Methionine Intake in Rainbow Trout

Nutrient Metabolism

Methionine Intake in Rainbow Trout (Oncorhynchus my kiss), Relationship to Cataract Formation and the Metabolism of Methionine1 ! COLIN B. COWEY2, C. YOUNG CHO, JACOB G. S/VAK,* JUDY A. WEERHEIM* AND DARRIN D. STÃœART* Department of Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada NIG 2W1 and *School of Optometry, university of Waterloo, Waterloo, Canada N2L 3G1

INDEXING KEY WORDS: â€¢methionine â€¢cataract â€¢fish â€¢transulfuration â€¢requirement

Recent studies indicate that the dietary re quirement for methionine in rainbow trout (On corhynchus mykiss] is in the range 0.85-1.0% of the diet (1-3). This requirement may be spared by cystine, the requirement for which is -0.3% with a diet mar ginally deficient in methionine. These values were obtained from dose-response curves in which the re sponse measured was weight gain. The main abnormality seen in trout deficient in methionine is bilateral cataracts (4); however, no at

1Supported by Ontario Ministry of Agriculture and Food, Natural Sciences and Engineering Research Council and Medical Research Council of Canada. To whom correspondence should be addressed. Abbreviations used: AdoMetDC, S-adenosylmethionine decar boxylase; CS, cystathione synthase; GSHred, glutathione reducÃ-ase; MAT, methionine adenosyltransferase; ODC, ornithine decarboxy lase.

0022-3166/92 $3.00 Â©1992 American Institute of Nutrition. Received 3 October 1991. Accepted 30 December 1991. 1154

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tempt has yet been made to accurately measure the relationship between methionine intake and the ap pearance of cataracts. The association between methi onine deficiency and the development of cataracts in trout is of general interest in that age-related, oxidative changes in protein-bound sulfur-containing amino acids from the mammalian lens have been known for some time. Oxidative damage is apparent from the occurrence of increased disulfide formation and increased oxidation of methionine to the sulfoxide in protein isolated from cell membranes of older, normal, mammalian lenses (5). Glutathione, which is present in high concentrations in the mam malian lens, may be a key element in the repair of oxidative damage. In the first place it may react with protein disulfides to form mixed disulfides; these may then be fully reduced by glutathione acting together with glutathione reducÃ-ase(GSHred, EC 1.6.4.2)3 and NADPH. Glutathione also protects against oxidative damage through the glutathione peroxidase system, which has been well characterized in several tissues. Methionine is the first limiting amino acid for rainbow trout in many food proteins,- consequently it is important to know how methionine catabolism in the fish is controlled, if at all. The predominant pathway of methionine catabolism in mammalian liver is by transmethylation and transulfuration (6). This provides a very effective regulatory system, al lowing conservation or breakdown of methionine de-

ABSTRACT Young rainbow trout were given diets con taining graded levels of methionine for 16 wk. Analysis of the weight gain and food efficiency data showed the methionine requirement to be not more than 0.76% of the diet (1.9% of dietary protein). Activities of regulatory enzymes of the transulfuration pathway, methionine adenosyltransferase and cystathionine synthase in trout liver were not altered by changes in methionine intake. Concentrations of free serine in liver and plasma of the trout were high at low levels of methionine intake but fell as dietary methionine increased. This implied decreased flux through cystathionine synthase at low methionine intakes. Large increases in liver and plasma taurine oc curred at high methionine intakes, implying enhanced transulfuration activity. Liverornithine decarboxylase ac tivity was reduced at the lowest level of dietary methi onine used but the activity of S-adenosylmethionine decarboxylase was unchanged. Eye lenses of the trout given these diets were examined by a scanning lens monitor. Analysis of focal length variability with this equipment demonstrated that, if abnormality of the lens is to be avoided, a higher concentration of dietary methi onine (0.96% or 0.6% methionine + 0.36% cystine) is needed than that required to maximize growth. J. Nutr. 122: 1154-1163, 1992.

METHIONINE

METABOLISM AND CATARACTS

pending on whether dietary intake is marginal or well in excess of requirement. A second, transaminative, pathway may operate in mammals when the diet contains large amounts of methionine (7). The indica tions are that methionine catabolism in fish is mainly via the former pathway (2). The objects of the present work were 1}to examine the relationship between methionine intake and the development of pathology in the eye and 2} to measure the activity of enzymes involved in methi onine catabolism and the effect on them of variation in methionine intake.

