.0 394.0 ± 55.1 88.5 ± 8.4 4 50 45.1 ±4.0* n.a. n.a. 98.8 ± 6.4 250 74.5 ± 4.5" n.a. n.a. 95.6 ± 8.2 1250 74.6 ± 1.9" 257.4 ±7.8 445.0 ± 11.1 115.0 3:11.1 One unit refers to 1 nmol AA formed/mg soluble protein • h at 25”C. 'One unit refers to 1 nmol AA formed/mg soluble protein in dialyzed enzyme preparation • h at 25°C. o\ o One unit refers to 1 pmol nitrophenol formed/1 of blood plasma • min at 25"C. ^Not analyzed. Data are shown as means ± SEM for 3 replicate tanks. GLO activities not sharing a common letter are significantly different (P < 0.05). Table 11 : L-Gulonolactone oxidase (GLO) in posterior kidney, dehydroascorbic acid (DHAA) reductase in posterior kidney and liver, and total alkaline phosphatase in blood plasma of juvenile lake sturgeon fed graded levels of AA 90 Posterior kidney Head kidney ao 80 2 5 0 80 □ 1230 1250 250 70 250 ■ 250 a 60 60 2a Q. 50 0Û CO 50 50 B < 40 = 0.584 40 = 0.476 < P<0.01 50 P<0.02 3 0 1------1------1------1------1------1------1_ 30 I 400 500 600 700 800 900 1000 600 700 800 900 1000 1100 5 Total ascorbic acid (nmol/g tissue) > Î 90 90 ma Liver Blood plasm a rt #250 ^250 T32 80 80 X 1250 • . A o 1250 o • • c 70 250 1250 70 c ^ e 250 u □ • 250 ca A ^ "o 60 60 c _c 3 50 50 a *50 40 - 1^=0.222 40 - r =0.418 50* P = 0.145 ^50 P = 0.031 30 30 300 350 400 450 500 550 600 10 20 30 40 50 60 70 Total ascorbic acid (nmol/g tissue or |j,mol/l plasma) Fig. 13: Effect of dietary AA on GLO activity. Correlations between posterior kidney GLO activity and tissue total AA concentrations in lake sturgeon fed graded levels of AA. Values represent means per tank. One degree of freedom was assigned to each tank. Labels indicate AA supplement expressed as mg/kg diet 61 6 0 " c ■s 50- obH CL ÛÛ 40- < Qio= 1.65 < ,— 20 - ’> u y = 1.758X + 0.695 o 1 0 - r: = 0.998 o “T“ 0 5 10 15 20 25 30 35 40 Assay temperature C’C) Fig. 14: Effect of assay temperature on sturgeon GLO activity. In vitro synthesis of AA was measured at four temperatures in crude extract prepared from lake sturgeon posterior kidney. Data are shown as mean ± SD for 3 replicate measures. was calculated between 15 and 25°C following linear regression. 62 (A) 200-1 150- Z : 100- y = 36.717x + 55.369 ^ = 0.962 Kg, = 0.66 mM V___ = 0.02 mM/min -5 -4 -3 -2 -1 0 1 2 3 4 5 1/S 250 - 200 - 150- 100 - y = 40.826x-4-48.181 r = 0.992 Kg,= l.I8mM ^max — 0-0- mM/min -2 1 0 1 2 3 4 6 Fig. 15: Effect of L-giilonolatone (S) concentration on sturgeon GLO velocity (V) at 25°C. In vitro rate of AA synthesis as catalyzed by GLO purified from white sturgeon posterior kidney. (A) Lineweaver-B urk double reciprocal plot. (B) S/V against S plot. Data represent means for 2 replicate measures. Standard deviations lie within the symbols. 63 REFERENCES Bânhegyi, G., Csala, M„ Braun, L., Garzô, T. and Mandl, J., 1996. Ascorbate synthesis- dependent glutathione consumption in mouse liver. FEBS Lett., 381: 39-41. Bimey, E.G., Jenness, R. and Ayaz, K.M., 1976. Inability of bats to synthesise L- ascorbic acid. Nature, 260:626-628. 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VITAMIN C-YTTAMIN E INTERACTION IN JUVENILE LAKE STURGEON (ACIPENSER FULVESCENS R.), A FISH ABLE TO SYNTHESIZE ASCORBIC ACID INTRODUCTION Free radical damage constitutes a constant threat to aerobic organisms which are continuously exposed to oxygen toxicity. Aerobic organisms are protected from oxygen toxicity by an array of defense mechanisms; 1) enzyme systems preventing oxidants formation, such as superoxide dismutase, catalase and glutathione peroxidase, that reduce the generation of free radicals, and 2) chain-breaking antioxidants that scavenge radicals in order to terminate free radical chain reactions, such as vitamin C, vitamin E, uric acid, cysteine, and reduced glutathione. Ascorbic acid (vitamin C) and a-tocopherol (vitamin E) are among the major chain- breaking antioxidants. Ascorbic acid operates in the aqueous environment, whereas tocopherols scavenge reactive oxygen species (ROS) within the membranes. Since vitamin E may be the most abundant protective agent present within the membranes, vitamin E may be the last line of defense against membrane phospholipid peroxidation. To be an effective antioxidant and prevent the formation of malondialdehyde (MDA), each oxidized tocopherol (vitamin E radical) must be recycled, i.e. reduced back to a-tocopherol (Fig 1). 68 But at present, enzymes capable of^reducing oxidized tocopherol have not been described. The demonstration that vitamin C may interact with vitamin E radical in vitro, i.e. regenerating the antioxidant form of a-tocopherol at the water-membrane interface (Niki 1987), suggests that vitamin C and vitamin E interact in vivo to protect membrane components susceptible to free radical damage. Vitamin C is a likely candidate because oxidized monodehydroascorbate (vitamin C radical) is efficiently reduced back to ascorbic acid by enzymatic (DHAA reductase) and non-enzymatic systems. Additionally, ascorbic acid can be endogenously synthesized by gulonolactone oxidase (GLO) from L- gulonolactone, a molecule derived from D-glucose. We previously showed that Siberian, white and lake sturgeon have GLO activity located in the kidney (Dabrowski 1994; Moreau et al. 1996; Moreau and Dabrowski 1996a), therefore sturgeon can synthesize ascorbic acid de novo unlike most other living fishes. Thus, sturgeon represent a unique model to study the relationships between endogenously-produced ascorbic acid and vitamin E-mediated protection against hpid peroxidation. The objective of the study was to investigate the effects of the presence or absence of vitamin E in the diet concomitantly with no vitamin C or high vitamin C dietary level on growth, survival, phospholipid peroxidation, muscle and liver fatty acids compositions, a-tocopherol and ascorbic acid tissue concentrations, ascorbic acid biosynthesis, and oxidized ascorbate regeneration in juvenile lake sturgeon. MATERIALS AND METHODS Feeding trial Ten-month old lake sturgeon, Winnebago Lake system strain (Wisconsin, USA), were used. Prior to the feeding trial, sturgeons were fed a commercial diet. A week before the initiation of the experiment, sturgeon were offered a mixture of commercial diet and our 69 experimental casein-based diet devoid of vitamins C and E. Lake sturgeon were then divided into 3 weight classes, and sorted into a complete block design comprising 3 blocks of 4 tanks each at a density of 7 to 10 fish per tank. Large, medium and small fish weighed on average 68 , 34 and 20 g at the beginning of a feeding trial. AU procedures and handling of animals were conducted with care in compliance with the guidelines of the Institutional Laboratory Animal Care and Use Committee, The Ohio State University. We used a semi-purified casein-based diet devoid of vitamins C and E (basal diet OC-OE, Table 12), formulated according to previous work (Moreau and Dabrowski 1996b). The basal diet proved to be scorbutogenic to channel catfish (Dabrowski et al. 1996), and contained less than 1 mg a-tocopherol (a-T)/kg. Lipid sources were a-T- stripped com oü, a-T-stripped lard, and a n-3 series ethyl ester fatty acid concentrate containing 39.2% EPA (eicosapentaenoic acid, 20:5n-3) and 26.4% DHA (docosahexaenoic acid, 22:6n-3) courtesy of Dr. P.A. Fair (National Marine Fisheries Service, Charleston, SC). The fatty acid composition of the basal diet (OC-OE) is shown in Table 13. Vitamins C and E were added to the basal diet according to a 2 x 2 factorial design at the levels of 0 or 1250 mg/kg for vitamin C, and 0 or 200 mg/kg for vitamin E. Vitamin C source was in the form of ascorbyl- 2-monophosphate Mg (Phospitan C, Showa Denko America, Inc., NY). This form was chosen because it is stable (Dabrowski et al. 1994) and bioavaüable to fish (El Naggar and LoveU 1991; Matusiewicz and Dabrowski 1995; Saroglia et al. 1996). Vitamin E source was in the form of aU-rac-a-tocopheryl monophosphate Na (Showa Denko America, Inc., NY). The whole amount of feeds required for the feeding trial was processed at one time. Diets contained 30% water, and were sealed in plastic bags, and frozen at -15°C untü needed. Lake sturgeon were fed one of the four diets' in triplicate for 7 weeks. Weekly, feed rations were weighed. Fish were fed 7 days a week, 3 times a day. Feeding rates were adjusted to weight class at 0.8, 1.® and 1.5 g feeds (on dry weight basis) per 100 g fish wet weight for large, medium and 70 small fish, respectively. Water temperature was 17.0 ± 0.5°C, and dissolved oxygen was 7.7 ± 0.6 mg O 2/L throughout the experiment At the end of the feeding trial, fish which had showed the best weight gain were sampled, 24 hours after the last meal. Fish were stunned by a blow on the head and the spinal cord was sectioned. Liver and posterior kidney were removed. Tissues and carcasses were immediately frozen on dry ice, and stored at -80°C until analyzed. Ascorbic acid analysis Total ascorbic acid (TAA, i.e. combination of ascorbic acid and dehydroascorbic acid) and reduced ascorbic acid (AA) concentrations in liver and posterior kidney were determined using a HPLC-EC procedure. The HPLC system, set at 20°C, comprised two 15-cm X 4.6-mm, 5-(im particle size, Beckman Ultrasphere ODS columns in series, a LC- 4C amperometric detector (Bioanalytical Systems, Inc., West Lafayette, IN) and a cross- flow cell equipped with a glassy carbon electrode and an Ag/AgCl reference electrode. The applied potential was +0.4 volt (oxidative mode). The mobile phase consisted of 20 raM NaH2P0 4 , 0.06% (w/v) metaphosphoric acid, and 0.4% (v/v) acetonitrile (pH 3.0) delivered at a flow rate of 0.8 mL/min. Tissue samples were homogenized (10 mg tissue in 100 jiL) in cold 0.2% (w/v) metaphosphoric acid (MPA) containing 0.1 mM ethylenediaminetetra-acetic acid (EDTA) and 1 mM thiourea, pH 2.2, and centrifuged at 14,300 X g for 60 min at 4°C. The supernatants were kept at -80°C until analyzed. Tissue AA concentrations were determined by injecting subsamples diluted with homogenizing MPA-based solution, and filtered through 0.45-p.m nylon membrane prior to injection. Peak heights were compared to AA standards made in homogenizing MPA-based solution. TAA concentrations were measured after incubating 100 |iL of tissue extract with 100 |iL of 100 mM (3-mercaptoethanol, and 800 fiL of 50 mM phosphate buffer (pH 7.7-7. 