J. Sci. Food Agric. 1983, 34, 696-700 The Nutritional Role of 5'-Methyl-L-Cysteine Michael D. Eyrea, David E. Phillipsb, I. Marta Evansb and Alan Thompsona a Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, Newcastle upon Tyne NEI 7RU and b Department of Botany, University of Durham, Durham D H l 3 L E (Manuscript received 27 July 1981) Using the nitrogen balance technique, the effects of S-Methyl-L-cysteine on the pro- tein quality of egg albumin, extracted microbial protein and Maris Bead bean as well as in amino acid mixtures has been evaluated. No sparing effect was observed for methionine or cystine under any of the experimental conditions investigated. Equally, with a minor exception, no serious deleterious effect appears to result from the feeding of S-Methyl-L-cysteine. 1. Introduction S-Methyl-L-cysteine (SMLC) is a sulphur amino acid found in the non-protein fraction of legume seeds,l-3 which are generally deficient in both methionine and cystine. If SMLC exerts an anti- nutritional effect on these amino acids its effect could be critical on the protein utilisation of legumes. SMLC is readily oxidised in the living animal and in tissue culture,4* 5 one of the oxidation products being S-Methyl-L-cysteine sulphoxide (SMLCS). Crucifers, especially kale, contain SMLCS,6 which has been shown to be a cause of anaemia when fed to rurninants.7 SMLCS may have a prophylactic value against heart disease in humans8s9 and both SMLC and SMLCS have been isolated from human urine.lO, l1 The role of SMLC in nutrition is uncertain. Evans and Bandemer12 postulated that it could possibly act as an anti-metabolite for methionine or cystine in a similar way to other non-protein amino acids.16 No harmful effects were observed when SMLC was fed to rats12 and earlier work suggested that SMLC could not replace cystine in the diet of rats.14~ 15 SMLC has been used in toxicity studies where it was fed in excess16 and it appears that it can interfere in the metabolism of methionine when fed in large amounts.17~ l8 The substitution of various other sulphur-containing compounds for methionine and cystine has been investigated previously.19~ 20 In the present work the relationship of SMLC to rnethionine and cystine has been investigated using two protein sources deficient in sulphur amino acids and by using synthetic amino acid mixtures where methionine and cystine are replaced with SMLC, on an equimolar basis. 2. Materials and methods 2.1. Materials Maris Bead field beans (Viciafaba L. minor) were obtained from George Burlingham Ltd, Station Hill, Bury St. Edmunds, a microbial protein product (Pruteen) from ICI PIC, Wilton, Cleveland and amino acids from Sigma London Chemical Company Ltd, Poole Dorset. 2.2. Preparation of samples for rat feeding trial The Maris Bead beans were boiled for 1 h, freeze dried and then ground in a hammer mill to pass through a 1 mm sieve. The microbial protein product was in a powdered form which would be immediately used for feeding. 696 Nutritional role of S-methyl-L-cysteine 697 Table 1. Composition of the 19 experimental diets (g kg-1 of diet as fed) ~ Diets A B C D E F G H I J K L M N - S Component Maize starch Glucose Maize oil Cellulose Vitamin mixturea Mineral mixture0 Egg albumin Maris Bead bean Microbial protein L-Methionine L-Cystine S- Methyl-L-cys teine Amino acid mixture 1-6 610 540 535 160 160 160 100 100 100 80 80 80 20 20 20 20 20 20 10 80 80 420 160 100 80 20 20 200 - 417 417 417 417 160 160 160 160 100 100 100 100 80 80 80 80 20 20 20 20 20 20 20 20 200 200 200 200 2.99 - 2.22 - - 2.41 0.63 - - - - _ - _ _ _ - 2.71 - - - _ - - 516 160 100 80 20 20 - - 104 - 514 160 100 80 20 20 - - 104 2.34 514 514 160 160 100 100 80 80 20 20 20 20 - _ - _ 104 104 - 0.73 1.88 1.29 514 160 100 80 20 20 - - 104 - - 2.11 - 540 160 100 80 20 20 - - 80 