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METABOLISM AND NUTRITION |
Department of Animal Sciences, University of Illinois, Urbana 61801
1 Corresponding author: dhbaker{at}uiuc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: chick • cysteine • cystine • efficacy • methionine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been suggested that dietary cyst(e)ine may affect utilization of Met in chicks (Graber and Baker, 1971; Katz and Baker, 1975; Sell et al., 1980a). Moreover, evidence suggests this effect may result from cyst(e)ine affecting intestinal Met transport (Lerner and Taylor, 1967; Sell et al., 1980b). Methionine is an indispensable AA, and whereas cyst(e)ine can be synthesized in vivo from Met, the reverse is not possible. Therefore, poultry diets are supplemented with Met to meet the total SAA requirement, and cyst(e)ine content tends to fluctuate according to the ingredients used in diet formulation. Great variability exists in ingredient cyst(e)ine concentrations, ranging from ingredients low in cyst(e)ine (e.g., meat meal) to those extremely high in cyst(e)ine (e.g., hydrolyzed feather meal). Although cyst(e)ine can safely contribute 50% of the total SAA requirement (wt:wt) in young, rapidly growing poultry (Graber and Baker, 1971), it is still unclear how excess dietary cyst(e)ine affects utilization of different Met products in practical diets.
Thus, our objectives were to 1) compare the effectiveness of L-Met and DL-Met in diets with varying cyst(e)ine concentrations, and 2) characterize the effects of dietary cyst(e)ine on chick growth performance. Our approach used a purified diet severely deficient in SAA, but we also tested the effect of dietary cyst(e)ine in a practical-type diet using ingredients commonly used in poultry nutrition. Throughout this article we use the term "cyst(e-)ine" to indicate the sum of cysteine (reduced form) plus cystine (oxidized form). It has been clearly established that cysteine and cystine are equally efficacious on a weight or concentration basis when provided as low-level supplements to cyst(e)ine-deficient diets (Graber and Baker, 1971; Baker, 2006). In the pharmacologic dosing range, however, supplemental cysteine is far more toxic than cystine at levels exceeding 2% of the diet—for chicks, but not for pigs or rats (Dilger et al., 2007b).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All procedures were approved by the University of Illinois Animal Care and Use Committee. Three bioassays were conducted using male chicks (New Hampshire male x Columbian female) obtained from the University of Illinois Poultry Farm. Chicks were housed in thermostatically controlled starter batteries with raised-wire flooring in an environmentally controlled room with continuous lighting. From hatch to d 7 posthatch, chicks were fed a 23% CP corn-soybean meal starter diet adequate in all dietary nutrients (NRC, 1994). Following an overnight fast, chicks were weighed, wing-banded, and randomized to dietary treatments on d 8 such that average initial pen weights and weight distributions were similar across treatments.
Two separate basal diets were formulated to assess bioefficacy of DL-Met relative to L-Met. A purified crystalline amino acid-based diet formulated to be deficient in both Met and cyst(e)ine was used in assay 1 (Table 1
). This diet was analyzed (Dilger and Baker, 2007; Dilger et al., 2007b) and found to contain 0.12% Met and 0.05% cyst(e)ine. The supplemental Met requirement of chicks fed this purified diet was previously determined to be 0.12% (0.24% total Met) in the presence of adequate dietary cyst(e)ine (Dilger and Baker, 2007). The basal diet used for assays 2 and 3 was a low-protein corn-soybean meal-peanut meal diet (18.1% CP) analyzed to contain equally deficient concentrations of Met and cyst(e)ine (0.25% of each).
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Experimental diets and tap water were freely available to chicks at all times. Chicks were fed for a total of 9 d in assay 1 (d 8 to 17 posthatch) and 12 d in assays 2 and 3 (d 8 to 20 posthatch). Body weight of individual chicks and pen feed intakes were measured at the termination of each chick assay. Weight gain, feed intake, and feed efficiency (gain:feed ratio) were calculated for each replicate pen of chicks.
Assay 1
The objective of this 9-d assay was to compare the ability of supplemental L-Met and DL-Met to support chick growth when added to purified diets containing increasing levels of dietary cyst(e)ine. The basal diet (Table 1
) was supplemented with L-cystine at 4 levels (0, 0.07, 0.15, or 0.35%), and L-Met or DL-Met was added at 0 or 0.05% at each level of cystine by replacing dietary cornstarch. This assay used a factorial arrangement of 12 diets (4 levels of added cystine and 3 Met treatments), and growth performance was quantified using 5 replicate pens of 4 chicks (mean initial weight 83 g).
