J. exp. Biol. 189, 279–284 (1994) Printed in Great Britain © The Company of Biologists Limited 1994
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SHORT COMMUNICATION DETERMINATION OF PROTEIN SYNTHESIS IN RAINBOW TROUT, ONCORHYNCHUS MYKISS, USING A STABLE ISOTOPE C. G. CARTER1, S. F. OWEN1,2, Z.-Y. HE1, P. W. WATT2, C. SCRIMGEOUR2, D. F. HOULIHAN1 AND M. J. RENNIE2 1Department
of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, UK and 2Department of Anatomy and Physiology, University of Dundee, Dundee DD1 4HN, UK Accepted 7 January 1994 It has been suggested (Houlihan, 1991) that the consumption of 1 g of protein in a variety of species of fish stimulates the synthesis of, approximately, an equal amount of protein. Although synthesis of protein may account for as much as 40 % of the wholeanimal oxygen consumption (Lyndon et al. 1992), only about 30 % of the synthesized proteins are retained as growth (Houlihan et al. 1988; Carter et al. 1993a,b). Thus, one focus of attention is the potential advantage gained by fish in allocating a considerable proportion of assimilated energy to protein turnover in contrast to relatively low-cost, low-turnover protein growth (Houlihan et al. 1993). Rates of protein synthesis in several species of fish have been measured using radioactively labelled amino acids, frequently given as a flooding dose (reviewed by Fauconneau, 1985; Houlihan, 1991). These measurements cannot be made for longer than a few hours because of the decline in specific radioactivity in the amino acid free pool. However, as protein synthesis rates vary during the course of a day as a result of the post-prandial stimulation, and since radiolabelled amino acid methodology is invasive, short-term and terminal, it has been difficult to be certain of the relationship between protein growth measured in the long term and protein synthesis rates measured in the short term. This paper addresses these problems by developing a method using 15N in orally administered protein to measure protein synthesis rates in fish over relatively long periods, the aim being to use procedures that are as non-invasive and repeatable as possible. The use of stable isotopes to measure protein metabolism is well established in terrestrial mammals (see Rennie et al. 1991; Wolfe, 1992), but to our knowledge the only published data for aquatic ectotherms are on the blue mussel (Mytilus edulis L.) (Hawkins, 1985). In the present study, rates of protein synthesis of individual rainbow trout [Oncorhynchus mykiss (Walbaum)] were calculated from the enrichment of excreted ammonia with 15N over the 48 h following the feeding of a single meal (dose) containing protein uniformly labelled with 15N by use of an endpoint stochastic model (Waterlow et al. 1978; Wolfe, 1992). Application of this type of Key words: Oncorhynchus mykiss, rainbow trout, protein synthesis, nitrogen metabolism, stable isotope, mass spectrometry.
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modelling would appear to be ideal for measuring ammonotelic fish nitrogen metabolism since, unlike the situation in mammals, the catabolic flux of amino acids through urea is very small. Further, ammonia is excreted directly into the surrounding water via the gills and is not stored for any length of time, in contrast to the situation in mammals, so the rate of tracer appearance is easily measurable. Rainbow trout (College Mill Trout farm, Almondbank, Perthshire, UK) of approximately 100 g were held in groups in stock tanks before being used. Selected fish were placed individually in 20 l tanks of aerated running water (pH 6.4; 14 ˚C) in an environmentally controlled room (12 h:12 h light:dark regime). The fish were acclimated to these conditions for 21 days. There were two experiments: the fish in experiment 1 were fed (Aqualine Trout starter, North Eastern Farmers) each day; the fish in experiment 2 were not fed during the 21 day acclimation period. The labelled diet was made using yeast protein enriched with 15N (a generous gift from Dr K. Wutzke, University of Rostock). The normal food was ground and sieved (250 mm) to produce a fine powder to which labelled protein was added at a concentration of 5 (experiment 1) and 10 (experiment 2) mg g21 dry food and then thoroughly mixed and re-pelleted using a Gallenkamp pill press. The diet contained 7.4 % dry mass nitrogen and had a protein to energy ratio of 27.7 mg protein kJ21. The aim of experiment 1 was to determine rates of protein synthesis in normally feeding rainbow trout. Four trout (116.8±3.7 g: mean ± S.E.M. given throughout) were transferred to tanks as described above and fed with the trout diet once a day at a ration of 1 % body mass (% BM). Once the fish had consumed all offered food (initial 7 days), they were fed once a day for a further 14 days at 1 % BM day21. To measure protein synthesis, each fish was transferred, by net, to a second tank containing a known volume (about 16 l) of fresh tap water. The water was aerated throughout. The fish were then fed and ate individually weighed pellets of the labelled diet; the exact