Analysis Crude protein (N x 6.25) 9.4 71.7 71.9 72.7 76.1 75.7 78.1 74.6 85.8 86.2 87.3 89.1 84.9 a Contained (per kg mixture): choline chloride, 49.34 g; ascorbic acid, 29.61 ; menadione, 3.29 g ; p-amino- benzoic acid, 3.29 g; inositol, 3.29 g; niacin, 2.96 g ; retinol, (200 000 i.u. g-l) 2.96 g, calcium pantothenate, 1.97 g; a-tocopherol, 0.99 g ; riboflavin, 0.66 g; pyridoxine, 0.66 g; thiamin, 0.66 g ; cholecalciferol, (400 000 i.u. g-1) 0.16 g, cobalamin, 0.09 g, folic acid, 0.06 g; biotin, 0.01 g; maize starch, 900 g. Contained (per kg mixture): CaC03, 272.7 g, KHzp0.1, 318.2 g, NaCI, 230.9 g, MgS04.7Hz0, 92.7 g, FeS04.7Hz0,4.5 g ; CuS04.5Hz0, 1 . 4 g ; MnS04.4Hz0, 1.5 g; ZnS04.7Hz0, 0 .3 g, KI03, 0.005 g; maize starch, 77.7 g. Table 2. Composition of the amino acid mixtures (g kg-1) Amino acid mixture Component 1 2 3 4 5 6 L-Aspartic acid L-Threonine L-Serine L-Glutamic acid L-Proline Glycine L-Alanine L-Valine L-Methionine L-Isoleucine L-Leucine L-Tyrosine L-Phenylalanine L-Histidine L-Lysine.HCI L- Arginine L-Cystine L-Tryptophan S-Methyl-L-cysteine Maize starch 82.2 36.3 55.5 118.9 26.3 30.5 50.1 57.4 31.3 43.4 72.0 36.0 50.0 19.0 70.5 41.6 23.1 15.0 140.9 - 82.2 36.3 55.5 118.9 26.3 92.7 50.1 57.4 43.4 72.0 36.0 50.0 19.0 70.5 41.6 23.1 15.0 110.0 - - 82.2 36.3 55.5 118.9 26.3 67.8 50.1 57.4 31.3 43.4 72.0 36.0 50.0 19.0 70.5 41.6 15.0 126.7 - - 82.2 36.3 55.5 118.9 26.2 30.5 50.1 57.4 43.4 72.0 36.0 50.0 19.0 70.5 41.6 23.1 15.0 28.4 143.8 - 82.2 36.3 55.5 118.9 26.3 30.5 50.1 57.4 31.3 43.4 72.0 36.0 50.0 19.0 70.5 41.6 15.0 26.0 138.0 _ 82.2 36.3 55.5 118.9 26.3 30.5 50.1 57.4 43.4 72.0 36.0 50.0 19.0 70.5 41.6 15.0 54.2 141.1 - - Analysis Crude protein (N x 6.25) 65.1 in the diet, as fed 69.1 68.5 65.1 68.9 b3.U 698 M. D. Eyre ef al. 2.3. Rat feeding trial The calculation of true digestibility and biological value and the statistical test used were as in Phillips et ~ 1 . ~ 1 2.3.1. Diets The diets used are shown in Table 1 and the composition of the amino acid mixtures are given in Table 2. Diet A was a semi-synthetic diet containing 10 g kg-l egg albumin (BDH Ltd) for the purpose of calculating metabolic faecal nitrogen and endogenous urinary nitrogen. Diet B contained 80 g kg-l of egg albumin as a reference diet and Diet C was the same with a supplementation of SMLC to the level of methionine and cystine found in egg albumin, on an equimolar basis. In diet D Maris Bead bean was the sole protein source. In diets E, F and H Maris Bead bean was supplemented with methionine, cystine and SMLC respectively to the level of total sulphur amino acids in egg albumin. In diet G there was supplementation of the bean with methionine and cystine to the level of methionine and cystine in egg albumin. The pattern of the diets D-H were followed in diets I-M using the microbial protein product instead of the bean. In diet N the nitrogen was provided by an amino acid mixture based on an amino acid analysis of egg albumin. In amino acid mixtures 2 and 3, where the methionine and cystine respectively were omitted, the deficiency in nitrogen content was made up using glycine. In the amino acid mixtures 4, 5 and 6 SMLC replaced methionine, cystine, and methionine plus cystine, respectively, on an equimolar basis. 2.3.2. Animals One hundred and five Wistar male albino rats of approximately 100 g liveweight and approxi- mately 5-6 weeks of age were used. Five rats were allocated to each diet except in the case of diet A, where 15 animals were used. Table 3. The mean true digestibility (%) and biological value of the 17 experimental diets, together with the reference 80 g kg-1 albumin diet Diet Egg albumin (8 %) Egg albumin (8 %)+SMLC Maris Bead bean Maris Bead bean+methionine Maris Bead bean + cystine Maris Bead bean + methionine + cystine Maris Bead bean+ SMLC Microbial protein Microbial protein +methionine Microbial protein+ cystine Microbial protein +methionine + cystine Microbial protein+ SMLC Complete amino acid mixture (1) Complete amino acid mixture - Complete amino acid mixture-cystine (3) Complete amino acid mixture- Complete amino acid mixture- Complete amino acid mixture - methionine (2) methionine+SMLC (4) cystine + SMLC (5) (methioninef cystine) + SMLC (6) True digestibility (% k s.e.) ______. 