Assay 2
This assay was designed to test the effect of supplemental cystine on growth performance of chicks consuming graded doses of L-Met in a practical-type diet. Chicks received the corn-soybean meal-peanut meal basal diet (Table 1) supplemented with 0, 0.025, 0.05, or 0.075% L-Met, or 0.05% DL-Met. At each level of Met, diets were additionally supplemented with 0 or 0.2% L-cystine, forming a factorial arrangement of 10 diets (5 Met treatments and 2 levels of added cystine). All dietary additions were made at the expense of cornstarch. Six replicate pens of 4 chicks (mean initial weight 92 g) consumed these diets for 12 d, after which weight gain, feed intake, and feed efficiency were quantified.
Assay 3
This assay compared the relative ability of L-Met and DL-Met to support growth of chicks consuming a practical-type diet with deficient or adequate dietary cyst(e)ine. The low-protein, corn-soybean meal-peanut meal basal diet (Table 1) was supplemented with 2 levels of L-cystine (0 or 0.2%) and either 0 or 0.03% L-Met or DL-Met, forming a factorial arrangement of 6 diets. Growth performance was quantified over a 12-d feeding period using 5 replicate pens of 4 chicks (mean initial weight 83 g).
Statistical Analysis and Calculations
All data were subjected to ANOVA using the GLM procedure of SAS/STAT software (SAS Institute, 2004). Data were analyzed using pen means with procedures appropriate for a completely randomized design. Data are presented as mean values with pooled SEM estimates, and significance was set at an 
level of 0.05. Chick growth responses to graded supplementation with either cystine or Met were evaluated using orthogonal polynomial single df contrasts. Polynomial contrast coefficients were calculated in assay 1 due to unequal spacing of cystine levels. A nonorthogonal set of contrasts (including 0 vs. added Met, linear and quadratic responses to L-Met, and L-Met vs. DL-Met) was tested in assay 2. Because the effect of L-Met vs. DL-Met was not significant in assay 1, data were pooled and reanalyzed as a 2 x 4 factorial (0 or 0.05% Met and 0, 0.07, 0.15, or 0.35% L-cystine).
Upon analysis of data from assay 2, it was evident that weight gain, feed intake, and gain:feed responded in a curvilinear fashion in that both linear and quadratic contrasts were significant (P < 0.01). Thus, these data were fitted to an exponential model because 1) it was evident that growth responses attained plateau, and 2) this procedure allowed simultaneous and separate modeling of responses to graded L-Met intake and the effect of added cystine. Exponential responses of weight gain and gain:feed to graded L-Met intake were fitted using the NLIN procedure of SAS/STAT software (SAS Institute, 2004). The statistical model allowed parameters to vary with supplemental cystine level, and took the following form:
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where Y = growth response (weight gain or feed efficiency), X = mg of supplemental L-Met intake, a and d = intercept (minimum performance), b and e = asymptotic response, c and f = steepness coefficient for supplemental L-Met intake, and C1 and C2 are indicator variables (0 or 1) representing 0 and 0.2% added L-cystine, respectively.
Quantitative estimates of DL-Met bioefficacy were obtained relative to L-Met using standard curve methodology in assay 2. The responses of weight gain and gain:feed (dependent variables) as functions of supplemental L-Met intake (independent variable) were fitted using the aforementioned exponential model. Supplemental DL-Met intake was calculated from the L-Met standard curve using the mean weight gain or gain:feed values for DL-Met. This calculated relative bioavailable Met intake was then divided by the mean supplemental Met intake and multiplied by 100 to obtain bioefficacy estimates for DL-Met relative to L-Met.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Weight gain, feed intake, and gain:feed were increased (P < 0.001) due to Met or cystine supplementation, suggesting the purified diet was first-limiting in cyst(e)ine (Table 2). Overall, L-cystine supplementation at levels ranging from 0.07 to 0.35% resulted in linear decreases in weight gain and feed intake (P < 0.001), as well as gain:feed (P < 0.05). Growth performance criteria were increased (P < 0.001) by the addition of 0.05% Met regardless of cystine level. However, there were no differences (P > 0.63) in growth responses due to L-Met vs. DL-Met. Thus, data were pooled and reanalyzed as a 2 x 4 factorial arrangement (Figure 1). From this analysis, it was evident that chick weight gain was improved to a greater extent (M x C1 interaction, P < 0.001) due to concomitant supplementation of cystine and Met compared with Met supplementation alone. Additionally, there was an interaction between Met supplementation and the decreasing linear response of weight gain (P < 0.05), but there was no such interaction with the quadratic response.