8 ) 71 containing 0.1 mM EDTA and I raM thiourea for 20 rain at 20°C. The reduction reaction was stopped by adding 100 jiL of cold 10% MPA. Twenty-piL aliquots were injected into the HPLC system after filtration through 0.45-|ira nylon membrane. Gulonolactone oxidase activity (GLO, EC 1.1.3.8) GLO activity was measured in the posterior kidney (Moreau and Dabrowski 1996a) using the direct spectrophotometric assay (Dabrowski 1990). Samples were homogenized in cold 0.25 M sucrose (1 g tissue in 5 mL sucrose) at low speed for 2 periods of 10 sec using a spin homogenizer Omni 5000 (Omni International, Inc., Waterbury, CT). Homogenates were centrifuged at 15,000 x g for 15 min at 4°C. Pellets were discarded and supernatants were centrifuged at 100,000 x g for 60 min at 4°C. Pellets (or microsomes) were resuspended with 0.5 mL of 20 mM Tris-acetate (pH 8.0) containing 10 mM KCl, 1 mM EDTA and 0.2% (w/v) Na-deoxycholate using a glass rod and a spin homogenizer (Omni 5000). The suspensions were then centrifuged at 14,000 x g for 35 min at 4”C to solubilize the microsomes and release the enzyme into solution. The top fraction was assayed at 18°C for GLO activity as the microsomal fraction of the tissue preparation. GLO activity was expressed as p.g AA formed/mg microsomal protein • h. Dehydroascorbic acid reductase activity (EC 1.8.5.1) Livers were sliced and rinsed twice in cold 1.15% (w/v) KCl containing 10 mM EDTA (pH 7.4) to remove blood. One g of tissue was homogenized for 20 sec at a moderate speed using a spin homogenizer (Omni 5000) in 3 mL of cold 0.25 M sucrose, 0.05 M Tris-HCl (pH 7.5) containing 1 mM EDTA. The horaogenate was centrifuged at 7,300 X g for 10 mih at 4°C, then 14,300 x g for 20 min at 4°C. Supernatant (SI) was further centrifuged at 100,000 x g for 60 min at 4°C. Two-hundred-and-fifty-piL aliquots 72 of supernatant (S2) were dialyzed for 1 hour at 4°C against 0.05 M Tris-HCl (pH 7.5) containing 1 mM EDTA using an oscillating motion microdialyzer (OMS 101, National Labnet Company) and cellulose membranes of 12,000-molecular weight cut-off. The enzymatic formation of AA was measured based on the change of absorbance at 265 tun (Stahl et al. 1983). The reaction mixture contained 885 |iL of 50 mM phosphate buffer (pH 6.7) supplemented with 0.147 raM EDTA, 50 |LiL of 100 mM reduced glutathione, and 15 pL of dialyzed enzyme preparation. The reaction was initiated by adding 50 pT of 25 mM dehydroascorbic acid (DHAA) (Aldrich Chemical Company, Inc., Milwaukee, WI). A blank was carried out simultaneously without enzyme preparation in order to determine the non-enzymatic reduction of DHAA. The increase in absorbance at 265 nm was recorded at 18°C for 5 min. We used the molar extinction coefficient of AA at 265 nm, i.e. 14,700 L/(mol • cm), for calculation. The DHAA reductase activity was expressed as nmol of AA formed/ mg soluble protein in the dialyzed enzyme preparation • h. a-Tocopherol analysis Liver and basal diet (OC-OE) a-T concentrations were measured by HPLC according to previous work (Cort et al 1983, Zaspel and CsaUany 1983). The HPLC system comprised a Model 506 Beckman autosampler equipped with a 20-p.L injection loop, a 7.5-cm x 4.6-mm Phenomenex Ultremex 3 Silica column, a 3-cm x 4.6-mm Ultremex 3 Süica guard column, and a FL3000 fluorescence detector (Thermo Separation, San Jose, CA) with excitation wavelength set at 294 nm and emission wavelength at 326 nm. The mobile phase consisted of iso-octane/tetrahydrofuran (98.5:1.5), and was delivered at 2 mL/min. Frozen livers were pulverized in aluminum castings covered in liquid N2. Five mL of 1% ascorbic acid prepared in ethanol/water (92:8) and 2 mL of hexane were added to weighed samples, vortexed and centrifuged at 1,800 x g for 10 rain 73 at 15°C. The hexane layer was collected, and the sample was extracted a second time with 2 mL hexane, vortexed and centrifuged at 1,800 x g for 5 min at 15°C. Hexane extracts were combined and evaporated under a stream of high purity N 2. The residue was dissolved in iso-octane to within the range of calibration, and injected into the HPLC system. Chromatogram peak area was compared to standards made of d-a-T (United States Biochemical Corp., Cleveland, OH) to a range of 0.25 to 10 |xg/mL. Phospholipid peroxidation analysis Phospholipid peroxidation was assessed from liver microsomes by using the thiobarbituric acid (TEA) reaction for malondialdehyde (MDA). One mole of MDA generated during the peroxidation of unsaturated fatty acids will react with 2 moles of TEA in acid solution to form a trimethine colored substance with an absorption maximum at 532- 533 nm. Livers were sliced and rinsed twice in cold 1.15% (w/v) KCl containing 10 mM EDTA (pH 7.4) to remove blood. One g of tissue was homogenized for 20 sec at a moderate speed using a spin homogenizer (Omni 5000) in 3 mL of cold 0.25 M sucrose, 0.05 M Tris-HCl (pH 7.5) containing 1 mM EDTA. The homogenate was centrifuged at 7,300 X g for 10 min at 4°C, then 14,300 x g for 20 min at 4°C. Supernatant (81) was further centrifuged at 100,000 x g for 60 min at 4°C. Pellets (P2, or microsomes) were washed twice in 1.15% KCl containing 10 mM EDTA (pH 7.4), resuspended with 400 |iL of 50 mM phosphate buffer (pH 7.4), and frozen at -80°C until assayed by TEA reaction. Phospholipid peroxidation was promoted using a combination of ferrous sulfate and AA (Aitken et al. 1993). Two-himdred-and-sixty pL of microsome suspension were incubated with 8 |iL of 0.025 mM FeSO^. 7 H 2O and 8 |iL of 0.2 mM AA for 2 hoiurs at 37°C (Liu et al. 1997). Two-hundred pL of incubate were added to 360 p.L of TEA reaction mixture, and boiled for 30 min in a water bath. The TEA reaction mixture (pH 0.6) contained 200 74 |iL of 243 mM sodium dodecyl sulfate, 2 mL of 0.1 M HCl, 300 jiL of 10% (w/v) phosphotungstic acid, I mL of 46.5 mM TEA, and 100 |iL of 0.2 mM butylated hydroxytoluene. After boiling, the tubes were cooled down under tap water, and 500 |iL of n-butanol were added. The tubes were vortexed and centrifuged for 5 min at 3,000 x g to extract the TEA adducL Absorbance of TE A-reactive substances was read at 533 nm. MDA standards were prepared by overnight hydrolysis of 1,1,3,3-tetraraethoxypropane in 0.1 M HCl. Protein determination Protein content was measured by the Eradford procedure (Eradford 1976) using commercial Coomassie protein assay reagent (Pierce Chemical Company, Rockford, IL). Eovine serum albumin was used as the standard. Fatty acids analysis One-g samples of dorsal white muscle tissue were dissected out from sturgeon’s trunk behind the head. When two fish were pooled together, two 0.5-g samples from each individual were combined. Liver (0.2-1 g samples) and basal diet (OC-OE) were also analyzed for fatty acid composition. Total lipids were extracted from homogenized samples according to the procedure of Folch et al. (1957). Crude lipid extracts were separated into polar (phospholipids) and neutral (mostly triglycerides) fractions using Süica Sep-Pak cartridges (Waters, Division of MiUipore Corp., Bedford, MA). The residual oü was applied to silica füter, and neutral lipids were eluted with 30 mL of chloroform. After elution of neutral lipids, polar lipids were eluted with 30 mL of methanol. Solvents were evaporated under N 2, and amounts of polar and neutral lipids were determined gravimetrically. Fatty acid methyl esters mixtures were prepared from phospholipid and 75 triglyceride fractions according to the method described by Metcalfe and Schmitz (1961). Triglyceride fraction was saponified with NaOH in methanol, and fatty acid methyl esters were prepared by transmethylation with BF 3 in methanol. Phospholipid fraction was transmethylated with BF 3 in methanol directly. Fatty acid methyl esters were analyzed using a Varian 3300 gas chromatograph. The carrier gas was helium held at a pressure of 80 psi. The thermal gradient was performed as follows; 175°C for 26 min, increased to 205“C (2°C/min), and held at 205°C for 30 min. Fatty acid methyl esters were identified by comparing their retention times with those of fatty acid methyl ester standards (Nu-Chek- Prep, Inc., Elysian, MN). Fatty acids were quantified by comparing the areas of their peaks with the peak area of a known amount of internal standard (nonadecatone, C19:0). Unsaturation index (UI) was calculated as follows; UI = Z(Xa/YaXZa) where X is the number of double bonds in fatty acid (a), Y is the total number of carbon atoms in fatty acid (a), and Z is the proportion of fatty acid (a) in the total pool of fatty acids. Statistical analysis The effects of vitamin C and vitamin E dietary supplementation on ascorbate, a-T and TBA-reactive substances concentrations, and on GLO and DHAA reductase activities in selected tissues were tested by two-way ANOVA followed by Newman-Keuls’ multiple comparison procedure. Data were logarithmically transformed before ANOVA (log x). Correlations between GLO activities in the posterior kidney and TAA concentrations in the posterior kidney and the Hver were assessed by linear regression using the StatView (Brain Power, Inc., Calabasas, CA) package for the Macintosh. One degree of freedom was assigned to each tank. The accepted level of significance was 0.05. 