Effects of Methionine and Cystine on the Cholesterol Concentrations in the Serum and Liver of Cholesterol-Fed

Transcription

1 Effects of Methionine and Cystine on the Cholesterol Concentrations in the Serum and Liver of Cholesterol-Fed Chicks Hiroshi UEDA and Wataru FUKUI College of Agriculture, Ehime University, Matsuyama-shi 790 (Received October 17, 1995) Abstract Experiments were conducted to study the effects of methionine and cystine on the cholesterol concentrations in the serum and liver of chicks fed diets containing 1% cholesterol. Soybean protein isolate (SPI) and casein were used as protein sources providing 14% and 20% crude protein in diets. Casein was fortified with arginine and glycine. Thus, all experimental diets were deficient in sulfur-containing amino acids. Increasing the dietary protein content decreased serum and liver cholesterol concentrations, irrespective of protein source. The addition of methionine to SPI diets lowered the serum cholesterol concentration, but not the liver cholesterol content. The effect of methionine was not observed in casein-fed chicks. Serum cholesterol concentrations were decreased by the addition of methionine, homocystine, N-acetylmethionine and cystine to the SPI diet containing 14% crude protein. This effect was accompanied by the improved growth rate with the exception of cystine. On the other hand, homoserine, taurine, potassium sulfate and choline chloride did not have a hypocholesterolemic action nor a growth-promoting effect. While adding methionine beyond the requirement had little effect on the serum cholesterol concentration, excess cystine tended to lower them. SPI contains more cystine than casein does. However, feeding SPI diets did not always show a hypocholesterolemic action compared with casein diets. These results indicated that methionine and cystine were not positively involved in the control of serum cholesterol concentrations in chicks. Anim. Sci. Technol. (Jpn.) 67 (6): , 1996 Key words: Serum cholesterol, Dietary protein, Dietary methionine, Dietary cystine The quality and quantity of dietary protein affect blood cholesterol concentrations in various animal species3,5,9-11). Substituting soybean protein isolate (SPI) for casein in a diet suppresses the increase in plasma cholesterol concentrations in cholesterol-fed chicks16,17). Increasing dietary protein content is also effective in alleviating hypercholesterolemia induced by the dietary addition of cholesterol even when casein is used as a protein source15,16). A number of trials have been carried out to determine which components in dietary proteins are responsible for their different effects. Since dietary proteins vary in amino acid composition3), it is reasonable to consider that amount or proportion of certain amino acids is a determinant affecting blood cholesterol concentration. Arginine acts as hypocholesterolemic whereas lysine as hypercholesterolemic in cholesterol-fed chicks when these amino acids are supplemented in excess18). Sulfur-containing amino acids (SAA) have received attention, because casein contains