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Chick growth performance criteria were improved (P < 0.001) by Met supplementation and showed linear and quadratic responses (P < 0.001; Table 3). Weight gain and gain:feed responses were best described using an exponential model that allowed parameters to vary according to supplemental cystine level (Figure 2). Overall, addition of 0.2% L-cystine depressed (P < 0.01) weight gain 3.6% and feed intake 6.9%. This led to an improvement (P < 0.001) in gain:feed of 3.5% due to addition 0.2% L-cystine. The reduction in weight gain and feed intake due to added cystine occurred predominantly in the absence of supplemental Met (added Met x added cystine interaction, P < 0.05), and the negative effect of added cystine on weight gain was lessened as Met intake increased (Figure 2).
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Assay 3
Using the corn-soybean meal-peanut meal diet, this assay showed that growth performance criteria were positively affected (P < 0.001) by Met supplementation regardless of added cystine (Table 4). Similar to results from assay 1, there were no differences in weight gain, feed intake, or gain:feed due to feeding L-Met vs. DL-Met. Addition of 0.2% L-cystine reduced (P = 0.03) weight gain of chicks in both the absence and presence of supplemental Met. No interactive effects for weight gain or feed intake were present when Met and cystine were supplemented together in this intact-protein diet. However, the gain:feed response to cystine was greater (P = 0.03) in the presence than the absence of supplemental Met.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well established that the chick can use D-Met with greater than 90% efficacy relative to L-Met (Wretlind and Rose, 1950; Berg, 1959; Sugahara et al., 1967; Sunde, 1972; Baker, 1986). Because DL-Met is produced via chemical synthesis, it contains a racemic (50:50) mixture of D- and L-isomers. L-Methionine is the form that is incorporated into body proteins, and it is assumed to be 100% efficacious, whereas D-Met must be converted to L-Met in vivo. After metabolic conversion of D-Met into 2-keto-4-(methylthio)butyric acid (i.e., keto-Met), catalyzed by D-amino acid oxidase (EC 1.4.3.3 [EC] ), a subsequent transamination reaction results in synthesis of L-Met. Therefore, it has been traditionally accepted that DL-Met is 95% efficacious relative to L-Met due to equal dietary contributions of the D- and L-isomers (Baker, 2006). We recently reported evidence that the effectiveness of DL-Met to support weight gain and gain:feed was not less than that of L-Met (Dilger et al., 2007a) in chicks fed low-protein diets of varying SAA content from 8 to 20 d of age. The data we present herein provide further evidence to support such a claim. Qualitative estimates of bioefficacy from assays 1 and 3 clearly show that DL-Met was as effective as L-Met in both purified and practical-type diets severely deficient in total SAA. Moreover, bioefficacy of DL-Met was not different than L-Met regardless of whether dietary cyst(e)ine was deficient, adequate, or in excess.
When expressed on a Met intake basis, responses of both weight gain and gain:feed were curvilinear in assay 2. This is a common problem in SAA nutrition studies and is related to the metabolic conversion of Met into cyst(e)ine. Quantitative estimates of DL-Met bioefficacy from assay 2 concurred with data from assays 1 and 3 to confirm that DL-Met was at least as efficacious as L-Met. To our knowledge, this is the first example in which a practical-type diet, markedly deficient in SAA, was used to concurrently evaluate chick growth responses to graded supplementation of L-Met and L-cystine. Data from assay 2 also provide compelling evidence as to the effect of dietary cyst(e)ine on Met utilization and the interrelationship between these nutrients. Perhaps most striking was the fact that just 0.2% L-cystine depressed chick growth in diets where cyst(e)ine was deficient and equally first-limiting (with Met), but this cystine effect was absent when as little as 0.025% L-Met was added.