76 RESULTS Growth and survival were not significantly affected by dietary treatments after 7 weeks of feeding. Depending on the initial body weight, lake sturgeon which were sampled for analyses grew at least by 27, 50 and 74% for large, medium and small weight classes, respectively. In the posterior kidney, TAA and reduced AA concentrations were significandy higher (P < 0.05) in fish fed vitamin C-supplemented diets (Fig 17A). Vitamin E supplementation lowered TAA concentration by 25% among the groups fed a diet devoid of vitamin C. GLO activity was stimulated by 63% in sturgeon fed vitamin C-supplemented diets when compared to sturgeon fed vitamin C-deprived diets (Fig 17B). In the liver, TAA and reduced AA concentrations increased significantly (P < 0.05) as a result of vitamin C supplementation (Fig 18A). When vitamin C was withdrawn from the diet, vitamin E supplementation significantly lowered (P < 0.05) reduced AA concentration by 30%. DHAA reductase activity increased by 44% in sturgeon fed diets supplemented with vitamin C when compared to sturgeon fed vitamin C-deprived diets (Fig 18B). When both vitamins C and E were withdrawn from the diet, tissue TAA concentrations amounted to 37 and 60 |ig/g in the liver and posterior kidney, respectively. GLO activity positively correlated with TAA concentrations in the posterior kidney (P = 0.01, r^ = 0.539) (Fig. 19). The positive correlation was not as strong between GLO activity and liver TAA concentrations (P = 0.025, ir = 0.444). In the hver, the highest a-T concentration (99.5 ± 22.9 pg/g) was found in the group supplemented with both vitamins (Fig 20A). Vitamin E supplementation increased a- T concentration by 46% in sturgeon fed the diet devoid of vitamin C compared to sturgeon fed the diet devoid of both vitamins. Dietary vitamin E supplementation increased significantly (P < 0.05) a-T concentration by 90% in the liver of fish fed the diets 77 supplemented with vitamin C. When vitamin E supplement was absent from the diet, vitamin C supplementation signiScandy lowered (P < 0.05) a-T concentration in the hver by 68 %. Concomitantly, TBA-reactive substances concentration increased by 39% (Fig 20B). When vitamin E was added to the diet, vitamin C supplementation increased the hver a-T concentration by 41%. Proportions of major polyunsaturated fatty acids in white dorsal muscle phosphohpids and triglycerides were not significantly (P > 0.05) affected by dietary treatments (Tables 14 and 15). Total saturated, total n- 6 , total n-3, n-3/n-6 ratio, and unsaturation index were not significantly (P > 0.05) different among dietary groups. In phosphohpids, EPA and DHA amounted to 5 and 15%, respectively. Fatty acid profiles of hver phosphohpids were not significantly (P > 0.05) affected by the dietary treatments (Table 16). DISCUSSION The results suggest that a-T status in the hver of juvenile lake sturgeon was markedly affected by dietary vitamins C and E. Feeding high vitamin C levels (10-25 times the vitamin C requirement for teleosts, NRC 1993) along with a vitamin E supplement contributed to an increased a-T concentration in the hver as compared to feeding no vitamin C. This supports the hypothesis that a-T is regenerated by AA in vivo (Igarashi et al. 1991; Hamre et al. 1997). In sturgeon fed the high vitamin C supplementation, endogenous a-T oxidation and in vitro phosphohpid peroxidation were promoted in the hver when vitamin E was withdrawn from the diet, suggesting that high doses of vitamin C may be prooxidanL The adverse effects of high vitamin C supplementation have been previously reported in the rat, a mammal able to synthesize ascorbic acid. In rats fed a high level of vitamin C (1500 mg AA/kg diet) and a minimum dietary level of vitamin E (50 mg a-T/kg diet in the form of aU-rac-a-tocopheryl acetate), red blood ceU hemolysis and hver hpid peroxidation were 78 significantly increased, whereas red blood cell level of reduced glutathione and plasma level of a-T were significantly lowered after 1-month feeding (Chen 1981). Elevated AA- induced lipid peroxidation of liver microsoraes was also observed in channel catfish fed diets lacking supplemental vitamin E (Gatlin et al. 1986), but the tissues concentrations of AA and a-T were not measured in this study. The adverse effects of high vitamin C supplement were overcome by increasing the vitamin E diet level. One reason may be that dietary vitamin C is absorbed in the form of AA or DHAA, the latter is then reduced to AA in the intestinal mucosa by reduced glutathione-dependent chemical and enzymatic mechanisms (Rose et al. 1988; Welch et al. 1995). Vitamin C absorption in the intestine may create an oxidative debt proportional to the quantity of DHAA absorbed. DHAA reductase is one of the enzymatic mechanisms which contributes to reduce DHAA to AA. Because DHAA reductase requires reduced glutathione as a co-factor the enzyme activity will depend in vivo on the size of reduced glutathione pool, which is one important component of the overall antioxidant capacity of the animal. Another reason may be that a high serum level of reduced AA induced the conversion of ferritin ferric iron to ferrous iron (Herbert et al. 1996), and released ferrous iron generated free radicals via Fenton reaction. The reason why GLO activity was stimulated by vitamin C supplementation is unclear. No vitamin C supplement should be preferred since sturgeon fed vitamin C-free diet grow normally. Indeed, sturgeon are able to maintain tissue ascorbate concentrations via de novo synthesis (Dabrowski 1994; Moreau et al. 1996). When both vitamins C and E were withdrawn from the diet, total ascorbate concentrations in the liver and posterior kidney of lake sturgeon (37 and 60 |ig/g, respectively) remained much higher than the concentrations reported in scurvy-prone fish deprived of vitamin C (<10 p.g/g) (Matusiewicz et al. 1995). In fish tissues, lipids are rich in n-3 polyunsaturated fatty acids, especially EPA and DHA. Since com oil and lard are low in n-3 polyunsaturated fatty acids, we supplemented the basal diet with a n-3 series fatty acid concentrate in order to meet EPA and DHA 79 requirements of the lake sturgeon. The analysis of dorsal muscle fatty acids in the lake sturgeon fed the experimental diets showed that EPA and DELA values were similar to those previously reported in the muscle tissue of the white sturgeon fed a control oil diet (Xu et al. 1996). Linoleic acid and arachidonic acid (two major n -6 series fatty acids) also were detected at levels consistent with those found in that study. Such similarities suggested that fatty acids requirements of the lake sturgeon were met. Neither vitamin C nor vitamin E dietary supplementation had any significant effect on the fatty acid compositions in the dorsal muscle tissue and the hver of the lake sturgeon under the experimental conditions. Yet, Stephan et al. (1995) reported that peroxidation of polyunsaturated fatty acids was almost complete in muscle tissue but not in the hver of 55-g turbot fed a diet containing 19 mg a-T/kg dry diet and 5% cod hver oil for 34 weeks. Feeding trial duration and fish species physiology may have contributed to the discrepancies between the two studies. Liver and posterior kidney ascorbate concentrations were correlated with dietary vitamin C irrespective of vitamin E level in the dieL An interpretation may be that ascorbate status was not affected by vitamin E in sturgeon fed a high dose of vitamin C. However, when vitamin C was withdrawn from the diet, high vitamin E supplementation (4 times the vitamin E requirement for teleosts, NRC 1993) lowered ascorbate concentrations in the tissues. This suggested a direct or indirect prooxidant effect of a-T on AA when tissue AA concentrations were low. Like any antioxidant, vitamins C and E are redox agents protecting against free radicals in some circumstances, but promoting free radicals generation in others (Herbert 1996). Pharmacological amounts of both antioxidant vitamins may have caused an oxidative stress to sturgeon Ulustrated by the increased phosphohpid peroxidation and the increased DHAA reductase activity. Excessive supplementation with either of vitamins C or E was hkely to produce unbalanced diets which adversely affected fish physiology. 80 Ingredient Concentration (g/kg dry diet) Casein (vitamin-free) ' 360 Dextrin (80% water-soluble) ' 150 Com starch ' 105 Fish protein concentrate 50 Egg white ' 50 Gelatin ^ 40 Com oil (tocopherol-stripped) ' 60 Lard (tocopherol-stripped) ' 50 Bulk n-3 ethyl ester fatty acid concentrate 5 Vitamin mixture^ 40 Bemhart Tomarelh salt mixture ^ + selenium ^ 30 Choline chloride (99%) ' I Carboxymethyl cellulose (sodium salt) ' 20 L-Methionine ^ 5 Cellulose (Alphacel) ' 34 ' ICN Biomedicals, Inc., Costa Mesa, CA. ' A.E. Staley MFG. CO., Decatur, XL. ^ CPSP 90, Sopropêche S.A., Boulogne-sur-mer, France. NTH/NOAA, National Marine Fisheries Service, Charleston, SC. ^ Roche Performance Premix composition per g of the vitamin mixture : vitamin A (retinyl acetate), 2645.5 lU; vitamin D,, 220.46 lU; vitamin Bj^, 13 |ig; riboflavin, 13.23 mg; niacin, 61.73 mg; d-pantothenic acid, 22.05 mg; menadione, 1.32 mg; folic acid, 1.76 mg; pyridoxine, 4.42 mg; thiamin, 7.95 mg; d- biotin, 0.31 mg, Hoffman-La Roche, Inc.. Nudey, NJ. ^ Five mg Se/kg salt mixture in the form of sodium selenite. Degussa Corporation, Chemicals Division, Ridgefield Park, NJ. Table 12: Composition of the basal diet 81 Fatty acid Percentage of Percentage total fatty acid in dry diet 14:0 4.33 0.426 16:0 15.10 1.485 I6:ln-9 1.40 0.138 I6:4n-3 0.23 0.023 18:0 6.91 0.680 I8:ln-9 31.11 3.059 18:2n-6 35.13 3.455 18:3n-3 CL80 0.079 18:4n-3 0.78 0.077 20:0 0.31 0.030 20:2n -6 0.27 0.026 20:3 n -6 0.09 0.009 20:4n-3 0.01 0.001 20:4n-6 0.39 0.038 20:5n-3 1.76 0.173 22:4n-6 0.01 0.001 22:5n-3 0.01 0.001 22:6n-3 1.34 0.131 Total saturated 26.65 2.621 Total monoenoic 32.51 3.197 Total n -6 35.89 3.529 Total n-3 4.93 0.485 100.00 9.833 Table 13: Fatty acid composition of the basal diet (OC-OE) 82 Fatty acid Diet OC-OE OC - 200E 1250C - OE 1250C - 200E Saturated 14:0 0.23 ± 0.04 0.23 ± 0.05 0.19 ±0.07 0-18 ±0.05 16:0 12.73 ± 0.88 14.82 ± 3.39 13.29 ±0.12 12.06 ± 1.92 18:0 5.45 ± 0.52 6.17 ± 1.40 5.87 ± 0.72 5.52 ± 0.93 20:0 0.34 ± 0.14 0.32 ±0.10 0.22 ± 0.06 0.33 ± 0.09 Monoenoic 16:ln-9 1.21 ±0.15 1.28 ± 0.36 1.05 ±0.15 0.95 ± 0.01 18:In-9 16.79 ± 1.50 18.88 ±3.90 17.03 ± 1.00 16.10 ±0.80 n -6 18:2n-6 7.36 ± 1.07 9.79 ± 2.27 8.84 ± 0.29 9.21 ± 1.24 20:2n -6 1.21 ± 0.37 1.45 ± 0.29 1.63 ± 0.36 1.41 ± 0.24 20:3n-6 1.15 ± 0.33 1.15 ±0.35 1.05 ± 0.08 1.09 ± 0.22 20:4n-6 3.78 ± 0.83 3.74 ± 1.09 3.47 ± 0.34 3.56 ± 0.06 22:4n-6 0.24 ± 0.05 0.23 ± 0.09 0.23 ± 0.01 0.21 ± 0.01 22:5n-6 0.48 ± 0.14 0.50 ±0.31 0.38 ± 0.07 0.56 ± 0.21 n-3 16:4n-3 0.40 ± 0.36 0.13 ±0.04 0.18 ±0.09 0.22 ± 0.03 18:3n-3 0.63 ± 0.09 0.60 ± 0.20 0.73 ± 0.28 0.53 ± 0.01 20:4n-3 0.22 ± 0.04 0.25 ± 0.10 0.20 ± 0.01 0.20 ± 0.01 20:5n-3 4.93 ± 0.46 5.69 ± 1.25 5.11 ± 0.67 4.31 ± 1.51 22:5n-3 1.21 ±0.08 1.36 ± 0.23 1.37 ±0.18 1.17 ± 0.44 22:6n-3 13.93 ± 1.37 15.91 ±2.73 15.89 ± 2.93 13.05 ±4.17 Total n -6 14.60 ± 2.33 17.16 ±4.18 15.79 ± 0.52 16.19 ± 1.62 Total n-3 21.32 ± 1.27 23.94 ±4.13 23.48 ± 3.98 19.48 ±6.14 n-3 / n -6 1.48 ±0.25 1.42 ± 0.22 1.48 ± 0.20 1.23 ± 0.5 UI 11.90 ±0.10 11.79 ±0.18 12.06 ± 0.57 11.64 ±0.75 Data are expressed as mg fatty acid/100 mg phospholipid. Values are means ± SD for 3 replicates except dietary treatment 1250C-0E for which data are shown for 2 replicates. Table 14: Phospholipid fatty acid composition of lake sturgeon dorsal muscle 83 Fatty acid Diet OC-OE OC - 200E 1250C - OE 1250C-200E Saturated 14:0 11.14 ±4.85 9.95 ± 5.79 6.55 ± 0.06 12.91 ±9.41 16:0 11.79 ± 1.72 10.73 ± 0.57 12.52 ± 0.64 11.57 ±2.27 18:0 1.20 ± 0.15 1.25 ±0.19 1.51 ±0.16 1.25 ±0.15 20:0 0.45 ±0.11 0.33 ± 0.05 0.41 ±0.10 0.36 ± 0.20 Monoenoic 16:ln-9 5.66 ± 0.88 4.99 ± 0.28 6.61 ± 0.46 5.60 ± 1.86 18:ln-9 18.98 ± 2.29 16.64 ± 1.04 20.90 ±0.61 17.85 ±2.71 20:ln-9 0.87 ± 0.15 0.81 ± 0.05 1.10 ± 0.16 0.92 ± 0.34 n -6 18:2n-6 10.27 ± 2.74 9.28 ± 0.74 11.10 ± 1.55 9.38 ± 4.50 20:2n -6 0.52 ± 0.17 0.50 ±0.10 0.67 ±0.11 0.44 ± 0.29 20:3n-6 0.42 ± 0.05 0.32 ± 0.07 0.53 ±0.13 0.33 ±0.12 20:4n-6 1.25 ± 0.04 0.83 ±0.12 1.31 ±0.29 1.09 ±0.27 22:4n-6 0.51 ± 0.10 0.35 ± 0.03 0.47 ±0.11 0.41 ±0.17 22:5n-6 0.38 ± 0.10 0.29 ±0.10 0.42 ±0.18 0.34 ±0.14 n-3 16:3 n-3 0.24 ± 0.07 0.19 ±0.05 0.26 ± 0.01 0.27 ±0.13 16:4n-3 0.72 ± 0.20 0.62 ± 0.05 0.89 ±0.11 0.76 ±0.31 18:3n-3 0.78 ± 0.24 0.59 ± 0.05 0.78 ± 0.06 0.67 ±0.21 18:4n-3 1.76 ± 0.28 1.29 ±0.11 1.75 ± 0.09 1.57 ±0.38 20:4n-3 0.85 ±0.18 0.71 ±0.04 1.08 ± 0.26 0.84 ± 0.41 20:5n-3 3.74 ± 0.63 3.36 ±0.21 4.68 ± 0.61 3.85 ± 1.71 22:5n-3 1.34 ± 0.16 1.17 ±0.01 1.76 ± 0.32 1.39 ±0.64 22:6n-3 4.59 ± 0.29 4.09 ± 0.28 5.65 ± 0.42 4.75 ± 1.76 Total n -6 13.34 ±2.92 11.56 ±0.95 14.49 ± 0.73 11.98 ±4.56 Total n-3 14.02 ± 0.95 12.02 ± 0.42 16.86 ± 1.86 14.11 ± 5.32 n-3 / n -6 1.09 ± 0.28 1.04 ±0.08 1.17 ± 0.19 1.39 ± 0.95 UI 8.45 ± 0.37 8.32 ± 0.85 9.30 ± 0.49 8.30 ± 1.60 Data are expressed as mg fatty acid/100 mg triglyceride Values are means ± SD for 3 replicates except dietary treatment 1250C-OE for which data are shown for 2 replicates. Table 15: Triglyceride fatty acid composition of lake sturgeon dorsal muscle 84 Fatty acid Diet OC-OE OC - 200E 1250C - OE 1250C - 200E Saturated 14:0 ND ND ND ND 16:0 10.80 ±0.52 10.49 ± 0.56 8.24 ± 1.34 9.81 ± 1.0 18:0 6.92 ± 0.67 7.71 ±0.99 5.97 ± 0.69 7.11 ± 1.15 20:0 0.10 ± 0.01 0.21 ±0.13 0.14 ± 0.10 0.11 ±0.04 Monoenoic 16:ln-9 0.41 ±0.05 0.66 ± 0.05 0.46 ± 0.09 0.48 ± 0.07 18:ln-9 9.72 ± 0.39 10.30 ± 1.65 8.26 ± 0.54 9.43 ± 1.22 n -6 18:2n-6 0.85 ± 0.01 1.08 ± 0.26 0.83 ± 0.16 1.11 ±0.39 20;2n -6 0.25 ± 0.03 0.29 ±0.10 0.24 ± 0.09 0.29 ± 0.07 20:3n-6 0.12 ± 0.01 0.15 ± 0.01 0.08 ± 0.02 0.14 ±0.06 2G:4n-6 3.26 ± 0.21 3.27 ± 0.48 2.34 ± 0.22 2.89 ± 0.35 22;4n-6 0.14 ±0.04 0.18 ±0.03 0.12 ±0.04 0.14 ±0.02 22:5n-6 0.17 ± 0.04 0.19 ±0.05 0.11 ± 0.03 0.13 ± 0 n-3 16:4n-3 0.22 ± 0.01 0.27 ± 0.04 0.20 ± 0.06 0.22 ± 0.01 18:3n-3 0.80 ± 0.08 0.93 ± 0.23 0.71 ±0.11 0.85 ± 0.08 20:4n-3 0.31 ± 0.03 0.37 ±0.14 0.25 ± 0.05 0.34 ± 0 20:5n-3 7.09 ± 0.34 7.33 ± 0.24 6.57 ± 0.35 7.36 ± 0.69 22:5n-3 1.16 ±0.25 1.35 ±0.22 0.79 ±0.18 1.15 ± 0.23 22:6n-3 16.31 ±0.12 17.48 ± 2.35 12.93 ± 2.13 15.19 ±0.57 Total n -6 4.91 ± 0.45 5.42 ± 0.99 3.91 ± 0.54 4.92 ± 0.88 Total n-3 25.88 ± 0.82 27.73 ± 3.23 21.43 ± 2.88 25.11 ± 1.12 n-3 / n -6 5.29 ± 0.32 5.15 ±0.35 5.49 ± 0.02 5.17 ±0.70 UI 13.92 ±0.16 14.01 ±0.04 13.88 ± 0.02 13.90 ± 0.43 Data are expressed as mg fatty acid/100 rag phospholipid. Values are raeans ± SD for 2 replicate tanks. ND = none detected. Table 16: Phospholipid fatty acid composition of lake sturgeon hver 85 MDA LOOM vîtO ^ gulonoiactone GLO I membrane cytosol 1 ' 1 microsomes Fig. 16: Pathways of vitamin E antioxidant action and its regeneration by vitamin C in the cell. MDA, malondialdehyde; DHAA, dehydroascorbic acid; GLO, gulonoiactone oxidase. 8 6 Posterior kidney 200 150 IW) BCQ 1 0 0 - 1O cn(U _ C3 T3 — X o 2 1------r OC-OE 0C-200E I250C-0E 1250C-200E Dietary treatment Fig. 17: (A) Total ascorbic acid (whole column) and ascorbic acid (black column) concentrations, and (B) gulonoiactone oxidase activity in posterior kidneys of juvenile lake sturgeon fed the four experimental diets. Two diets were devoid of vitamin C and supplemented with either 0 or 200 mg vitamin E/kg dry diet Two other diets were supplemented with 1250 mg vitamin C/kg dry diet and either 0 or 200 mg vitamin E/kg dry diet Data are shown as mean + or - SEM (n = 3 except dietary treatment 1250C-0E for which n = 2). Means not sharing a common letter are significantly different (P < 0.05). Vitamin C supplementation had a significant effect on ascorbate concentrations in the posterior kidney. Vitamin E supplementation and vitamin C-vitamin E interaction had no significant effect on ascorbate concentrations in the posterior kidney. Neither nutrient had any significant effect on GLO activity in the posterior kidney. 87 L iver OC-OE 0C-200E 1250C-0E 1250C-200E Dietary treatment Fig. 18: (A) Total ascorbic acid (whole column) and ascorbic acid (black column) concentrations, and (B) dehydroascorbic acid reductase activity in livers of lake sturgeon fed the four experimental diets. Two diets were devoid of vitamin C and supplemented with either 0 or 200 mg vitamin E/kg dry diet. Two other diets were supplemented with 1250 mg vitamin C/kg dry diet and either 0 or 200 mg vitamin E/kg dry dieL Data are shown as mean -i- or - SEM (n = 3 except dietary treatment 1250C-0E for which n = 2). Means not sharing a common letter are significantly different (P < 0.05). Vitamin C supplementation had a significant effect on ascorbate concentrations in the liver. Vitamin E supplementation and vitamin C-vitamin E interaction had no significant effect on ascorbate concentrations in the liver. Neither nutrient had any significant effect on DHAA reductase activity in the liver. 88 o Diet OC-OE □ Diet 1250C-0E # Diet 0C-200E ■ Diet I250C-200E 70 1 Posterior kidney "a 6 0 ” B 50 - a.2 ojo B 4 0 - < 30 - r: = 0.539 20 - < O 0 25 50 75 100 125 150 175 200 (D C o o Liver C3 "o G 0 1 o c 73 O r2 = 0.444 Î03 P = 0.025 o cu 20 40 60 80 100 Total ascorbic acid (jxg/g tissue) Fig. 19: Correlations between GLO activity in the posterior kidney and total ascorbic acid concentrations in the posterior kidney and the liver of juvenile lake sturgeon. Data are shown as means per tank. One degree of freedom was assigned to each tank. 89 Liver oû OC-OE 0C-200E 1250C-0E 1250C-200E Dietary treatment Fig. 20: (A) a-Tocopherol, and (B) thiobarbituric acid-reactive substances concentrations in livers of lake sturgeon fed the four experimental diets. Two diets were devoid of vitamin C and supplemented with either 0 or 200 mg vitamin E/kg dry diet Two other diets were supplemented with 1250 mg vitamin C/kg dry diet and either 0 or 200 mg vitamin E/kg dry dieL Data are shown as mean -t- SEM (n = 3 except dietary treatment 1250C-0E for which n = 2). Means not sharing a common letter are significantly different (P < 0.05). Vitamin E supplementation and vitamin C-vitamin E interaction had a significant effect on a- tocopherol concentrations in the liver. Neither nutrient had any significant effect on thiobarbituric acid-reactive substances concentrations in the liver. 90 REFERENCES Aitken, R.J., Harkiss, D. and Buckingham, D.W. 1993. Analysis of lipid peroxidation mechanisms in human spermatozoa. MoL Reprod. Develop. 35: 302-315. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72: 248. Chen, L.H. 1981. An increase in vitamin E requirement induced by high supplementation of vitamin C in rats. Am. J. Clin. Nutr. 34: 1036-1041. Cort, W.M., Vicente, T.S., Waysek, E.H. and Williams, B.D. 1983. Vitamin E content of feedstuffs determined by high performance hqitid chromatograpic fluorescence. J. Agric. Food Chem. 31: 1330-1333. Dabrowski, K. 1990. Gulonoiactone oxidase is missing in teleost fish - The direct spectrophotometric assay. Biol. Chem. Hoppe-Seyler 371: 207-214. Dabrowski, K. 1994. Primitive Actinopterigian fishes can synthesize ascorbic acid. Experientia 51: 745-748. Dabrowski, K., Matusiewicz, M. and Blom, J.H. 1994. Hydrolysis, absorption and bioavailabüity of ascorbic acid esters in fish. Aquaculture 124: 169-191. Dabrowski, K., Moreau, R. and El-Saidy, D. 1996. Ontogenetic sensitivity of channel catfish to ascorbic acid deficiency. J. Aqua. Animal Health 8 : 22-27. El Naggar, G O. and Lovell, R.T. 1991. L-Ascorbyl-2-monophosphate has equal antiscorbutic activity as L-ascorbic acid but L-ascorbyl-2-sulfate is inferior to L-ascorbic acid for channel catfish. J. Nutr. 121: 1622-1626. Folch, J., Lees, M. and Stanley, G.H.S. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509. Gatlin, D.M., Poe, W.E., Wilson, R.P., Ainsworth, A.J. and Bowser, P.R. 1986. Effects of stocking density and vitamin C status on vitamin E-adequate and vitamin E- deficient fingerling channel catfish. Aquaculture 56: 187-195. Hamre, K., Waagb 0, R., Berge, R.K. and Lie, 0. 1997. Vitamins C and E interact in juvenile Atlantic salmon {Salmo salar L.). Free Rad. Biol. Med. 22: 137-149. 91 Herbert, V. 1996. Introduction to the Symposium: Prooxidant effects of antioxidant vitamins. At the Experimental Biology ‘95 meeting, April 13, 1995, Atlanta, GA. J. Nutr. 126: 1197S-1200S. Herbert, V., Shaw, S. and Jayatilleke, E. 1996. Vitamin C-driven free radical generation from iron. In Symposium: Prooxidant effects of antioxidant vitamins. At the Experimental Biology ‘95 meeting, April 13, 1995, Atlanta, GA. J. Nutr. 126: 1213S- 1220S. Igarashi, O., Yonekawa, Y. and Fujiyama-Fujihara, Y. 1991. Synergistic action of vitamin E and vitamin C in vivo using a new mutant of Wistar-strain rats, ODS, unable to synthesize vitamin C. J. Nutr. Sci. Vitaminol. 37: 359-369. Liu, L, Ciereszko, A., Czesny, S. and Dabrowski, K. 1997. Dietary ascorbyl monophosphate depresses lipid peroxidation in rainbow trout spermatozoa. J. Aqua. Animal Health (in press). Matusiewicz, M. and Dabrowski, K. 1995. Characterization of ascorbyl esters hydrolysis in fish. Comp. Biochem. Physiol. HOB: 739-745. Matusiewicz, M., Dabrowski, K., VoUcer, L. and Matusiewicz, K. 1995. Ascorbate polyphosphate is a bioavaüable vitamin C source in juvenile rainbow trout: tissue saturation and compartLmentahzation model. J. Nutr. 125: 3055-3061. Metcalfe, L.D. and Schmitz, A.A. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33: 363-364. Moreau, R. and Dabrowski, K. 1996a. The primary localization of ascorbate and its synthesis in the kidneys of acipenserid (Chondrostei) and teleost (Teleostei) fishes. J. Comp. Physiol. B. 166: 178-183. Moreau, R. and Dabrowski, K. 1996b. Feeding stimulants in semipurified diets for juvenile lake sturgeon, Acipenserfidvescens Rafinesque. Aquacult. Res. 27: 953-957. Moreau, R., Kaushik, S.J. and Dabrowski, K. 1996. Ascorbic acid status as affected by dietary treatment in the Siberian sturgeon {Acipenser baeri Brandt): tissue concentration, mobilisation and L-gulonolactone oxidase activity. Fish Physiol. Biochem. 15: 431-438. National Research Council 1993. Nutrient requirements of fish. National Academy Press, Washington, D.C. 92 Niki, E. 1987. Interaction of ascorbate and a-tocopheroL Ann. N.Y. Acad. Sci. 498: 186- 199. Rose, R.C., Choi, J.-L. and Koch, M.J. 1988. Intestinal transport and metabolism of oxidized ascorbic acid (dehydroascorbic acid). Am. J. Physiol. 254: G824-G828. Sarogha, M., Terova-SarogUa, G., Papp Gy, Z. and Jeney, Z. 1996. Comparative biological avaüabilily of some commercial ester forms of ascorbate utilized in fish feed. Anim. Biol. 5: 99-103. Stahl, R.L., Liebes, L.F., Farber, C.M. and Silber, R. 1983. A spectrophotometric assay for dehydroascorbate reductase. Anal. Biochem. 131: 341-344. Stephan, G., Guillaume, J. and Lamour, F. 1995. Lipid peroxidation in turbot {Scophthalmus maximus) tissue: effect of dietary vitamin E and dietary n -6 or n-3 polyunsaturated fatty acids. Aquaculture 130: 251-268. Welch, R.W., Wang, Y., Crossman, A.Jr., Park, J.B., Kirk, K.L. and Levine M. 1995. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J. Biol. Chem. 270: 12584-12592. Zaspel B.J. and CsaUany, A.S. 1983. Determination of a-tocopherol in tissues and plasma by high-performance liquid chromatography. Anal. Biochem. 130: 145-150. Xu, R., Hung, S.S.O. and German, J.B. 1996. Effects of dietary lipids on the fatty acid composition of triglycerides and phospholipids in tissues of white sturgeon. Aquaculture Nutrition 2: 101-109. 93 CHAPTERS CONCLUSION Recent (since a decade ago) research work in fish biology has shed new Light on the ability to synthesize ascorbic acid (AA) in vertebrates, its origin and phylogeny (Appendix A). These new data have initiated this dissertation in which earlier and present states of knowledge have been presented. Since methodology has played a critical role in generating misleading data, the techniques frequently employed to measure L-gulonolactone oxidase (GLO) activity in fish have been reviewed (Appendix B). Research into the mechanism of AA biosynthesis in fish has been pursued in this dissertation and compared to the mammalian model. Further studies of the regulation of AA biosynthesis in fish wiU have implications in nutrition and aquaculture, possibly in determining the vitamin C requirement associated with a specific physiological condition. Distribution of GLO activity among vertebrates The first part of this dissertation focused on the distribution of AA biosynthesis capacity in vertebrates with an emphasis on extant fishes. At first look, vertebrates seem particularly unequal with respect to AA nourishment. While many vertebrates can synthesize the vitamin, some cannot and thus depend upon a dietary source. To date, no rule has been established that could explain such disparity. Some data published in fishes have added to the confusion by their controversial nature (see Table 17). 94 Twenty years ago, it was reported that some teleost fish may synthesize AA (Yamamoto et al. 1978; Dykhuizen et al. 1980). Before this work, scientists believed that all fish lack GLO activity, and that the capacity to synthesize AA (by acquisition of GLO) first appeared in amphibians and reptiles (Roy and Guha 1958; Chatteijee 1973). Although these early reports bore a significant importance, today some of these data appear suspicious and misleading from the evolutionary perspective. In their studies, Yamamoto et al. (1978) and Sato et al. (1978), and later, Soliman et al. (1985) and Thomas et al. (1985) used the DNPH methods described by Ikeda et al. (1963) or Terada et al. (1978) to measure GLO activity in teleosts, and claimed to have detected GLO in some teleosts including the common carp (Cypriniis carpio) and the goldfish (Carassius auratus). As judged by the lack of appropriate correction for interfering DNPH-derivatives the data reported in the forementioned studies were overestimated (see Appendix B). Since the levels of GLO activity found were among the lowest in fishes (< 1 |imol AA/ g tissue/ h. Table 18), their results are most likely analytical errors and, in fact, the teleosts do not have GLO activity. Repeated analyses of GLO in C. carpio in our laboratory using improved methodology, both enzymatic and immunologic, have revealed no detectable activity (Dabrowski 1990; 1991; Chapter 4 of this dissertation). On the contrary, substantial evidence indicates that, while teleosts cannot synthesize AA, aU non-teleost taxa analyzed thus far, including the lungfishes (Dykhitizen et al. 1980; Touhata et al. 1995), the sharks and rays (Touhata et al. 1995; Mæland and Waagb 0 1998), ± e sturgeons and paddlefishes (Dabrowski 1994; Moreau et al. 1996; Mæland and Waagb 0 1998), the bowfin, the gars, Polyptems (Chapter 4), and the lampreys (Touhata et al. 1995; Moreau and Dabrowski 1998) can produce AA de novo in the kidney. Fish research is in agreement with the view that kidney is the site of AA biosynthesis in poiküothermic vertebrates, such as amphibians and reptiles. The fact that teleosts, which account for -96% of extant fish species, cannot synthesize AA has overshadowed the remaiiting 4% for a long time and was responsible 95 for overstatement or belief that “fishes are incapable of synthesizing AA” (Chatteijee 1973b). As additional non-teleost species are tested for GLO activity, it becomes clear that a variety of fish taxa possess the ability to synthesize AA (Fig 21). The survey of modern- day fish is still incomplete, however, as representatives of hagfish, chimaera and coelacanth taxa are yet to be investigated for GLO activity. Based on our data confirming that modem teleosts have lost the ability to syn±esize AA, given the monophyly of teleosts, and invoking the principle of parsimony, we argue that the loss is the result of a single event that occurred in the ancestor of modem teleosts. Considering the bowfin {Amia calva) as being the living sistergroup to teleost, the time of divergence between bowfin and teleost should provide a good estimate of the date the trait was lost in teleosts. The genus Amia is known as early as the Upper Cretaceous (-140 million years ago) but the earhest known teleosts date back to the late Triassic (-210 million years ago) (Carroll 1988). However, other members of the Amiidae family, today extinct, are known from the Upper Jurassic (-200 million years ago). Therefore, as a first approximation, the loss of the AA synthesis pathway in teleosts may have occurred 200- 210 million years before present. The presence of GLO activity in the Austrahan lungfish (Neoceratodus forsten) and African lungfish {Protoptenis aethiopicus) bears a considerable importance in linking terrestrial and aquatic vertebrates with respect to the evolution of AA biosynthesis. There is no doubt that terrestrial vertebrates have evolved from lobe-finned fish (Sarcopterygii, represented today by the lungfishes and the coelacanth) early in the Devonian period (-400 million years ago). It can be argued, therefore, that AA biosynthesis is homologous among the vertebrates, aquatic and terrestrial, and that the origin of the trait dates back to the Cambrian (500-590 million years ago) when the ancestor of present-day lampreys lived. Achieving the purification of GLO from sturgeon kidney using a methodology originally 96 designed for chicken GLO implies that the fish and bird enzymes have structural similarities likely passed on by common ancestry, and supports the character homology. The inability to synthesize AA is rather the exception in vertebrates (Fig 22). Many higher vertebrates can synthesize the vitamin due to the presence of a functional GLO associated with the microsomal fraction in either