amount of food consumed by each fish was calculated (approximately 1 % BM). After 6 h each fish was transferred, by net, to a further identical tank of aerated water. To keep stress to a minimum, the transfer of fish was rapid, of the order of a few seconds. Water samples of 4 l were then taken and used for analysis of [14N]- and [15N]ammonia-nitrogen (see below). In order to measure the cumulative excretion of isotope, this procedure was repeated after 12, 24, 36 and 48 h. Two 50 ml samples were taken for the analysis of ammonia before feeding and at each transfer (Carter and Brafield, 1991). Ammonia was not lost from tanks without fish and spiked with ammonium chloride (see Wilkie and Wood, 1991). In experiment 2, the rate of protein synthesis was measured in seven rainbow trout (99.7±4.4 g) which were fed a known weight of labelled diet equal to 1 % BM day21 after 21 days without food. Three further rainbow trout (106.6±2.8 g) were used to calculate the relative amounts of ammonia- and urea-nitrogen excreted (Carter and Brafield, 1991). Isotopic enrichment was measured in samples taken from each tank before the fish were placed in the tanks and after 24 and 48 h. The collected water was used to concentrate the ammonia in preparation for analysis of the 15N enrichment. Four litres of sample water was transferred to a 5 l flask to which 50 ml of 10 mol l21 NaOH was added with anti-bump granules. The water was heated and ammonia was distilled into 100 ml of 1 mol l21 hydrochloric acid (experiment 1). The
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method was improved in experiment 2 in which 80 ml of 0.1 mol l21 EDTA in 10 mol l21 NaOH was added to the 4 l sample which was distilled into 10 ml of 1 mol l21 boric acid. The concentrated samples were then frozen (220 ˚C) and freeze-dried (Edwards Super Modulyo, UK). Replicate 20 mg borate samples were weighed into ‘Tin capsules’ (Europa Scientific Ltd, Crewe, UK) for enrichment analysis, which was carried out using an ANCA Roboprep-CN linked to a tracer mass isotope ratio mass spectrometer (Europa Scientific Ltd). The samples were combusted and the evolved nitrogen-containing compounds were then reduced to nitrogen gas. The 15N enrichment (APE; atom per cent excess) in the samples was estimated by running samples of known nitrogen enrichment alongside the experimental samples. The 15N enrichment of the labelled diets was 0.49 % (experiment 1) and 0.99 % (experiment 2). The 15N enrichment of ammonia was used to estimate whole-animal rates of protein synthesis by adopting the end-point stochastic model presented by Waterlow et al. (1978). A criterion for using the model is that the end product is formed from the same precursor pool used for protein synthesis. Ammonia and urea are the major nitrogenous excretory product of teleosts (Randall and Wright, 1987). In the present study, nitrogen attributable to urea was equivalent to 15.37±1.14 % of the ammonia-nitrogen. However, in rainbow trout, ammonia is the only quantitatively important end product of amino acid oxidation and, consequently, ammonia was used as the only end product in our calculations. Thus: Z = Q 2 ETN , where Z (mmol N) represents protein synthesis, Q (mmol N) represents the nitrogen flux and ETN (mmol N) represents the total nitrogen excretion (ammonia plus urea) over a period t and: e*/d* = Ex/Q = Ex/(Z + ETN) , where e* (mmol 15N) is the cumulative excretion of [15N]ammonia over time t after a dose d* (mmol 15N) of 15N and Ex (mmol N) is the total ammonia excreted over time t (Waterlow et al. 1978). Rates of synthesis calculated thus were 397±102 (N=4) and 236±10 (N=7) mg protein g21 day21 for experiments 1 and 2, respectively, and were not significantly different from each other (two-sample t-test: P=0.215). The decrease in the enrichment of ammonia was described by: APE = 0.558e20.05t (N=20; r2=0.83; F=96.1; P<0.0001), where APE is the [15N]ammonia enrichment (atom per cent excess) and t is the time after feeding (the time of maximum [15N]ammonia enrichment). Thus, the maximum [15N]ammonia enrichment was 0.558 % and the turnover time, the time taken for enrichment to decrease to less than 5 % of the initial value, was 60 h. The turnover time indicated the time taken for the free amino acid pool to be cleared of isotope (Hawkins, 1985). Hourly rates of synthesis calculated over 24, 36 and 48 h periods were 22.34±6.23, 18.31±4.91 and 16.53±4.26 mg protein g21 h21, respectively, and were not significantly different from each other (ANOVA: P=0.729). The decline in synthesis over the second day was to be expected since the fish were not re-fed after receiving the labelled meal. The relationship between the cumulative [15N]ammonia excretion and time after feeding in experiment 1 was estimated using a non-linear regression model (‘Regression’, Blackwell Scientific Software, UK) and described by ce*=
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Fig. 1. The mean (± S.E.M.) cumulative excretion of 15N as a percentage of the dose over 48 h after feeding a labelled meal to four rainbow trout in experiment 1.