MATERIALS AND METHODS

could be performed. Livers and kidneys were also removed for assay of substrates and enzymes; for each intended assay tissues were removed from 12 fish per treatment (four from each of the three tanks used for each diet group), immediately frozen in liquid ni trogen, then held at -80Â°Cuntil analyzed, a period of not more than 6 wk. Diets. The basal diet is shown in Table 1. Herring meal, soybean meal and wheat middlings were ob-

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TABLE 1 Composition

of the basal diet

Ingredient

Amount g/kg

Herring meal (680 g crude protein/kg; 100 g fat/kg) Soybean meal (490 g crude protein/kgl Wheat middlings (170 g crude protein/kg) Gelatin Starch Agar (binder) Amino acid premix1 Vitamin premix2 Mineral premix^ Fish oil4

100 110 160 50 160 10 200 20 40 150

'Supplied (as L-racemer| the following (g/kg diet): arginine HC1, 8.53; isoleucine, 1.49; leucine, 3.64; lysine HC1, 8.09; phenylalanine, 4.71; threonine, 2.33; tryptophan, 0.26; valine, 2.12; glycine, 23.74; proline, 12.66; tyrosine, 7.91; alanine, 30.08; aspartic acid, 28.49; glutamic acid, 50.12; serine, 15.83. 2Supplied the following (mg/kg diet, except as noted): retinyl acetate, 1.6; cholecalciferol, 0.05; all-rac-a-tocopheryl acetate, 100; menadione sodium bisulphite, 30; ascorbic acid, 250; cyanocobalamin, 0.2; biotin, 0.3; choline dihydrogen citrate, 7 g; folie acid, 12; inositol, 300; niacin, 200; D-Ca-pantothenate, 25; pyridoxine HCl, 10; riboflavin, 5; thiamin HCl, 10. 3Supplied the following (mg/kg diet, except as noted); CaHPO4-2H2O, 15 & CaCO3, 1.5 & NaCl, 7.5 g; MgSO4, 5 & FeSO4-7H2O, 350; MnSO4-H2O, 300; ZnSO4-H2O, 250; CuSO4-5H2O, 80; CoCl2-6H2O, 12; Kl, 8; Na2SeO3, 1. 4De-aerated with nitrogen and stabilized with 0.05% ethoxyquin. For Experiment 1, diets were supplemented with L-methionine as follows (g/kg): Diet 1, 0; Diet 2, 2.0; Diet 3, 4.0; Diet 4, 5.5; Diet 5, 7.0; Diet 6, 11.0; Diet 7, 15.0; Diet 8, 2.0. Diet 8 was also supplemented with 2.0 g of L-cystine/kg. For Experiment 2, diets were supplemented with L-methionine as follows (g/kg): Diet 1, 0; Diet 2, 2.0; Diet 3, 0; Diet 4, 4.0; Diet 5, 0, Diet 6, 6.0. Diets 7 and 8 were supplemented with 4.0 and 6.0 g of DL-methionine, respec tively. Diets 3 and 5 were supplemented with 1.6 and 3.2 g Lcystine/kg, respectively.

tained from Martins Feed Mills (Elmira, Ontario), as was the fish oil, a high quality herring oil. Gelatin, agar and cornstarch were from U.S. Biochemical (Cleveland, OH) and the amino acids were from Biokyowa, Cape Girardeau, MO. The basal diet con tained 400 g crude protein/kg, supplied 22 g digestible protein/MJ digestible energy and met the nutrient (other than amino acids containing sulfur) require ments for salmonids as specified by National Re search Council (8). The commercial diet (MNR 98G), manufactured under contract by Martins Feed Mills for use at fish culture stations of the Ontario Ministry of Natural Resources, contained 410 g crude protein/kg and supplied 22 g digestible protein/MJ digestible energy. The main components were (g/kg): fish meal, 200; blood meal, 90; corn gluten meal, 170; soybean meal, 120; wheat middlings, 200; and herring oil, 130. Two experiments were conducted, the eight


Fish. Young rainbow trout of -2.5 g mean weight were obtained from Spring Valley Trout Farm (Peters burg, Ontario). They were acclimated to laboratory conditions for ~2 wk before being randomly dis tributed between 27 rectangular polyethylene tanks of 60-L capacity at 80 fish/tank. Three tanks were used for each of eight dietary treatments. The trout in the remaining three tanks were given a commercial diet. The tanks were part of a flow-through water system in which water was supplied as a mixture (50:50 blend) of dechlorinated City of Guelph supply and well water. The water temperature was thermostati cally maintained at 15 Â±1Â°Cand measured daily. Flow rate of water to each tank was ~2 L/min. Dis solved oxygen content and pH of the water were measured weekly. They were in the range 7.5-8.5 mg OX/Land 7.4-7.7, respectively, throughout the experi ment. A photoperiod of 12 h light, 12 h dark was used. The fish were fed to satiation three times daily, any dead fish were removed from the tank and re corded, and the fish were weighed collectively every 28 d. At the end of 16 wk the fish were recounted and weighed. All fish were cared for in accordance with the provisions of the University of Guelph Animal Care Committee. At the end of the experiment, blood samples were taken from the caudal vein of 12 fish from each treatment after anaesthetization; EDTA was used as anti-coagulant. Fish were killed by cervical section directly after blood sample taken. The red blood cells were removed by centrifuging (4000 x g for 10 min) and plasma from three fish per treatment was com bined; the resulting four samples per treatment were stored at -80Â°Cuntil analyses of free amino acids

IN TROUT

COWEY ET AL.