the hver or the kidney or in both organs (Bimey et al. 1979; 1980). Among reptiles and amphibians analyzed to date, all have been shown to possess GLO activity in the kidney (Roy and Guha 1958; Chatteijee 1973). In birds, while sixteen of the twenty eight species of passerines examined were reported incapable of producing AA (Chaudhuri and Chatteijee 1969), twelve passerine species and all eleven non-passerines under study had GLO activity in the kidney and/or the liver. A recent phylogenetic reanalysis of Chaudhuri and Chatteijee (1969) data showed that the occurrence of the capacity to synthesize AA in passerines was unpredictable and enigmatic (del Rio 1997). In mammals, the primates, the guinea pigs (Bums 1957), and the bats (Bimey et al. 1976) cannot produce AA de novo. The enzyme deficiency was the result of missense mutations within the gene for GLO (Kawai et al. 1992; Nishikimi et al. 1992; 1994). The date of GLO losses was estimated at -20 and -70 million years before present in the ancestors of the guinea pigs and humans, respectively (Nishikimi et al. 1992; Nishikimi and Yagi 1991). It was demonstrated that GLO is the only enzyme missing in the AA synthetic pathway of scurvy-prone animals (Sato et al. 1976). Administering chicken GLO to the guinea pig induced the production of AA as revealed by elevated plasma levels (Sato and Grahn 1981). Is the rate of ascorbic acid synthesis in a fish adequate to meet the requirement? All animals have a nutritional requirement for vitamin C whether or not they can synthesize AA. Many fish species raised in aquaculture, such as the salmonids, ictalurids, 97 cichlids, and cyprinids, are teleosts and thus require a dietary source of vitamin C. Their vitamin C requirements vary with species, strain (Matusiewicz et al. 1994), age, environmental conditions (Wahli et al. 1995), induced stressors (Wise et al. 1988). All aforesaid factors could significantly affect GLO activity and/or expression. Studying the rate of AA production in a fish like sturgeon under controlled conditions should allow nutritionists to identify factors that affect AA requirements in teleosts as well. In order to meet this requirement, teleost species cultured for economic purposes typically are fed compounded diets supplemented with 50-100 mg AA/kg diet. Daily a salmonid should ingest 1 mg AA/kg body weight to meet its vitamin C requirement. The recent finding that some fish synthesize AA poses the question of whether they are able to produced enough vitamin C to meet their requirement. Among the fish species able to synthesize AA, a few are easily available for experimentation. Since several species of sturgeon have been domesticated and their progeny raised in tanks and fed commercial or semipurifed diets, sturgeon stand as the most available model today. The lengthy juvenile stage in sturgeon would allow us to distinguish between life stages prior to sexual maturity. Alternatively, lamprey could become a valuable model at the time of reproduction as we found that AA status among upstream migrants was consistent over two successive years. Using in vitro kinetics data (Chapters 3 & 6), we have applied to adult sea lamprey (Moreau and Dabrowski 1998) and juvenile lake sturgeon (Moreau et al. 1999b) the mathematical procedure used by others (Rucker et al. 1980) to quantify the rate of AA synthesis in mammals. The rate of AA synthesis per day in fish was calculated at I5°C based on GLO maximum velocity (V ^ ) measured at 25°C. The in vitro Michaehs constant (K^) of white sturgeon GLO for L- gulonolactone (0.92 mM, Moreau et al. 1999b) was used for calculation. Projections show that a 1-kg sea lamprey synthesizes 4 mg AA daily, and a 1-kg sturgeon, 3 mg at 15°C. These estimates compare favorably with the daily requirement of a 1-kg rainbow trout (1 98 mg AA). Therefore, it appears that a juvenile sturgeon produces adequate amounts of AA to meet its needs. A comparison with a mammal able to synthesize AA, such as the rat, reveals, however, that the rate of synthesis in fish is approximately 7 tunes slower than in the rat per unit of weight. Indeed, a 0.1-kg rat was shown to produce 2.6 mg AA per day (Bums et al. 1954). Another major difference between rodent and fish AA metabolism is that AA turnover is faster in these mammals than in fish as judged by half-lives of 3 days in the guinea pig (Bannay and Dimant 1962) versus 60 days in the rainbow trout (Blom and Dabrowski 1996). In short, rodent AA metabolism is characterized by fast rate of synthesis and short half-life (or fast tumover), whereas in sturgeon both the rate of synthesis and the tumover are slower. Overall the rate of AA synthesis in vertebrates may be correlated positively with the basal metabolic rate. How important is it for a fish to synthesize ascorbic acid Throughout the animal kingdom, natural diets of AA-synthesizing species do not differ dramatically from those of scurvy-prone species with respect to AA intake. Since vitamin C occurs in significant amounts (up to 3 g/lOOg in the Australian fruit Tenninalia femandiana. Brand et al. 1982) in food sources (animal organs, fruits, and vegetables except in grains and nuts), feral species are rarely exposed to long periods of AA deprivation if at all, and being incapable of synthesizing AA does not undermine fitness. However, in situations where access to food is restricted as a result of human activities, such as for domesticated animals, scurvy-prone species may suffer the consequences of vitamin C deficiency (impaired bone formation, retarded wound healing), whereas species synthesizing AA will not be affected. But such situations are exceptional, anthropogenicaUy driven, and most importantly unrelated to the evolutionary process that led to the loss of the function long before the origin of humans. We suggest that -500 million years ago, the 99 ancestor of present-day vertebrates was able to synthesize AA, and that the trait was lost secondarily on at least five separate occasions, in the teleosts, the passerine birds, the guinea pigs, the bats, and the primates. Despite the character loss, these vertebrate taxa have been and continue to be among the most successful to radiate and colonize the earth, as evidenced by the explosive spéciation in African cichlids. Is being able to synthesize ascorbic acid adaptive? The pathway for AA synthesis may likely have arisen in fish when adequate supphes were unavailable in the food (590-500 million years agao; Appendix A). But in the course of evolution AA may have become more available in the food chain or vertebrates may have modified their diets ti include food items rich in AA to a point where retaining the pathway did not benefit to its bearer in the sense of promoting fitness. As a consequence, secondary reversal could be functionally neutral (King and Jukes 1969). If having GLO activity has become superfluous, natural selection is no longer capable of correcting point mutations, and they wül tend to accumulate as in the guinea pigs (Nishikimi et al. 1992) and the humans (Nishikimi and Yagi 1991). Modem fishes may also provide a convincing illustration that retaining the pathway is a vestigial character. Indeed, while fish species that have changed httle since their origins have retained the AA synthesis pathway (the lampreys, the sharks, the sturgeons), recently evolved teleosts have lost the function. In spite of the loss, supposedly attributable to the inevitable degeneration by mutation of unneeded function, teleosts account for -96% of today’s fish biodiversity. Furthermore, in the sturgeon, a species that synthesize AA, the absence of regulation of the key enzyme GLO by dietary AA could demonstrate the funtional neutrality of the pathway being abandoned. 100 Is the loss of ascorbic acid biosynthesis adaptive or non-adaptive? The reasons why the teleosts have lost the ability to synthesize AA are unknown. So are the explanatory mechanism for its disappearance in some passerines and mammals. Was it the consequence of selection or genetic drift? It is expensive for an organism to maintain its genes in good working condition. Retaining the ability to synthesize AA while dietary AA is abundant for many generations does not seem most cost-effective. Adaptive change, wherein the new allele increases to fixation because the carrier of the new form, in the present case a variant unable to carry out the synthesis of AA, is more fit than the homozygote of the original form, can be inferred to have occurred at the molecular level. A mutation shutting down the pathway could be beneficial, thus selected for. Although instances of rat (Mizushima et al. 1984) and swine mutants unable to synthesize AA have been reported, no benefit has been associated with the reversal. On the contrary, homozygote osteogenic-disorder rat {odiod) were found to be sterile. Consequently, the adaptive hypothesis (Pauling 1970) for the loss of AA biosynthesis is not well documented, perhaps because fitness is so difficult to measure. Genetic drift, however, can be an important evolutionary force. Its effects within population are the random fixation and loss of alleles. Because genetic drift is a sampling process, its effects are most pronounced in small populations. Most species are thought to have originated with low population sizes. Although the population may subsequently grow in size and later consist of large number of individuals, like for the teleosts, the gene pool of subsequent generations derive from the genes present in the original founders. A mutation that occurred in the founding population of modem teleosts, passerines, bats, or primates may have become fixed and was passed on to the subsequent generations. Jukes and King (1975) suggested that the loss of AA biosynthesis in primates and guinea pigs was non-adaptive; in the feral state these vertebrates consume large quantities of AA. 101 However, even beneficial functions may be lost by genetic drift provided that the selective advantage and/or the population size becomes small. 