2 more methionine and less cystine than SPI does3). Sugiyama et al.9-14) have carried out the systematic studies with rats in this regard, and they emphasized that methionine was hypercholesterolemic. However, conflicting results were obtained by the same group10,13). Yagasaki et al.21) also reported that the methionine effect with rats varied with the supplementary levels of the amino acid as well as with the dietary protein content. In chicks, supplementary methionine is effective in mitigating the hypercholesterolemia produced by feeding diets deficient in this amino acid4,6), but a hypercholesterolemic action of methionine has not been reported yet. Cystine can spare partly the methionine requirement. However, the effect of cystine on plasma cholesterol concentrations in rats is not always the same as that of methionine8,10,13). A partial replacement of methionine with cystine in the amino acid mixture simulating casein reduced plasma cholesterol concentrations in cholesterol-fed rats11). Sugiyama et a1.12) considered that the differences in effects between methionine and cystine depended on whether methyl group was included or not. However, no information is available on the cystine effect in chicks. Besides, the effect of cystine as well as that of methionine on plasma cholesterol concentrations is modified by other dietary components in rats7,21). The objective of this study was to investigate the effects of supplementary methionine, cystine and other related compounds on the cholesterol concentrations in the serum and liver of cholesterolfed chicks. Materials and Methods Day-old Single Comb White Leghorn male chicks were maintained in a room with con- ing. They were fed 4g of a commercial chick starter ration per head daily for 6 days in wire-mesh cages. The merit of the restricted pre-feeding program was described by Yoshida22). At 7 days of age, the necessary number of chicks with close body weight was selected from the stock. They were housed individually in metabolism cages and distributed into experimental groups of six chicks each. Chicks were given free access to diets and water for 8 days in Experiment 1 and for 10 days in Experiments 2 and 3. At the end of the feeding experiment, chicks were deprived of feed for 5h, and their body weights and feed intake were recorded. Blood was taken by heart puncture in all experiments and centrifuged at 3,500rpm for 10min after coagulation to separate serum. Chicks were killed by decapitation and their livers were excised in Experiments 1 and 2. They were rinsed with sodium chloride solution (8.5g/l), analysis. Table 1 shows the composition of basal diets containing 1% cholesterol. As protein sources providing 14% and 20% protein in the diets, SPI (soybean protein isolate, Fujipro-RB, Fuji Oil Co., Osaka), and casein (Wako Pure Chemical Ind., Osaka) fortified with arginine and glycine were used. These diets were referred to as diets S14, S20, C14 and C20 according to the source and content of dietary protein. All diets were deficient in SAA. In Experiment 1, 0.21% or 0.3% DLmethionine was supplemented to basal diets containing 14% and 20% protein, respectively. To exaggerate the hypercholesterolemia due to exogenous cholesterol20), 0.25% sodium cholate was added to the half of the experimental diets. In Experiment 2, methionine and its metabolites or related compounds were added to the S 14 diet. When sulfur is contained, the added amount was on the isosulfurous basis. DL-Homoserine and choline chloride were added on the equimolar basis to methionine. N-acetyl-L-methionine is known to have a methionine-sparing value of 100% in chicks1). In Experiment 3, different levels of DLmethionine (0.6% and 1.2%) and L-cystine (1.2%

3 Sulfur-Containing Amino Acids and Serum Cholesterol Table 1. Composition of basal diets and 2.4%) were added supplemented to the S20 diet already with 0.3% DL-methionine. Total cholesterol concentrations in serum and liver were measured with enzymatic procedures as described previously19). Data were analysed using the analysis of variance, and comparisons among treatments were made by Tukey's multiple range test23). Results The results of Experiment 1 are summarized in Table 2. Irrespective of sodium cholate added, body weight gain, feed intake and gain/feed ratio in chicks fed the S14 diet were improved by adding 0.21% methionine and increasing dietary protein content to 20%. Adding 0.3% methionine to the S20 diet also improved body weight gain and gain/feed ratio, but these alleviatory effects were small compared to chicks fed the S14 diet. Adding sodium cholate to the S14 and S20 diets depressed body weight gain and feed intake. However, this adverse effect disappeared when methionine was supplemented. Increasing dietary protein content was effective in improving body weight gain and gain/ feed ratio in chicks fed the C14 diet. Contrary to chicks fed SPI diets, the growth performance in chicks fed the C14 and C20 diets was not altered by the supplementation of methionine or sodium cholate. Without addition of methionine, body weight gain and gain/feed ratio were higher in chicks fed casein diets than in those fed SPI diets at both protein levels. However, the effect of protein source was insignificant when methionine was added to each diet. Adding sodium cholate to every SPI diet increased serum cholesterol concentrations in chicks (Table 2). Increasing both methionine and protein contents in the S14 diet lowered serum cholesterol concentrations independent of the presence or absence of sodium cholate. Methionine added to the S20 diet also reduced the serum cholesterol concentration. In chicks fed casein diets, serum cholesterol concentrations were reduced by increasing the dietary protein content, and elevated by adding sodium cholate as found in chicks fed SPI diets. However, in contrast to chicks fed SPI diets, the addition of methionine to casein 535

4 UEDA and FUKUI Table 2. Effects of dietary methionine, protein and sodium cholate on growth performance and cholesterol concentrations in the serum and liver of cholesterol-fed chicks (Experiment 1)1) diets did not affect serum cholesterol concentrations. Consequently, the effect of protein source on serum cholesterol concentrations was complicated. Contrary to the general consensus that SPI acts as hypocholesterolemic as compared to casein15-18), serum cholesterol concentrations were much higher in chicks fed the S14 diet than in those fed the C14 diet. This difference was more evident when sodium cholate was added to the diets. However, supplementing methionine to the S 14 diet reversed the effect of the dietary protein source, irrespective of the absence or presence of sodium cholate. On the other hand, there were no differences in serum cholesterol concentrations between chicks fed the S20 and C 20 diets even when methionine or sodium cholate was added. Increasing the dietary protein content reduced the liver cholesterol content, irrespective of the protein source and the presence or absence of sodium cholate, whereas supplementation of methionine had little effect on liver cholesterol. The difference in effect of the protein source was only observed between chicks fed the S14 and C14 diets. Table 3 shows the results of Experiment 2. Body weight gain in chicks fed the S14 diet was increased by supplementing methionine, homocystine and N-acetylmethionine, which 536

