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Amelioration of Ethionine Toxicity in the Chick
Amelioration of Ethionine Toxicity in the Chick KAREN R. LOWRY and DAVID H. BAKER1 Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801 (Received for publication May 29, 1986)
1987 Poultry Science 66:1028-1032 INTRODUCTION
The toxic effects of ethionine, the ethyl analog of methionine, have been demonstrated in rats, mice and poultry (Hsu et al., 1968; Friedman et al., 1977; Christensen and Anderson, 1980). However, neither the specific mechanism by which ethionine exerts its toxic effects nor the best means of ameliorating the toxicity have been clearly defined. Some investigators have suggested that the toxic effect of ethionine results from metabolites produced in a transaminative pathway of ethionine catabolism (Steele and Benevenga, 1979; Steele, 1982; Benevenga and Steele, 1984) but other workers have suggested that the primary effect of ethionine is to react with ATP and thereby trap adenosine as S-adenosylethionine (Shull et al., 1966). Thus, both methionine and adenine administration have been found to ameliorate ethionine toxicity in mice (Friedman et al., 1977) whereas only methionine was found to ameliorate ethionine toxicity in poultry, although adenine enhanced the response due to methionine supplementation (Yamada and Takahashi, 1977). If the mechanism by which ethionine exerts its toxic effects can be elucidated, it may be possible to use ethionine as a
'To whom reprint requests should be addressed.
model to study sulfur amino acid metabolism, methyl donor activity and methionine bioavailability in practical corn-soy diets without fear that ethionine is acting elsewhere to confound the results. To examine the effects of ethionine and means of ameliorating its toxicity, a series of experiments were conducted. The objectives of the experiments were to determine if ethionine toxicity could be completely alleviated in chickens by the administration of either methionine or choline and to examine the effect of ethionine toxicity on plasma free amino acids. A third experiment was conducted to determine if the changes observed in plasma amino acid levels were due to secondary amino acid deficiencies caused by ethionine toxicity. MATERIALS AND METHODS
New Hampshire x Columbian chicks were used in each of the three experiments. Female chicks were used in Experiment 1 and male chicks were used in Experiments 2 and 3. The chicks were fed a corn-soybean meal starter diet during the first 7 days posthatching. On the 8th day posthatching, following an overnight fast, chicks were weighed and allotted to experimental groups so that all groups had similar mean initial weights and weight distributions. Triplicate groups of five chicks were placed on the experimental diets at 8 days posthatching. The
1028
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
ABSTRACT Several chick bioassays were conducted to evaluate means of ameliorating ethionine toxicity. Supplementing a corn-soy diet marginally deficient in sulfur amino acids (methionine + cystine) with .075% D,L-ethionine reduced weight gain in 8-day-old chicks by 70% compared to gains of unsupplemented controls. Dietary addition of .50% DL-methionine prevented reduction in weight gain and feed intake resulting from ethionine supplementation whereas feeding supplemental L-cystine was without effect. Supplementation of the ethionine-containing diet with either choline or betaine ameliorated the growth depression, although neither compound was able to completely overcome the toxic effects of ethionine. Dietary ethionine did not affect plasma levels of free methionine or cystine but did increase plasma free glycine 6-fold. Dietary addition of .50% DL-methionine caused normalization of plasma glycine levels whereas it elevated plasma methionine concentration. Although results suggested the possibility of ethionine-induced serine or threonine deficiency, dietary additions of .75% L-serine or .75% L-threonine failed to improve chick weight gain. These studies suggest that ethionine, in addition to affecting transsulfuration and transmethylation activity, may exert specific effects on certain amino acids in tissue pools. (Key words: ethionine, methionine, choline, betaine, plasma glycine)
ETHIONINE TOXICITY IN CHICKS TABLE 1. Composition of the basal diet Ingredient
1
52.13 37.00 2.00 1.00 4.00 2.20 1.00 .40 .05 .01 .10 .10 .01
Crude protein.