26.2(1 2 e20.079t), where ce* is the cumulative excretion of [15N]ammonia (percentage of dose) t hours after receiving a single dose of [15N]protein (Fig. 1). The relationship between ce* and time suggested a slow rate of recycling of 15N once it had been incorporated into protein. Assuming that the nitrogen content of fish protein was 17.09 % (Gnaiger and Bitterlich, 1984) and the whole-animal protein contents of fed and fasted trout were 20 and 15 %, respectively (McCarthy, 1993), the fractional rates of protein synthesis (ks, grams of protein synthesised per gram fish protein expressed as a percentage per day) were 1.70±0.46 (N=4) and 1.59±0.06 (N=7) % day21 for experiments 1 and 2, respectively, and were not significantly different from each other (two-sample t-test: P=0.814). The fractional rate of synthesis measured over the first 24 h following the labelled meal was 2.30±0.67 % day21. Using the conventional flooding dose methodology for measuring protein synthesis with L-[2,6-3H]phenylalanine (Garlick et al. 1980), the relationship between fractional rates of protein consumption (kc, grams of protein consumed per gram of fish protein expressed as a percentage per day) and protein synthesis (ks) for feeding rainbow trout (40–100 g; 14 ˚C) was described by ks = 1.69 + 0.45kc (McCarthy, 1993). This relationship predicted a mean ks of 2.60 % day21 for the trout in experiment 1, which was similar to that calculated using the stable isotope method. Furthermore, wholeanimal fractional rates of protein synthesis in rainbow trout ranged between 2.3 % day21 (108 g; 10 ˚C) and 2.9 % day21 (118 g; 18 ˚C) when calculated from the specific radioactivity of the plasma free L-[U-14C]leucine (Fauconneau and Arnal, 1985). In the present study, the protein intake (kc) of the continuously fed trout (experiment 1) was 2.04±0.01 % day21 and indicated that 0.83 g of protein was synthesised per gram consumed. Thus, an equality between protein consumption and synthesis was approached, in agreement with previous studies on fish (e.g. Houlihan et al. 1988; Carter et al. 1993a,b).
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In conclusion, whole-animal rates of protein synthesis were calculated for juvenile rainbow trout by adopting the stochastic end-point model and measuring the [15N]ammonia collected after feeding [15N]protein. Rates of protein synthesis obtained over 48 h using a stable isotope or calculated over several hours using other methods for juvenile rainbow trout of approximately the same size were similar. The similarity between whole-animal rates of protein synthesis measured over 2 days and over a few hours, as by flooding dose methodology, further supports the validity of calculating rates of protein degradation as the difference between synthesis and growth (Millward et al. 1975) and, therefore, the estimates of protein turnover made for fish (e.g. Carter et al. 1993a,b; Houlihan et al. 1993). We are grateful to Dr I. D. McCarthy for allowing us to use his unpublished data. This work was supported by the Natural Environmental Research Council and the Agriculture and Food Research Council.