1156

e u u

Focal

Length

(mm)

FIGURE 1 Focal length profile for a single rainbow trout lens from a fish fed Diet 1 (0.40% methionine, 0.16% cystine). Abscissa indicates focal length (mm). Ordinate refers to laser beam distance (mm) from the optical center (0.0) of the lens. The plus signs (+) indicate the focal points for each incident beam away from the optic center. The array of lines coming to a poor focus represents the direc tions of all the refracted beams.

Methionine Deficient Diet

Length

(mm)

FIGURE 3 Focal length profile for a single rainbow trout lens from a fish fed Diet 2 (0.6% methionine, 0.16% cys tine). The plus signs (+) indicate the focal points for each incident beam away from the optic center. I

mounted on an X-Y table controlled by stepping mo tors. By appropriate positioning of the X-Y table and a specially designed lens cell, the laser beam, deflected upwards by mirrors, can be projected through a lens that is maintained in physiological saline (9 g NaCl/ L). The refracted beam is video recorded and digitized, and this information is used to compute focal length for each laser position. The spherical shape of the fish lens and the radial symmetry of its focal power (11, 12) obviate the need to orient the lens in a particular direction. The focal profile produced (Fig. 1-4) consists of a vertical line of plus signs that outline the focal lengths of peripheral to central lens zones. Straighter profile lines (e.g., Fig. 4) indicate lenses of better quality (i.e., minimal spherical aberration). Because the lens develops throughout life, with new growth at the periphery, the focal profile can be considered anal ogous to a cross-sectional growth ring profile of a tree. In healthy trout lenses there is relatively little focal length variation (Fig. 4, Fig. 5). The apparatus was first programmed to find the optical center of the lens in the lens container. This indicated that the lens was intact after the dissection procedure. Equivalent focal length was measured from the principal plane (intercept of incoming beam with exiting beam) to the intercept of the beam with

I OÃC 03

_1 i-I ID U

0.2

O

Diet FIGURE 2 Summary of focal length variability (SEM)for rainbow trout lenses from fish fed Diets 1 to 9, Experiment 1. Each data point represents eight or nine lenses. Diet 1 was significantly different (P < 0.05) from the other eight diets; Diet 2 was significantly different (P < 0.05) from Diet 9. Honestly significant difference of Tukey (P < 0.05) was 0.22.

3.0

4.0

Focal

Length

5.0

(mm)

FIGURE 4 Focal length profile for a single rainbow trout lens from a fish fed Diet 8 (0.6% methionine, 0.36% cys tine). The plus signs (+) indicate the focal points for each incident beam away from the optic center.


experimental dietary treatments in each formulated by substituting L-methionine, L-cystine or DL-methionine for glutamic acid in the basal diet as shown in Table 1. The concentrations of methionine and cyst(e)ine in the basal diet were measured after performic acid oxidation and hydrolysis (9). Analysis of lenses. At the end of Experiment 1, 10 fish from each treatment were killed by cervical section and the lens from each eye was separated from any attachment by gentle scraping with a blunt probe. Optical quality of the lenses was then assessed using a scanning lens monitor device (10). This system consists of a 2 mW helium-neon laser

Focal

METHIONINE METABOLISM AND CATARACTS IN TROUT

1157

assays were conducted at 15"C. They were shown to be linear over the time course used and proportional to the amount of supernatant used in the assay. The enzymes assayed were ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) (18), methionine adenosyltransferase (MAT, EC 2.5.1.6) (19), cystathione synthase (CS, EC 4.2.1.22) (20) and GSHred (21). Statistical methods. Data were analyzed by ANOVA and the statistical significance of differences between treatment groups was assessed at the 5% level of probability by calculating the honestly signif icant difference of Tukey (22).

RESULTS

the optic axis. Changes in this distance with beam eccentricity are influenced by the presence of coma and longitudinal spherical aberration, but spherical aberration is the dominant factor. The laser then scanned across the lens in 0.1-mm steps. The results are recorded as focal length (in millimeters) on either side of the optical center. Analytical methods. Free amino acids were mea sured in aqueous extracts of liver and in plasma by an HPLC method following deproteinization by mem brane filtration and pre-column derivitization (13). Liver polyamines were extracted and measured essen tially as described by Bedford et al. (14). Cysteine (15), glutathione (16) and cytosolic protein (17) in liver were measured by conventional methods. Enzyme

Growth data. The measured methionine and cystine contents of the basal diet (Table 1; Table 2, Diet 1) were 0.40% and 0.16%, respectively. These values agree well with those given by tables of food composition, and the contents of sulfur-containing amino acids in the remaining diets were taken to be this basal level together with the supplementary me thionine and/or cystine added to that diet. Table 2 shows the growth of fish in Experiment 1; other than for fish fed the basal diet there were no significant differences in final weight between treatments given experimental diets 1-8. We inferred that, in the presence of 0.16% cystine, the methionine re quirement of rainbow trout did not exceed 0.60% of the diet. Total requirement for amino acids con taining sulfur would not exceed 0.76% of the diet on


FIGURE 5 Trout lens in physiological saline, focusing an array of parallel laser beams.