5 1 Sulfur-Containing Amino Acids and Serum Cholesterol Table 3. Effects of methionine and its related compounds on growth performance and cholesterol concentrations in the serum and liver of cholesterol-fed chicks (Experiment 2)1) Table 4. Effects of methionine and cystine on growth performance and serum cholesterol concentrations in cholesterol-fed chicks (Experiment 3)1) 1) Values are means for six chicks each. Means with different superscript are significantly different (p<0.05). was attributed to the increased gain/feed ratio. Feed intake was not changed by the dietary treatment (data were not shown). Effects on growth performance of cystine, homoserine, taurine, potassium sulfate and choline chloride were insignificant. The addition of methionine, cystine, homocystine or N-acetylmethionine to the S14 diet decreased serum cholesterol concentrations, although it did not affect liver cholesterol content. Homoserine, taurine, potassium sulfate and choline chloride were not effective in reducing cholesterol concentrations of the serum and liver. The results of Experiment 3 are given in Table 4. Body weight gain was significantly depressed only when 1.2% methionine was added to the control diet (S20 diet already supplemented with 0.3% methionine). Since gain/feed ratio was not different between the two diets, this adverse effect was apparently attributed to the reduced feed intake. Adding 0.6% and 1.2% methionine to the control diet did not change serum cholesterol concentrations. Supplementing cystine at 1.2% and 2.4% levels tended to lower serum cholesterol concentrations although the effect was not statistically significant. 537

6 UEDA and FUKUI Discussion Serum cholesterol concentrations in chicks decreased with the increase of the dietary protein content, irrespective of the dietary protein source15,16). The addition of methionine to SPI diets caused a similar hypocholesterolemic effect. Seidel et al.8) reported that the hypocholesteroiemic action of protein supplements was due largely to methionine they provided. However, the addition of methionine to the casein diets did not alter the serum cholesterol concentration in this experiment. Besides, adding methionine to S20 diet beyond the requirement had little effect on serum cholesterol concentrations. We therefore considered that methionine itself did not have a hypocholesterolemic action and could not account for the hypocholesterolemic effect of protein supplements. The addition of methionine, homocystine and N-acetylmethionine to the S14 diet reduced serum cholesterol concentrations. Such hypocholesterolemic effect was accompanied by the improved growth rate. With exception of cystine, on the other hand, homoserine, taurine, potassium sulfate and choline chloride did not have a hypocholesterolemic action nor a growth-promoting effect. Lysine and arginine are also effective both in alleviating growth retardation and in lowering blood cholesterol concentrations in chicks given diets deficient in the corresponding amino acid4,6,18,19). These results indicated that the cholesterol-lowering effect of methionine observed in the present experiment was attributed to the growth-promoting effect as the limiting amino acid; the increase of body weight by adding limiting amino acids might cause the dilution of body cholesterol and consequently lower the serum cholesterol concentration. The effect of cystine on the serum cholesterol concentration was not negligible in this experiment. According to Japanese Feeding Standard for Poultry2), the requirement of SAA for starting chicks is 0.6%, at least half of which should be supplied as methionine. In this experiment, cystine did not promote the growth rate of chicks fed the S14 diet, which was accounted for by the fact that the S14 diet contained less than 0.3% methionine (Table 1). Nevertheless, cystine lowered the serum cholesterol concentration. Excess cystine also tended to lower the serum cholesterol concentration in chicks as reported with rats8,10,13). Sugiyama et al.11) observed a negative correlation between plasma cholesterol and cystine content in several protein sources in rats, and they14) suggested that the hypocholesterolemic effect of cystine was attributed to the enhanced bile acid excretion by taurine derived from cystine. However, in contrast to the result of Sugiyama et al.12), taurine did not alter the serum cholesterol concentration in this experiment. In spite of the different cystine content between SPI and casein, serum cholesterol concentrations were similar between chicks fed the S20 and C20 diets in this experiment. Furthermore, as shown in chicks fed 14% protein diets, cholesterol concentrations were rather higher in chicks fed the SPI diet than in those fed the casein diet. These results suggested that the participation of cystine in the hypocholesterolemic effect might be a little, if any. References 1) Baker DH. Efficacy of the D- and L-isomers of N-acetylmethionine for chicks fed diets containing either crystalline amino acids or intact protein. J. Nutr., 109: ) Japanese Feeding Standard for Poultry, National Research Council of Agriculture, Forestry and Fishery, Central Association of Livestock Industry. Tokyo ) Jaques H, Deshaies Y, Savoie L. Relationship between dietary proteins, their in vitro digestion products, and serum cholesterol in rats. Atherosclerosis, 61: ) Johnson Jr. D, Leveille GA, Fisher F. Influence of amino acid deficiencies and protein level on 538

