chapter  15
30 Pages

In vitro methods and results of ascorbic acid absorption in epithelial tissues of fish

ByMichele Maffia, T. Verri, C. Storelli

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 15.2 AA and its derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 15.3 AA homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 15.4 AA absorption in fish intestine: in vitro methods . . . . . . . . . . . . . . . 216

15.4.1 Isolated intestinal segments . . . . . . . . . . . . . . . . . . . . . . . . . . 217 15.4.2 Intestinal rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 15.4.3 Schultz chambers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 15.4.4 Membrane vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218

15.5 AA absorption in fish intestine: transport processes . . . . . . . . . . . . 220 15.5.1 Na-dependent AA transport: kinetic characteristics . . . . 222 15.5.2 Na-dependent AA transport: electrogenicity . . . . . . . . . . 222 15.5.3 DHA facilitated transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 15.5.4 Hydrolysis and absorption of AA derivatives . . . . . . . . . . 225

15.6 AA reabsorption in teleost kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 15.7 Perspectives in the study of ascorbic AA in fish:

the cloning approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

15.7.1 Toward the molecular structure of plasma membrane transport system(s) . . . . . . . . . . . . . . . . . . . . . . . 228

15.7.2 Expression of epithelial plasma membrane Na-dependent AA transporters . . . . . . . . . . . . . . . . . . . . . . 229

15.7.3 Na-independent transporters involved in DHA absorption in animal cells . . . . . . . . . . . . . . . . . . . . . . 231

15.8 Perspectives in the study of AA absorption in fish: the transgenic approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 15.8.1 Methods for gene transfer in fish . . . . . . . . . . . . . . . . . . . . . 232 15.8.2 Transgenic expression of L-gulono--lactone

oxidase in the scurvy-prone medaka fish . . . . . . . . . . . . . . 233 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

Abstract L-ascorbic acid (AA) is an essential nutrient for several teleost species. It plays an important role in many enzymatic reactions to maintain prosthetic metal ions in their reduced form (for example, Fe2, Cu) and for scavenging free radicals to protect tissues from oxidative damage. In mammals, it has recently been shown that the facilitative sugar transporters of the GLUT type (GLUT1 and GLUT3) can transport the oxidized form of the vitamin dehydro-L-ascorbic acid. However, the bulk of the vitamin, which is present in the plasma essentially in its reduced form, is carried by the Na-dependent AA transporters SVCT1 (sodium-dependent vitamin C transporter 1) and SVCT2 (sodium-dependent vitamin C transporter 2), which have recently been functionally expressed in Xenopus oocytes, cloned and sequenced.78 SVCT1 is mainly confined to epithelial tissues such as intestine, kidney, and liver. In fish, many results, as obtained by different in vitro techniques, have detailed the presence of an electrogenic Na-coupled AA transport mechanism at the brush border membrane of enterocytes, with kinetic characteristics similar to those found in mammals (apparent Km ranging between 0.22 and 0.75 mM), when measured using the same experimental approaches. At the basolateral level of fish intestinal-absorbing epithelial cells, transport of dehydro-Lascorbic acid (DHA) is mediated by Na-independent transport pathway(s), presumably belonging to the GLUT type family as found in mammals, although this has not been demonstrated so far. We report data on both the kinetic characteristics of vitamin C transport through biological membranes of epithelial cells and the experimental approaches used over the time to study AA absorption in fish. The possibility of getting new theoretical information on ascorbic acid absorption and metabolism in fish by using the Xenopus laevis expression system and the perspective to develop new biotechnological applications in aquaculture by gene transfer are also pointed out.