Some aspects of lipid transport in the rainbow trout.

In mammals, the bulk of the fatty acids is transported as triacylglycerol in the plasma chylomicrons and very-low-density lipoproteins. Uptake of the fatty acids by the extra-hepatic tissues is effected by the action of lipoprotein lipase (EC 3.1.1.34). We have shown that the rainbow trout (Salmo gairdneri Rich.) possesses a plasma lipoprotein system similar to that of mammals (Skinner & Rogie, 1978). In addition, lipoprotein lipase activity has been demonstrated in trout plasma after treatment of the fish with heparin (Skinner et al., 1979). It would therefore appear that ,the trout has a lipid-transport system that closely resembles that of mammals, although the existence of an alternative mechanism in which lipid is transported as non-esterified fatty acid in the plasma has been reported (Robinson & Mead, 1973; Kayama & Iijima, 1976). The nature of the fatty-acid carrier has not been determined. In this communication we report on the characterization of the chylomicron-like particles in the plasma of trout that have been maintained on a high-lipid diet, and also provide evidence that suggests that the high-density-lipoprotein fraction of trout plasma binds non-esterified fatty acids. Trout, fed on a high-lipid diet over a period of 2 weeks, were anaesthetized in fresh water containing 0.03% of ethyl maminobenzoate methanesulphonate (Sigma) and blood was withdrawn from the caudal vein by means of a syringe containing EDTA and trisodium citrate to give a final concentration in the blood of 0.01% EDTA and 15mg of citrate/ml. Chylomicrons were prepared by centrifuging trout plasma for 70 min at 10°C and 80000g,,~ in the type 5OTi rotor of the Beckman model L2 ultracentrifuge and collecting the upper turbid layer by tube-slicing. This fraction was mixed with NaBr solution of density 1.020g/ml and re-centrifuged under the same conditions. A portion of the chylomicron fraction was further washed by addition of 3vol. of 30% (w/v) sucrose. This solution (3 ml) was overlayered with 0.15 M-NaCI (2ml) and centrifuged for 45min at 10°C and 5OOOOg,,, in the SW65 Ti rotor of the Beckman model L2 ultracentrifuge. The upper fraction was removed by means of the Buchler Autodensiflow and the fraction washed by repeating this procedure three times. The chylomicrons were further fractionated on a continuous sucrose gradient (28-350/0, w/v) by a modification of the method of Pinter & Zilversmit (1962). The chylomicron fraction (2ml). adjusted to contain 60% (w/v) sucrose, was placed in a 5 ml centrifuge tube, overlayered with the sucrose gradient (3.21111) and centrifuged for 30min at 10°C and 7280g,,, in the SW65 Ti rotor. Three fractions (Iml) and a final bottom fraction (2ml) were collected from each tube by means of the Buchler Autodensiflow and dialysed against 0.15 M-NaCI before analysis. For the determination of the fatty-acid-binding properties of trout plasma, 13H]oleic acid (5pCi) was added to trout serum (3 ml). Lipoproteins were prepared and electrophoresis procedures were carried out as previously described (Skinner & Rogie, 1978). The trout very-low-density-lipoprotein fraction contained particles of 35-60nm diameter, which were electron-lucent, and, in addition, some particles of 300-800nm diameter were detected. It was found that a 50-fold increase in the number of particles of diameter greater than 300nm was detected by electron microscopy in the chylomicron fraction when the diet of the fish was changed from standard fish pellets (10% lipids) to a diet containing 20% lipid. These particles were spherical and electron-lucent, resembling mammalian chylomicrons. The distribution of chylomicron particle sizes between the different fractions was as follows: fraction 1 (top fraction), >400nm; fraction 2, 150-400nm; fraction 3, 100-150nm; fraction 4 (bottom fraction), 60-100nm. The largest proportion of particles appeared in fraction 4 with a diameter of approx. 100nm. Delipidation of the washed chylomicrom fraction with methanol/diethyl ether (Herbert et al., 1978) yielded an apoprotein that, when dissolved in 8 M-urea and characterized by polyacrylamide-gel electrophoresis, showed two bands, one near the origin and the other near to the buffet front. When [3H]oleic acid was mixed with trout serum, 90% of the radioactivity was recovered in the total lipoprotein fraction of which 86% was present in the high-density-lipoprotein fraction, both when the lipoproteins were separated by polyacrylamide-gel electrophoresis and by sequential flotation in the ultracentrifuge. It was also found that 88% of the radioactivity in the high-density-lipoprotein lipid was recovered as nonesterified fatty acid. When a portion of freshly drawn serum was treated with 5,5’dithiobis-2-nitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase (EC 2.3.1.43) activity, no significant change was found in the distribution of radioactivity within the lipid or lipoprotein classes. These observations suggest that under certain conditions, the trout has the ability to transport lipid by means of chylomicron-like particles in a manner similar to that of mammals and that the high-density lipoproteins appear to act as the carriers for the transport of fatty acids.