In agreement with Katz and Baker (1975), our data suggest that the growth-promoting effect of adding Met to a diet markedly deficient in SAA was reduced in the presence of just 0.2% L-cystine. The exact mechanism by which this phenomenon occurs is still unclear, though it has been suggested that the presence of cyst(e)ine in the alimentary tract may directly hinder Met absorption (Lerner and Taylor, 1967; Sell et al., 1980a,b). To this end, Featherston and Rogler (1978) observed that addition of 0.2% L-cystine to chick diets containing suboptimal dietary Met reduced the rate and efficiency of weight gain, which they hypothesized was attributable to an antagonism of Met by dietary cyst(e)ine. However, while investigating the dietary SAA requirement of puppies, Hirakawa and Baker (1985) showed strong evidence that this effect was mediated via reduced voluntary feed intake. Therefore, it was concluded that cyst(e)ine caused a nutritional imbalance because the depression in weight gain was caused by a reduction in voluntary feed intake (i.e., anorexia; Gietzen et al., 2007). Our data concur with this conclusion; supplementation with just 0.2% L-cystine reduced chick feed intake an average of 6.2% in assays 2 and 3. The fact that added cystine caused no difference in maximal weight gain due to Met supplementation (i.e., assay 2) is intriguing and difficult to explain, but ultimately resulted in improved efficiency of chick weight gain.
Addition of just 0.2% L-cystine to a corn-soybean meal-peanut meal diet containing no supplemental Met (i.e., basal diet contained 0.25% Met and 0.25% cyst(e)ine) caused a reduction in weight gain. In this scenario, Met and cyst(e)ine each provided 50% of the total SAA content in the basal diet. Addition of 0.2% L-cystine increased the proportion provided by cyst(e)ine to 64%. It has been well established in many species (Graber and Baker, 1971; Finkelstein et al., 1988; Chung and Baker, 1992; Di Buono et al., 2003) that cyst(e)ine can safely provide up to 50% of the total SAA requirement. However, when cyst(e)ine contributes a majority of the total SAA content, detrimental effects on growth performance have been observed, especially under conditions of suboptimal SAA intake (Featherston and Rogler, 1978). We observed that addition of even small amounts of Met could ameliorate the growth depressive effect of supplemental cystine at low SAA intakes. For instance, added cystine depressed weight gain and feed intake 11.5 and 11.9%, respectively, in the absence of supplemental Met in assay 2. However, addition of just 0.025% Met ameliorated this effect; 0.2% added L-cystine depressed weight gain and feed intake just 3.2 and 5.3%, respectively, with 0.025% added Met. Interestingly, this level of added Met only reduced the contribution of cyst(e)ine to the total SAA content from 64 to 62%. Thus, a stark threshold seems to exist in the chick regarding the proportion of total SAA content that can be provided by cyst(e)ine.
Titration of weight gain and gain:feed responses to graded Met intake, and the effect of supplemental cystine thereupon, led to intriguing outcomes in our chick model. Whereas rate of gain was unaffected when chicks received supplemental Met plus cystine, efficiency of gain continued to show improvement (Figure 2). This resulted from depressed voluntary feed intake due to the addition of 0.2% L-cystine. It is plausible that cyst(e)ine served as a physiological depressor of feeding behavior, a notion supported by previous research (Featherston and Rogler, 1978; Sell et al., 1980a; Hirakawa and Baker, 1985). Our data are interesting, however, because the presence of a small excess of cystine depressed feed intake without a proportional reduction in weight gain. This depression in feed intake appears to be a unique nutritional imbalance and is not a complete surprise considering cyst(e)ine contributed between 58.1 and 64.3% of the total SAA content in diets with added cystine in assay 2. However, improvement in efficiency of gain mediated through reduced feed intake is a rare event in nutritional studies. Thus, more research is warranted to provide a physiological basis for this intriguing phenomenon.
Results from assay 1, where chicks consumed graded levels of supplemental cystine (Figure 1), suggested a much greater response to Met supplementation in the presence, rather than absence, of 0.07% L-cystine. Moreover, the linear decrease in weight gain due to added cystine was less pronounced in the presence (31% decrease), rather than absence (36% decrease), of 0.05% Met. It should be noted that this purified diet contained only 0.12% Met and 0.05% cyst(e)ine; it was most deficient in cyst(e)ine. Thus, when the purified diet was supplemented with Met alone, some Met had to be converted to cyst(e)ine to meet the physiological growth requirement of the chick. However, when Met and cyst(e)ine were added, more Met could be used as Met per se because dietary cyst(e)ine was present to meet physiological needs. In short, the presence of dietary cyst(e)ine reduced the metabolic demand for conversion of Met into cyst(e)-ine [i.e., cyst(e)ine partly spared Met]. Thus, our data concur with previous research (Graber and Baker, 1971) to suggest a beneficial effect of satisfying the total SAA requirement using a combination of Met plus cyst(e)ine. It may be concluded, therefore, that by partly satisfying the total SAA requirement with Met, the detrimental effect of cyst(e)ine providing a majority of the total SAA content may be somewhat lessened.