98.14f0 .42b 100.39 20. 73ab 87.03k0.56" 86 .85+1.0Id 85.65+1.44d 87 .24f0 .31d 88.00+0.79" 92.24+0.43c 94.31+0.55c 93.75 0.49" 92.09 + 1.24" 101.28 f 0 . 87a 99.09f0.80ab 93.62 + O . 92c 99.22+0.65nb 98.56f1.28ab 99.10fO. 8 2 ' I b 99.50f1.1SUb Biological value (% k s . 4 99.15 f0.75ab 93.05fl.9Ob 42.09 i 2 . 69e 85.52 k2.09c 69.51 f2 .99" 85.71 +0.43c 40.33 k 2 . 3 P 63.80 f 2.72" 92.37+0.57b 91.02 f0. 55bC 90.95fI.OObc 69.06+1.87& 104.33 k 0 . 3 Y 27.32 k 3.89s 88.59+ 1.05bC - 1 . 9 4 i 2 . 6 1 S 90.43 + 1.01 bC ~ Any values under a stated parameter, which carry different superscripts (a, b, c, etc.), differ significantly ( P i O . 0 5 ) . Nutritional role of S-methyl-L-cysteine 699 2.3.3. Management and treatment of samples The management of the experimental animals and the treatment of the feeds, faeces and urine were as detailed in Phillips et aL21 3. Results The results are given in Table 3. Supplementation of egg albumin with SMLC did not have a statistically significant effect on the biological value when compared with the egg albumin reference diet. The supplernentation of Maris Bead bean with methionine and with methionine plus cystine doubled the biological value but with cystine alone the increase was considerably less. Supple- mentation with SMLC had no effect on biological value. The microbial protein product when supplemented with methionine and cystine and methionine plus cystine also gave an increase in biological value over the unsupplemented diet. Supplementation of the microbial protein product with SMLC did not statistically alter the biological value. There was no difference in true digesti- bility due to supplementation in any of the diets. In the case of the amino acid mixtures all the true digestibilities were similar. The reference amino acid mixture, diet N, gave a biological value very similar to the egg albumin reference diet. The mixtures without cystine but with either methionine or methionine plus SMLC gave biological values only about 10% lower than the reference diets. The mixture with SMLC but no methionine or cystine and the mixture with cystine and SMLC but no methionine gave similar results, the biological values approximating zero, whilst the mixture with cystine but no methionine or SMLC gave a biological value of 27.32%. 4. Discussion The supplementation of Maris Bead bean and the microbial protein product with methionine and cystine resulted in a considerable increase in the biological value of both. However, the increase in the biological value of Maris Bead bean supplemented with cystine alone was not as great as with either methionine or methionine plus cystine. This indicates that the Maris Bead bean was deficient in methionine per se rather than in total sulphur amino acids. All methionine and cystine supple- mentations of the microbial protein product gave similar increases in biological value, showing deficiency in total sulphur amino acids but not specifically in methionine. In general the supplementation of the Maris Bead bean and microbial protein product with SMLC did not significantly alter their biological value. Supplementation with SMLC of the reference egg albumin protein similarly resulted in no significant alteration in biological value. The addition of SMLC to an amino acid mixture with methionine but no cystine had no effect on the utilisation of the methionine, the biological