References CARTER, C. G. AND BRAFIELD, A. E. (1991). The bioenergetics of grass carp, Ctenopharyngodon idella (Val.): energy allocation at different planes of nutrition. J. Fish Biol. 39, 873–887. CARTER, C. G., HOULIHAN, D. F., BRECHIN, J. AND MCCARTHY, I. D. (1993a). The relationships between protein intake and protein accretion, synthesis and retention efficiency for individual grass carp, Ctenopharyngodon idella (Val.). Can. J. Zool. 71, 392–400. CARTER, C. G., HOULIHAN, D. F., BUCHANAN, B. AND MITCHELL, A. I. (1993b). Protein-nitrogen flux and protein growth efficiency of individual Atlantic salmon (Salmo salar L.). Fish Physiol. Biochem. 12, 305–315. FAUCONNEAU, B. (1985). Protein synthesis and protein deposition in fish. In Nutrition and Feeding in Fish (ed. C. B. Cowey, A. M. Mackie and J. G. Bell), pp. 17–45. London: Academic Press. FAUCONNEAU, B. AND ARNAL, M. (1985). In vivo protein synthesis in different tissues and the whole body of rainbow trout (Salmo gairdneri R.). Influence of environmental temperature. Comp. Biochem. Physiol. 82A, 179–187. GARLICK, P. J., MCNURLAN, M. A. AND PREEDY, V. R. (1980). A rapid and convenient technique for measuring the rate of protein synthesis in tissues by the injection of 3H phenylalanine. Biochem. J. 217, 507–516. GNAIGER, E. AND BITTERLICH, G. (1984). Proximate biochemical composition and caloric content calculated from elemental CHN analysis: a stoichiometric concept. Oecologia (Berlin) 62, 289–298. HAWKINS, A. J. S. (1985). Relationships between the synthesis and breakdown of protein, dietary absorption and turnovers of nitrogen and carbon in the blue mussel, Mytilus edulis L. Oecologia (Berlin) 66, 42–49. HOULIHAN, D. F. (1991). Protein turnover in ectotherms and its relationship to energetics. Adv. comp. env. Physiol. 7,1–43. HOULIHAN, D. F., HALL, S. J., GRAY, C. AND NOBLE, B. S. (1988). Growth rates and protein turnover in cod, Gadus morhua. Can. J. Fish. aquat. Sci. 45, 951–964. HOULIHAN, D. F., MATHERS, E. AND FOSTER, A. (1993). Biochemical correlates of growth rate in fish. In Fish Ecophysiology (ed. J. C. Rankin and F. B. Jensen), pp. 45–71. London: Chapman and Hall. LYNDON, A. R., HOULIHAN, D. F. AND HALL, S. J. (1992). The effect of short term fasting and a single meal on protein synthesis and oxygen consumption in cod. J. comp. Physiol. B 162, 209–215. MCCARTHY, I. D. (1993). Feeding behaviour and protein turnover in fish. PhD thesis, University of Aberdeen, 153pp. MILLWARD, D. J., GARLICK, P. J., STEWART, J. C., NNANYELUGO, D. O. AND WATERLOW, J. C. (1975). Skeletal-muscle growth and protein turnover. Biochem. J. 150, 235–243. RANDALL, D. J. AND WRIGHT, P. A. (1987). Ammonia distribution in fish. Fish. Physiol. Biochem. 3, 107–120.
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RENNIE, M. J., CHIEN, P., TAYLOR, D. J., WATT, P. W. AND BENNET, W. M. (1991). Applications of stable isotope tracers in studies of human metabolism. In New Techniques in Nutritional Research (ed. R. G. Whitehead and A. Prentice), pp. 3–15. London: Academic Press. WATERLOW, J. C., GARLICK, P. J. AND MILLWARD, D. J. (1978). Protein Turnover in Mammalian Tissues and in the Whole Body. Amsterdam: Elsevier/North Holland Biomedical Press. WILKIE, M. P. AND WOOD, C. M. (1991). Nitrogenous waste excretion, acid–base regulation and ionoregulation in rainbow trout (Oncorhynchus mykiss) exposed to extremely alkaline water. Physiol. Zool. 64, 1069–1086. WOLFE, R. R. (1992). Radioactive and Stable Isotope Tracers in Biomedicine: Principles and Practice of Kinetic Analysis. New York: Wiley-Liss Inc.