In summary, the bioassays reported herein suggest DL-Met is at least as efficacious as L-Met when young chicks were fed either purified or practical-type diets deficient in SAA, regardless of dietary cyst(e)ine concentration. However, addition of just 0.2% L-cystine reduced chick growth due to a direct interrelationship between Met and cyst(e)ine. Maximal weight gain due to ingestion of graded Met was not affected by 0.2% L-cystine, but efficiency of gain was improved by including this small amount of dietary supplemental cystine.
Received for publication May 22, 2007. Accepted for publication August 4, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Baker, D. H. 2006. Comparative species utilization and toxicity of sulfur amino acids. J. Nutr. 136:1670S–1675S.
Berg, C. P. 1959. Utilization of D-amino acids. Pages 57–96 in Protein and Amino Acid Nutrition. A. A. Albanese, ed. Acad. Press, New York, NY.
Chung, T. K., and D. H. Baker. 1992. Maximal portion of the young pig’s sulfur amino acid requirement that can be furnished by cystine. J. Anim. Sci. 70:1182–1187.[Abstract]
Di Buono, M., L. J. Wykes, D. E. Cole, R. O. Ball, and P. B. Pencharz. 2003. Regulation of sulfur amino acid metabolism in men in response to changes in sulfur amino acid intakes. J. Nutr. 133:733–739.
Dilger, R. N., and D. H. Baker. 2007. Oral N-acetyl-L-cysteine is a safe and effective precursor of cysteine. J. Anim. Sci. 85:1712–1718.
Dilger, R. N., C. Kobler, C. Weckbecker, D. Hoehler, and D. H. Baker. 2007a. 2-keto-4-(methylthio)butyric acid (keto-analog of methionine) is a safe and efficacious precursor of L-methionine in the chick. J. Nutr. 137:1868–1873.
Dilger, R. N., S. Toue, T. Kimura, R. Sakai, and D. H. Baker. 2007b. Excess dietary L-cysteine, but not L-cystine, is lethal for chicks but not for rats or pigs. J. Nutr. 137:331–338.
Featherston, W. R., and J. C. Rogler. 1978. Methionine-cystine interrelations in chicks fed diets containing suboptimal levels of methionine. J. Nutr. 108:1954–1958.
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Gietzen, D. W., S. Hao, and T. G. Anthony. 2007. Mechanisms of food intake repression in indispensable amino acid deficiency. Annu. Rev. Nutr. 27:63–78.[CrossRef][Medline]
Graber, G., and D. H. Baker. 1971. Sulfur amino acid nutrition of the growing chick: Quantitative aspects concerning the efficacy of dietary methionine, cysteine and cystine. J. Anim. Sci. 33:1005–1011.
Hirakawa, D. A., and D. H. Baker. 1985. Sulfur amino acid nutrition of the growing puppy: Determination of dietary requirements for methionine and cystine. Nutr. Res. 5:631–642.[CrossRef][Web of Science]
Katz, R. S., and D. H. Baker. 1975. Efficacy of D-, L- and DL-methionine for growth of chicks fed crystalline amino acid diets. Poult. Sci. 54:1667–1674.[Web of Science][Medline]
Lerner, J., and M. W. Taylor. 1967. A common step in the intestinal absorption mechanisms of D- and L-Methionine. Biochim. Biophys. Acta 135:990–999.[Medline]
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NRC. 1994. Nutrient Requirements of Poultry. 9th ed. Natl. Acad. Press, Washington, DC.
SAS Institute. 2004. SAS/STAT User’s Guide. Release 9.1. SAS Inst. Inc., Cary, NC.
Sell, D. R., W. R. Featherston, and J. C. Rogler. 1980a. Methionine-cystine interrelationships in chicks and rats fed diets containing suboptimal levels of methionine. Poult. Sci. 59:1878–1884.[Web of Science][Medline]
Sell, D. R., J. C. Rogler, and W. R. Featherston. 1980b. Influence of dietary cystine on intestinal absorption and tissue distribution of methionine in the chick. Poult. Sci. 59:1885–1891.[Web of Science][Medline]
Sugahara, M., T. Morimoto, T. Kobayashi, and S. Ariyoshi. 1967. The nutritional value of D-amino acid in the chick nutrition. Agric. Biol. Chem. 31:77–84.[Web of Science]
Sunde, M. L. 1972. Utilization of D- and DL-amino acids and analogs. Poult. Sci. 51:44–55.[Web of Science][Medline]
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