7 Sulfur-Containing Amino Acids and Serum Cholesterol the plasma cholesterol of the chicks. J. Nutr., 66: ) Kritchevsky D. Vegetable protein and atherosclerosis. J. Am. Oil Chem. Soc., 56: ) Leveille, GA, Shockley JW, Sauberlich HE. Influence of dietary protein level and amino acids on plasma cholesterol of the growing chicks. J. Nutr., 76: l962. K. Comparison between the plasma cholesterol-lowering effects of glycine and taurine in rats fed on high cholesterol diets. Agric. Biol. Chem., 53: ) Ueda H. Effect of dietary protein and soybean saponins on plasma cholesterol concentration in chicks. Anim. Sci. Technol. (Jpn.), 63: ) Ueda H. Effects of Gyphsophila saponins on 7) Oda H, Matsuoka S, Yoshida A. Effects of dietary performance and plasma cholesterol concenfate methionine, cystine and potassium sultration in chicks fed the diets different in on serum cholesterol and urinary ascorbic acid in rats fed PCB. J. Nutr., 116: casein content. Anim. Sci. Technol. (Jpn.), 63: ) Ueda H, Ohshima M. Hypocholesterolemic 8) Seidel JC, Nath N, Harper AE. Diet and effect of alfalfa leaf protein concentrate and cholesterolemia: V. Effects of sulfur-containing soybean protein isolate in chicks. Anim. Sci. amino acids and protein. J. Lipid. Res., 1: ) Technol. (Jpn.), 63: Ueda H, Fukumi R, Kumai S. Effect of dietary 9) Sugano M, Ishiwaki N, Nakashima K. Dietary arginine content on plasma cholesterol levels protein-dependent modification of serum chol in chicks. Anim. Sci. Technol. (Jpn.), 64: esterol level in rats. Significance of the arginine/lysine ratio. Ann. Nutr. Metab., 28: ) Ueda H, Imanishi T, Fukumi R, Kumai S. Effect of dietary lysine and arginine addition on 10) Sugiyama K, Kushima Y, Muramatsu K. growth performance and serum cholesterol Effects of sulfur-containing amino acids and level in chicks. Anim. Sci. Technol. (Jpn.), 66: glycine on plasma cholesterol level in rats fed on a high cholesterol diet. Agric. Biol. Chem., 20) Ueda H, Fukumi R, Kumai S. The effects of 49: sodium cholate and cholestyramine on the 11) Sugiyama K, Ohkawa S, Muramatsu K. Relationship lipid concentration of serum and liver in cho- between amino acid composition of lesterol-fed chicks. Anim. Sci. Technol. (Jpn.), diet and plasma cholesterol level in growing 66: rats fed a high cholesterol diet. J. Nutr. Sci. 21) Yagasaki K, Aoki T, Funabashi R. Serum and Vitaminol., 32: liver lipid responses to methionine and cystine 12) Sugiyama K, Akai H, Muramatsu K. Effects of in rats fed diets with different casein levels. methionine and related compounds on plasma Nutr. Rep. Int., 34: cholesterol level in rats fed a high cholesterol 22) Yoshida M. Improvement of the procedures to diet. J. Nutr. Sci. Vitaminol., 32: determine gross protein value with growing chick. II. Optimum protein level in assay diets and influence of pre-feeding program. Japan. Poult. Sci., 10: ) Yoshida M, Design of Experiments for Animal Husbandry Yoken-do. Tokyo

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