values being very similar, which appears to conflict with the results of Case and Benevenga'7 and Benevenga,ls who were, however, feeding SMLC in excess. However, in the extreme situation of a diet totally deficient in methionine, and where normally cystine would exert a limited sparing action, the presence of SMLC appears to interfere with this sparing activity. In view of the considerable amounts of SMLC in certain legumes, any appraisal of the protein quality based upon amino sulphur content must recognise that SMLC appears to have a largely passive nutritional role. References 1. Zacharius, R. M.; Thompson, J. F.; Morris, C. J. A new sulphur amino acid in beans. Plant Physiol. 1956, 31, xliv. 2. Thompson, J. F.; Morris, C. J.; Zacharius, R. M. Isolation of (-)S-Methyl-L-cysteine from beans (Phaseolus vulgaris). Nature 1956, 178, 593. 3. Evans, I. M.; Boulter, D. S-Methyl-L-cysteine content of various legume meals. Qual.Plant. 1975, 24,257-261. 4 . Homer, W. H.; Kuchinskas, E. J. Metabolism of methyl labelled S-Methylcysteine in the rat. J. B i d . Cfirm. 1959,234,2935-2931. 5. Kuchinkas, E. J. Oxidation of the S-Methyl group by rat tissue preparations. Arch. Biocfiem. Biopfiys. 1965, 112,605-609. 700 M. D. Eyre el al. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15 . 16. 17. 18. 19. 20. 21. Whittle, P. J.; Smith, R. H.; McIntosh, A. Estimation of S-Methylcysteine sulphoxide (kale anaemia factor) and its distribution among brassica forage and root crops. J. Sci. Food Agric. 1976, 27, 633-642. Smith, R. H.; Earl, C. R.; Matheson, N. A. The probable role of S-Methylcysteine sulphoxide in kale poison- ing in ruminants. Trans. Biochem. SOC. 1974, 2, 101-104. Nakamura, H. ; Ishikawa, M. Effect of S-Methyl-L-cysteine sulphoxide on cholesterol metabolism. Kanro 1971, 12, 673-677. Fugiwara, M.; Itokawa, Y . ; Uchino, H.; Inoue, K. Anti-hypercholesterolemic effect of a sulphur containing amino acid, S-Methyl-L-cysteine sulphoxide, isolated from cabbage. Experientiu 1972, 28, 254-255. Tominga, F.; Kobayashi, S.; Muta, I.; Takei, H.; Ichinose, M. On the isolation and identification of S- Methylcysteine from human urine. J. Biochem. 1963, 54, 220-221. Tominga, F.; Oka, K. ; Yoshida, H. The isolation and identification of 0-Xylosylserine and S-Methylcysteine sulphoxide from human urine. J. Biochem. 1965, 57, 717-720. Evans, R. J.; Bandemer, S. L. Nutritive value of legume seed proteins. J . Agric. Food Chem. 1967,15,439-443. Bell, E. A. Certain non-protein amino acids of plants and their effects on animals. Biochem. J. 1963, 88, Block, R. J.; Jackson, R. W. The metabolism of cystine and methionine. J . Biol. Chem. 1932, 97, cvi-cvii. Armstrong,M. D.; Lewis, J. D. Growth experiments with thioether derivatives of cysteine and homocysteine. J. Biol. Chem. 1951, 189,461-466. Benevenga, N. J.; Yeh, M.-H.; Lalich, J. J. Growth depression and tissue reaction to the consumption of excess dietary methionine and S-Methyl-L-cysteine. J . Nutr. 1976, 106, 1714-1720. Case, G. L.; Benevenga, N. J. S-Methylcysteine as a methionine analogue in methionine toxicity studies. Fed. Proc. 1972, 31, 715 (Abs.) Benevenga, N. J. Toxicity of methionine and other amino acids. J. Agric. Food Chem. 1974, 22, 2-9. Miller, D. S.; Samuel, P. D. Effects of the addition of sulphur compounds to the diet on utilisation of proteins in young growing rats. J. Sci. Food Agric. 1970, 21, 616-618. Gjeren, A. U. ; Njaa, L. R. Methionine sulphoxide as a source of sulphur-containing amino acids for the young rat. Br. J . Nutr. 1977, 37, 93-105. Phillips, D. E.; Eyre, M. D.; Thompson, A.; Boulter, D. Protein quality in seed meals of Phaseolus vulguris and heat-stable factors affecting the utilisation of protein. J. Sci. Food Agric. 1981, 32, 423-432. 58P-59P.
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