102 Reference______Results______Chatteijee 1973 •No GLO activity in Teleostei: Anabas testudineus, Apocryptes lanceolams, Chanos chanos, Caüa catla, Cirrhina mrigala, Clarias batrachus, Heteropneustes fossilis, Labeo calbasu, L. rohita, Lates calcarifer, Megalops cyprinoides, Mugil persia, Mystus bleekeri, Notopterus notopterus, Ophicephalus punctatus, O. striatus, Tilapia mossambica. Wilson 1973 •No GLO activity in Teleostei: Ictalurus frucatus, I. punctatus. Yamamoto et al. 1978 •No GLO activity in Teleostei: Anguilla japonica, Oncorhynchus masou, Pagrus major, Plecoglossus altivelis, Salmo gairdneri, Seriola quinqueradiata, Stephanolepis cirrhifer, TiIapia nilotica. •GLO activity reported in Teleostei: Carassius carassius, Cyprinus carpio, Parasilurus asotus, Tribolodon hakonensis. Sato et al. 1978 •GLO activity reported in Teleostei: Cyprinus carpio. Dykhuizen et ai. 1980 •GLO activity reported in Dipnoi: Neoceratodus forsteri. Soliman et al. 1985 •No GLO activity in Teleostei: Ctenopharyngodon idellus, Oreochromis niloticus, O. macrochir, O. mossanbicus, Salmo gairdneri, S. trutta, Salvelinus fontinatis, Sarotherodon galilaeus, Tilapia buttikoferi, T. zillii. •GLO activity reproted in Teleostei: Cyprinus carpio, Oreochromis aureus, O. spirulus. Thomas et al. 1985 •GLO activity reported in Teleostei: Carassius auratus, Fundulus grandis, Mugil cephalus, Tilapia aurea. Dabrowski 1990 •No GLO activity in Teleostei: Cyprinus carpio, Leuciscus cephalus. Dabrowski 1991 •No GLO activity in Teleostei: Cyprinus carpio. Dabrowski 1994 •GLO activity reported in Chondrostei: Acipenser transmontanus, A. hilvescens, Polyodon spathula. Touhataetal. 1995 •No GLO activity in Teleostei: Lophiomus setigerus, Paralichthys olivaceus; Neoptervgii: Lepisosteus oculatus; Chondrostei: Polypterus senegalus. •GLO activity reported in Dipnoi: Protopterus aethiopicus; Elasmohranchii: Dasyatis akajei, Mustelus manazo; Agnatha: Lampetra japonica. Mishra and Mukhopadhyay •No GLO activity in Teleostei: Clarias batrachus 1996 Moreau et al. 1996 •GLO activity reported in Chondrostei: Acipenser baeri. Moreau and •No GLO activity in Teleostei: Ictalurus punctatus, Oncorhynchus mykiss. Dabrowski 1996 •GLO activity reported in Chondrostei: Acipenser transmontanus. Mukhopadhyay et al. 1997 •No GLO activity in Teleostei: Labeo rohita Young et al. 1997 •No GLO activity in Teleostei: Morone saxatilis 103 Maeland and Waagbd 1998 •No GLO activity in Teleostei: Clupea harengus, Anguilla anguilla, Salmo salar, Gadus morhua. Scomber scombrus, Hippoglossus hippoglossus, Scophthalmus maximus. •GLO activity reported in Chondrostei: Acipenser ruthenus; Elasmohranchii: Squalus acanthias. Moreau and •GLO activity reported in Neoptervgii: Amia calva; Dabrowski 1998 Agnatha: Petromyzon marinus. Moreau et al. 1999a •GLO activity reported in Chondrostei: Acipenser hilvescens. Moreau 1999 • GLO activity reported in Neoptervgii: Amia calva, Lepisosteus osseus; (Chapter 4) Chondrostei: Polypterus senagalus. •No GLO activity in Teleostei: C. carpio, C. auratus. ______ Table 17: Contribution to the distribution of gulonolactone oxidase (GLO) among extant fishes 104 Clade/Species GLO activity Method Correction for (fimol AA/ g tissue/ h) DNPH interference Teleostei Cyprinus carpio 0.92% 0.95% 1.01“ DNPH“ No qU DNPH% UV’ Yes 0“ ÜV’ n.a. Carassius carassius 0.75* DNPH^ No C. ataratus 0.77" DNPH^ No 0“ UV^ n.a. Tribolodon hakonensis 0.17“ DNPH^ No Parasilurus asotus 0.24“ DNPH'* No Oreochromis spirulus 1.0“ DNPH^ No 0. aureus 0.80“, 0.07" DNPH^^ No Mugil cephalus 0.03" DNPH^ No 0" DNPH% UV" Yes Fundulus grandis 0.11" D N Ptf No Amiiformes Amia calva 2.5-4.6“ UV^ n.a. Semionotiformes Lepisosteus osseus 3-10“ u v ’ n.a. Acipenseriformes Acipenser transmontanus 1.4% 5.6“ DNPH% UV’ Yes A.fulvescens 2.6'", 2.8“ DNPH% UV’ Yes A. baeri 0.39k DNPH“ Yes Polyodon spathula 1.6“ DNPH“ Yes Polvpteriformes Polypterus senegalus 2.3“ UV"^ n.a. 105 Dipnoi Neoceratodus forsteri 0.05= DNPH', DCIP^ 7 Protopterus aethiopicus 0.37' Dipyridyl^ n.a. Elasmohranchii Mustelus manazo 0.46' Dipyridyl" n.a. Dasyatis akajei 0.14' Dipyridyl" n.a. Petromyzontiformes Lampetra japonica 0 . 12' Dipyridyl" n.a. Petromyzon marinus 4.6-5.S' UV’ n.a. “Yamamoto et al. 1978; ‘’Sato et al. 1978; =Dykhuizen et al. 1980; “Soliman et al. 1985; “Thomas et al. 1985; 'Dabrowski 1990; ^Dabrowski 1991; ‘'Dabrowski 1994; ‘Touhata et al. 1995; ^Moreau and Dabrowski 1996; ‘“Moreau et al. 1996; ‘Moreau and Dabrowski 1998; “Moreau et al. 1999a; “Moreau 1999 (Chapter 4). 'Roe and Kuether 1943; "Sullivan and Clarke 1955; ^Chatterjee et al. 1958; ''Ikeda et al. 1963; ^Terada et al. 1978; “Dabrowski and Hinterleitner 1989; ’Dabrowski 1990. n.a.= not applicable. Table 18: Gulonolactone oxidase (GLO) acüties reported in fish and methods used to determine the production of ascorbic acid in vitro 106 Cambrian Ordovician Silurian Devonian Carboniferous to presen t Teleosts (-) Bowfin (+) Gars (+) Sturgeons, paddlefish (+) Polypterus (+) Lungfishes (+) / Coelacanth (?) Sharks, rays (+) < Chimaeras (?) Lampreys (+) Hagfishes (?) Vertebrata 590 500 440 410 360 0 Million years Fig. 21: Distribution of GLO activity among extant fishes through time (Adapted from Nelson 1994). The continuous lines represent the fossil records and the dotted lines represent the inferred phylogeny (Greenwood 1984; Gardiner 1984; Schultze and Wüey 1984; Wiley and Schultze 1984; Carroll 1988; Nelson 1994). 107 kidney Pn mates liver and kidney liver Bats absent T3 Guinea pigs s § 3 Marsupials Monotremes Non-passerines —i Reptiles Amphibians Lungfishes Teleosts Sturgeons Lampreys Fig. 22: Phylogeny and tissue location of gulonolactone oxidase (GLO) in vertebrates. The cladogram implies that the most recent common ancestor to all vertebrates expressed GLO in the kidney. In the course of vertebrate evolution, the histological site of expression has shifted to the liver as evidenced by an intermediate state where GLO is expressed in the kidney and liver concomitantly. Ultimately, GLO was lost at more than one occasion, i.e. in teleost fish, passerine birds, guinea pigs, bats, and primates. 108 APPENDIX A FISH ACQUIRED ASCORBIC ACID SYNTHESIS PRIOR TO TERRESTRIAL VERTEBRATE EMERGENCE 109 Free RadiaJ Biology Jt Medicioe. Vol. 25. So. t . pp. 989-09<). I99g Copyngftt O 199S EÙevier Scieace loc. Pnsted la (he USA. All rigftu reserved QS9N5l49^S/$*see frool maucr liUSEVlER Letter to the Editor FISH ACQUIRED ASCORBIC ACID SYNTHESIS PRIOR TO TERRESTRIAL VERTEBRATE EMERGENCE R eg is M o r e a u and K onr a d D abrow ski School of Maiunl Reiources, The Ohio'State Uoiveatty, Coltintbtis, OH 43210. USA (fteeehtd 3 March 1998; Revised 20 April 1991: Accepted 23 April 1998) To the Etliiors: 1973) on the expression of LGO in fishes. In short, these studies indicate that while several representatives of Te The repon by Nandi el al. {Free Radie. BioL Med. 22:1047; leosts cannot synthesize ascorbic acttl due to the lack of 1997) auempied lo compare aquatic and terrestrial animals LGO activity Dl. fishes retaining numerous ancestral char oil the basis of their enzymatic systems of defense against acters such as lampreys [4,5], sharks [4], rays [4], lungfishes oxygen toxicity, namely t-gulonolactone oxidase (LGO) [4,6], sturgeons [7-91. paddlefishes [7], and bowfin [5] and superoxide dismutase (SOD). Nandi et al. concluded possess LGO activity in the kidneys and thus are able to that ( 1 ) terrestrial vertebrates developed a higher enzymatic synthesize ascorbic acid. Terrestrial vertebrates evolved defense system against oxirlation than aquatic vertebrates from lobe-finned fish ÇSarcopterygiî) early in the Devonian (including LGO) in response to higher partial pressures of period (—400 million years ago) [10]. Once a numerous and oxygen in the atmo^here than in water, and (2) high SOD diverse group of fish, the lobe-firmed fish are today repre activity compensates for low or missing LGO activity in sented by only three genera of lungfish, and by a single higher vertebrates. However, using enzyme activity data to qiedes of coelacanth XLaximeria chalumnae). Although draw conclusions about the level of defense against oxygen these living lobe-finned fish differ in many ways from the toxidty for the whole animal is inappropriate. To quantita lobe-finned fish that gave rise to terrestrial vertebrates, the tively interpret the results of LGO and SOD activities, smdy of lungfishes and die coelacanth has shed valuable enzyme activity ought to be corrected for metabolic body light on the fish-amphibian (aquatic-terrestrial) transition. size [1], that is, the rate of oxidant generation through There is no debate that limgfishes or the coelacanth or both, intrinsic metabolic processes. Also, an inverse relationship are rtiore closely related to tetrapods than ray-finned fishes of LGO and SOD, as claimed by Nandi et al, assumes that (ficiinopurygii) [11]. The presence of LGO activity in the across the animal kingdom a strict proportionality exists kidney of the Australian lungfish Qfeoceraiadus /brsteri) between the two enzyme activities in terms of antioxidant [6] and African lungfish {Protopterus aethiopicus)[4] prob defense equivalent. Yet, there is no proof for that. On the ably demonstrates the path by which the ancestral fish LGO contrary, LGO activity also generates hydrogen peroxide gene was passed on to amphibians 400 million years ago. [2], which is eliminated at the expense of the animal's Recent reports of LGO activity in lamprey [4,5] suggest that antioxidant defense. In addition, it is irrelevant to infer the this trait appeared even earlier in the evolutionary history of evolutionary history of an en^me from the measurement of fishes, back