TABLE 2 Growth and food efficiency of groups of rainbow trout fed diets containing different amounts of methionine for 16 wk (Experiment I)1 in

in

final

efficiency

weight2g38.99a46.59b47.72b48.43b47.24b46.10b45.80b47.59b80.08C0.984.88Food (gain/food)g/g0.65a0.74a0.74a0.74a0.71a0.67a0.67a0.75a0.9 eatenS4067a4556b4565b4540b4 diet'â€ž0.160.160.160.160.160.160.160.360.60Mean diet95%) was present in the reduced form. The physiological significance in the elevated activity of hepatic GSHred during methi onine deficiency is not clear. It presumably helps maintain cellular redox,- the Km value of the partially purified trout kidney enzyme for oxidized glutathione was found to be 0.067 mmol/L at 15Â°C,indicating

METHIONINE METABOLISM AND CATARACTS IN TROUT

32. Kim, K-I., Kayes, T. B. & Amundson, C. H. (1991) Require ments for sulfur-containing amino acids and utilization of Dmethionine by rainbow trout (Oncorhynchus mykiss). Aquaculture 101: 95-103. 33. Sivak, J. G. (1980) Accommodation in vertebrates: a contem porary survey. In: Current Topics in Eye Research (Davson, H. & Zadunaisky, J., eds.), vol. 3, pp. 281-330. Academic Press, New York, NY. 34. Cowey, C. B., Cooke, D. J., Matty, A. J. &. Adron, J. W. (1981) Effects of quantity and quality of dietary protein on certain enzyme activities in rainbow trout. J. Nutr. Ill: 336--34S. 35. Seiler, N. (1989) Acetylation and interconversion of the polyamines. In: The Physiology of Polyamines (Bachrach, U. & Heimer, Y. M., eds.), vol. 1, pp. 159-176. CRC Press, Boca Raton, FL. 36. Youngson, A., Cowey, C. B. & Walton, M. J. (1982) Some properties of serine pyruvate aminotransferase purified from liver of rainbow trout Salmo gaiidneri. Comp. Biochem. Physiol. 73B: 393^98. 37. Yokoyama, M. & Nakazoe, J-I. (1990) Induction of cysteine dioxygenase activity in rainbow trout liver by dietary sulfur amino acids. In: The Current Status of Fish Nutrition in Aquaculture (Takeda, M. & Watanabe, T., eds.), pp. 367-^372. Tokyo University of Fisheries, Tokyo, Japan.


25. Robinson, E. H., Wilson, R. P. & Poe, W. E. (1981 ) Arginine requirement and apparent absence of a lysine-arginine antag onism in fingerling channel catfish. J. Nutr. Ill: 46-51. 26. Walton, M. J., Cowey, C. B., Coloso, R. M. & Adron, ]. W. (1986) Dietary requirements of rainbow trout for tryptophan, lysine and arginine determined by growth and biochemical measurements. Fish. Biochem. Physiol. 2: 161-169. 27. Nose, T. (1971) Determination of nutritive value of food protein in fish. HI. Nutritive value of casein, whitefish meal and soybean meal in rainbow trout fingerlings. Bull. Fresh water Fish. Res. Lab. (Tokyo) 21: 85-98. 28. Rumsey, G. L. & Ketola, H. G. (1975) Amino acid supplemen tation of casein in diets of Atlantic salmon (Salmo salar] fry and of soybean meal for rainbow trout (Salmo gaizdnerÃ-)fingerlings. J. Fish. Res. Board Can. 32: 422-426. 29. McCallum, I. M. & Higgs, D. A. (1989) An assessment of processing effects on the nutritive value of marine protein sources for juvenile chinook salmon (Oncorhynchus tshawytscha}. Aquaculture 77: 181-200. 30. Andrews, J. W. & Page, J. W. (1974) Growth factors in the fish meal component of catfish diets. J. Nutr. 104: 1091-1096. 31. Adron, J. W. & Mackie, A. M. (1978) Studies on the chemical nature of feeding stimulants for rainbow trout, Salmo gairdnerÃ¬Richardson. J. Fish Biol. 12: 303^310.

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