Retinol and 3,4-dehydroretinol formation from xanthophylls in the goldfish, Carassius auratus

Abstract

Freshwater fish are unusual among animals in that they form 3,4-dehydroretinol (vitamin A,) as well as retinol (vitamin A,). Animals form retinol by reducing retinal, the product either of central cleavage of suitable carotenoids (provitamins A, e.g. /?,/?-carotene) or of a series of excentric oxidations which can ‘whittle down’ a provitamin A molecule to yield retinal. The reactions of carotenoid metabolism in animals are essentially oxidative. Thus dietary carotenoids are oxygenated by animals (carotenes can be converted into xanthophylls) or undergo oxidized cleavage (Davies, 1985). Contrary to this dogma, and over a number of years, there have been reports that highly oxidative carotenoids (e.g. astaxanthin; 3,3’-dihydroxy-/?,/?-carotene-4,4’-dione) can act as provitamins A in freshwater fish. Using vitamin A-depleted Gambusia holbrooki, a freshwater fish, Grangaud et al. (1956) showed that astaxanthin can be a precursor of both vitamins A, and A,. Gross & Budowski (1966) showed that a number of xanthophylls, including astaxanthin and canthaxanthin (B,/?-carotene4,4’-dione), can yield both A, and A, in two freshwater fish. Even a xanthophyll which is not a P-carotene derivative, lutein ([l,~-carotene-3,3’-diol), is converted into vitamin A, by the freshwater Saccobranchus fossilis (Barua et al., 1973); further studies implicated anhydrolutein (3,4-didehydro/?,~-caroten-3’-ol) as an intermediate (Barua & Das, 1975). The aim of the present study was to use biochemical radioactive-labelling techniques, rather than nutritional ones, to follow the metabolism of dietary carotenoids in the goldfish Carassius auratus. Radiolabelled /?-carotene and lutein were isolated and purified from etiolated seedlings which had ‘greened up’ in the presence of I4CO,. [14C]Zeaxanthin (j,P-carotene-3,3’diol) was prepared from [2J4 Clmevalonic acid as described by Britton et af . (1980). These biosynthetically prepared carotenoids were of comparatively low specific radioactivity ( 30pCi/mg). Goldfish, maintained individually in separate tanks, were trained to accept food pellets from forceps, thus ensuring that the intake of added carotenoid was quantitative. The carotenoid for each fish was added in dichloromethane to a 0.5 g cube of Petcraft freeze-dried ‘Magic Worms’ and the solvent evaporated. The cube was divided into 18 pellets and the fish was fed these, one per day, for 18 days. All fish were maintained on a non-labelled diet for a further 2 days and then killed using liquid nitrogen. The unsaponifiable lipid fractions from livers, eyes and intestines were analysed separately and for individual fish. Liquid chromatography was on neutral alumina (Brockmann grade 3); retinol and dehydroretinol, recognizable by