in the Cambrian (590-500 million years ago). its activity at the present time Levels of enzyme activity Consequently, the claim that colonization of the terrestrial that are measured in vertebrates living today do not provide environment rich in oxygen by amphibians coincided with a clue as to what the levels of activity were in the Devonian the first appearance of ascorbic add biosynthesis capability period when early tetrapods emerged. Enzyme activity is a (by acquisition of LGO activity) is not valid. Nandi et al. type of physiological variable charaaerized by fast feed may want to reevaluate their hypothesis considering fish back regulation that is induced or inhibited in a matter of evolution. days or hours. Consequently, statements like “The LGO Régis Moreau and Konrad Dabrowski acuvity decreased with phylogenetic evolution" or “SOD School of Natural Resources was not induced in the early tetrapods” are not justihed. The Ohio State University Most importantly, the report by Nandi et al. does not Columbus. OH 43210, USA reflect the current state of knowledge; it ignores an existing body of literature (at least 14 peer-reviewed articles since PII 50891-5849(98)00114-2 110 Ltfticr lo chc Editor REFERENCES (61 Dykhuizen. D. &: Harrison. K. M.: Richardson. B. J. Evolution tU Rucker. R. 8.; Oubiclc. M. A.; Mouriueo. J. Hypothetical calcu ary implications of ascorbic acid production in the Australian lations of ascorbic odd synthesis based on estimates to vitro. lungfish. E xp en a id a 36:945-946; 1980. Am. J. CUn. fiu tr. 33:961-964; 1980. [71 Dabrowski. K. Primitive Actioopcerygiaa fishes are capable of [21 Banbe^yi. G.; Ctala, M.; Braun. L : Cano. T.; Mandl. J. Ascor- ascorbic add synthesis. Expericntia 50:745-748; 1994. (81 Moreau. R.; Dabrowski. K. The primary localizatioo of ascorbate bate syathesis-dependeat glutathione consumption :n mouse liver. and its synthesis in the Iddneys of adpeoserid (Chottdrostctj and FEBS U tt. 381J 9 -4 1 ; 1996. teleost C T eteosui) fishes. / . Comp. Pkysiol. B 166:178-183; [31 Dabrowski. fC. Gulonolactone oxidase is mlssiog In teleost fish— 1996. the direct spectrophoconMtric assay. BioL CAem. H oppeSeyUr (91 Moreau, R.; fCausfait^ S. J.; Dabrowski. iC Ascorbic add status as 371:207-214; 1990. affected by dietary treatment in the Siberian sturgeon (A cip a u cr [41 Touhata. K.:Toyohara, H.; Mitani, T.; Kinoshita, M.; Saiou. M.; baeri Brandt): tissue concentraiioo. mobilization and L-gulooo- Sakagucfai. M. Distribution o f i.'guloao-t.4-lactooe okidase lactone oxidase activity. Fish PhynoL Bioehan. 15:431-438; among fishes. FUherUs ScL 61:729—730; 1995. 1996. [5[ Moreau. R.; Dabrowski. K. Body pool and synthesis of ascorbic . (101 Kelson, J. S. FoAes o/tfcs WO/Ü 3rd cd. New York: John Wiley add to adult sea lamprey {JPelronvyzpn marvcur):an agaathao fish' & Sons; 1994. with gulonolactone oxidase activity, froc. Had. Acad. ScL USA [11] Meyer. A. Molecular evidence oo the origin of tetrapods and the 95:10279-10282. relationships of the coelacanth. TREE 10:111-116; 1995. I l l APPENDIX B MEASUREMENT OF L-GULONOLACTONE OXIDASE ACTTVTTY IN FISH TISSUES 112 MEASUREMENT OF GLO ACTTVTTY IN FISH TISSUES Tissue preparation In fish, GLO is found in the kidneys only, but other tissues, such as Liver, hepatopancreas, spleen, intestine and muscle, have been analyzed following the same procedure (Dabrowski 1990). GLO activity can be assayed in both crude extracts and microsomes prepared from fresh or deep-frozen tissues. In white sturgeon, GLO activity recovery was not significantly different in frozen kidney (- 80°C) as compared to fresh kidney (Moreau and Dabrowski 1996). If the analysis is to be made on a crude enzyme preparation, a portion of tissue (0.3-0.5 g) is homogenized using a glass hand or spin homogenizer in 2-3 mL of 50 mM phosphate buffer (pH 7.4), containing 1 mM EDTA and 0.2% sodium deoxycholate, and centrifuged at 15,000g for 15 min at 4°C. To prepare microsomes, a tissue sample is homogenized in cold 0.25 M sucrose (1 g tissue in 4-6 mL sucrose) and centrifuged at 10,000g for 15 rain at 4°C. The supernatant is spun at 100,000g for 60 min at 4°C to obtain the microsomal pellet. The pellet is resuspended in 50 mM phosphate buffer (pH 7.4) containing 0.2% sodium deoxycholate. Quantitative activity assays Optimal assay conditions have been previously reported for fish GLO (Dabrowski 1990). In short, an aliquot of enzyme preparation is incubated in 50 mM phosphate buffer 113 (pH 7.4) in the presence of 10 mM L-guIonclactone and 2.5 mM reduced glutathione. Sturgeon and sea lamprey GLOs were not significantly affected by assay temperature up to 28°C. Fish GLO velocity was slowed down at further elevated temperature. Routinely, fish GLO activity is assayed at 25°C. between 15 and 25°C was found to be 1.73 and 1.65 for sea lamprey and lake sturgeon, respectively (Moreau and Dabrowski 1998; Moreau et al. 1999a). GLO activity assays most commonly used with animal tissues rely on the methods listed in Table 19. These methods can be grouped in four categories; i) dinitrophenylhydrazine-colorimetric method, ü) 2,2' -dipyridyl-colorimetric method, hi) UV spectrophotometry, and iv) oxygen consumption. Methods 1, 2 and 3 measure the production of AA. Method 1 quantitates total AA (reduced + oxidized AA), whereas methods 2 and 3 measure reduced AA. Method 4 follows the disappearance of oxygen, one of GLO substrates. Methods 1 and 2 are stopped reactions and require subsamples to be taken at the beginning and at the end of the enzymatic reaction (20-30 min). The enzyme velocity has to be linear throughout the reaction time. GLO activity is expressed as net production of AA. Methods 3 and 4 continuously monitor the course of the reaction. An advantage of these methods over stopped reaction methods is that the operator becomes aware of abnormal enzyme behavior. A disadvantage of method 4 is that the activities of Oj-requiring enzymes (oxidases, oxygenases) and spontaneous oxidation account for additional O, consumption in vitro especially when assaying a cmde tissue extract In their studies, Yamamoto et al. (1978) and Sato et al. (1978), and later, Soliman et al. (1985) and Thomas et al. (1985) used the DNPH methods (Method 1) described by 114 Ikeda et al. (1963) or Terada et al. (1978) to measure GLO activity. As judged by the lack of appropriate correction for interfering DNPH-derivatives the data reported in the forementioned studies were overestimated (Fig. 23). Qualitative activity assays A histochemical method for in situ identification of GLO was developed by Cohen (1961) and modified by Nakajima et al. (1969). The method was employed on tissue slices (Cohen 1961; Nakajima et al. 1969; Soliman et al. 1985). The sequence of events includes the oxidation of L-gulonolactone with transfer of electrons to intermediates and a final electron acceptor, nitroblue tétrazolium, which once reduced forms dark blue formazan at site of enzyme activity. Stronger staining was obtained when GLO was activated with menadione (Cohen 1961), 2,4-dinitrophenyl, and phenazine methosulfate (Nakajima et al. 1969). However several problems arose. Cytosolic xanthine oxidase (Nandi and Chatteijee 1991) and respiratory chain oxidases also react with phenazine methosulfate-nitroblue tétrazolium. Cyanide was used to inhibit cytochrome oxidase. Interfering reduction of nitroblue tétrazolium was reported when D-glucuronate and L-xylulose are produced from L-gulonolactone by aldonolactonase. In this case EDTA which inhibits aldonolactonase activity had to be added. 115 End product analysis Reference DNPH-derivatives Roe and Kuether 1943; Ikeda et al. 1963; Ayaz et al. 1976; absorbance (524-540 run) Terada et al. 1978; Dabrowski and Hinterleitner 1989 2,2 ’ -dipyridy 1-derivatives Sullivan and Clarke 1955; Zannoni et al. 1974 absorbance (525 nm) UV absorbance (265 run) Dabrowski 1990 Oxygen consumption Kiuchi et al. 1982; Dabrowski 1990 Table 19: Gulonolactone oxidase (GLO) activity assays using L-gulonolactone as a substrate 116 0 .2 5 Without correction 0 .2 0 - 20 min c 0 .1 5 - jQ(0 0 min n 0. 10- 0.05 0.00 4004 4 0 480 520 560 600 Net Absorbance: A20 - AO > 0 Wavelength (nm) 0.25 With correction 0 . 2 0 - 20 min 0 .1 5 - 0 min n 0 . 1 0 - r ^ 0.00 4004 4 0 480 520 560 600 Net Absorbance: A20 - AO = 0 Wavelength (nm) Fig. 23: Measurement of GLO activity in teleosts using the DNPH method (524 nm) with or without appropriate correction for interering DNPH-derivatives. 117 LIST OF REFERENCES Agrawal, N.K. and Mahajan, C.L. 1980. Comparative tissue ascorbic acid studies in fishes. J. Fish Biol. 17: 135-141 Aitken, R.J., Harkiss, D. and Buckingham, D.W. 1993. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol. Reprod. Develop. 35: 302-315 Artyuklin, E.N. 1995. Contribution on bio geography and relationships within the genus Acipenser. Sturgeon Quaterly 3(2): 6-8 Ayaz, K.M., Jenness, R., Bimey, E.C. 1976. An improved assay for L-gulonolactone oxidase. Anal. Biochem. 72: 161-171 Bânhegyi, G., Csala, M., Braun, L., Garzd, T. and Mandl, J. 1996. Ascorbate synthesis- dependent glutathione consumption in mouse liver. FEBS Lett 381: 39-41 Bannay, M. and Dim ant, E. 1962. On the metabohsm of L-ascorbic acid in the scorbutic guinea pig. Biochim. Biophys. Acta 59: 313-319 Barannikova, LA., Vasil’yeva, Ye.V., Trenkler, I.V. and Tsepelovan, P.G. 1978. The interrenal gland in the life of diadromous sturgeons (family Acipenseridae). J. 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High ascorbic acid content in the rat endocrine pancreas. Diabetologia 34: 839-842 135 IMAGE EVALUATION TEST TARGET (QA-3) / / V f % 1.0 La m iââ 12.2 ■ 4.0 12.0 l.i 1.8 1.25 1.4 1.6 1 50mm y^PPLIED ^ IIVUGE . Inc — 1653 East Main Street ■~~ = 'r Rochester. NY 14609 USA Phone: 716/482-0300 - = ~ - = Fax: 716/288-5989 O 1993. Applied Image, Inc.. Ail Rights Resen/ed