2
Vitamin premix provided per kilogram of diet: vitamin A, 4,400 IU; vitamin D 3 1,000 ICU; vitamin E, 11 IU; vitamin B , 2 , .01 mg; riboflavin, 4.41 mg; d-pantothenic acid, 10.0 mg; niacin, 22.0 mg; menadione sodium bisulfite, 2.3 3 mg.
chicks were housed in heated, thermostatically controlled wire-floor starter batteries, and a 24-h constant light schedule was maintained. Feed and water were provided ad libitum throughout the 14-day assay periods, and weight gain and feed intake were monitored at regular intervals. The composition of the basal 23% protein diet used in all three experiments is presented in Table 1. The basal diet was not supplemented with methionine in order to make it marginally deficient in sulfur amino acids (SAA). Experiment 1 was designed to examine the efficacy of SAA and methyl donors in ameliorating ethionine toxicity. Thus, isosulfurous dietary additions of DL-methionine (.25%) and L-cystine (.203%), as well as .20% choline-Cl and .20% betaine-HCl, were studied in terms of their capacity to overcome the growth depression caused by .075% DL-ethionine supplementation. In Experiment 2, the capacity of methionine and choline to completely alleviate the ethionine-induced growth depression was evaluated by adding graded levels of DLmethionine (.25% or .50%) or choline-Cl (.20%, .40% or .60%) to the ethionine-containing diet. At the termination of Experiment 2, the heaviest and lightest birds were culled from each replicate group and the three remaining chicks were bled by heart puncture using a heparinized syringe. The blood was pooled by replicate and plasma then prepared by centrifugation at 1300 x g for
15 min. The three pooled plasma samples for each dietary treatment were subsequently deproteinized with 3.5% sulfosalicylic acid, after which amino acid concentrations were quantified by ion-exchange chromatography using an automated amino analyzer (Model 6300, Beckman Instruments, Palo Alto, CA). Experiment 3 was conducted to determine if the elevated plasma glycine levels observed in Experiment 2 were indicative of an ethionine-induced deficiency of metabolic glycine precursors, i.e., serine or threonine. Thus, .75% additions of L-serine or L-threonine were evaluated in terms of their capacity to ameliorate ethionine toxicity in both the presence and absence of .30% supplemental DL-methionine (to assure that methionine deficiency would not limit the response to a second- or third-limiting amino acid). Pen means data for performance and plasma analyses were analyzed by analysis of variance procedures for a completely randomized design. Meaningful nonorthogonal single degree-offreedom comparisons were made to assess treatment effects (Steel and Torrie, 1980). RESULTS
The results of Experiment 1 are summarized in Table 2. Both weight gain and feed utilization were depressed severely by ethionine supplementation. Addition of methionine to the
TABLE 2. Performance of chicks fed ethioninecontaining diets supplemented with sulfur amino acids or methyl donors (Experiment l)1 Treatment
Gain2
Gain:feed 2
Basal (B) B + .075% DL-Ethionine As 2 + .25% DL-methionine As 2 + .203% L-cystine As 2 + .20% choline-Cl As 2 + .20% betaine«HCl
(g) 203 72 174 68 118 117
.579 .417 .615 .408 .547 .538
5
.011
Pooled standard error of the mean 1
Data represent means of triplicate groups of five female chicks fed experimental diets from 8 to 22 days posthatching; chicks weighed 66.9 g at Day 8. 2 Treatment means 1, 3, 5, and 6 are all significantly greater (P<.001) than that in Treatment 2; Treatment 4 mean is not significantly different (P>.05) from that of treatment 2.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
Ground corn Dehulled soybean meal (48% CP 1 ) Fishmeal, Menhaden (60% CP) Alfalfa meal, dehydrated (17% CP) Corn oil Dicalcium phosphate Ground limestone Iodized salt Manganese sulfate Zinc carbonate Choline chloride (60%) Vitamin premix 2 Lincomycin premix (4.4%)
Percent
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LOWRY AND BAKER
1030
TABLE 3. Performance of ethionine-intoxicated chicks fed graded levels of DL-methionine or choline (Experiment 2)1 Treatment
Gain 2
Gain:feed 2
(g) Basal (B) B + .075% DL-Ethionine As 2 + .25% DL-methionine As 2 + .50% DL-methionine As 2 + .20% choline°Cl As 2 + .40% choline-Cl As 2 + .60% choline'Cl Pooled standard error of the mean
230 64 167 231 138 148 144
.618 .417 .609 .679 .586 .585 .574
5
.011
1 Data represent means of triplicate groups of five male chicks fed experimental diets from 8 to 22 days posthatching; chicks weighed 67.6 at Day 8. 2 Significant (P<.001) linear effect due to methionine (Treatments 2 to 4); Treatment means 1, 3, 4, 5, 6, and 7 are all greater (P<.001) than that of Treatment 2.
TABLE 4. Plasma amino acid concentrations of ethionine-intoxicated chicks fed graded levels of DL-methionine or choline (Experiment 2) Plasma amino acid concentration Met 1
Treatment
Cys 1
Gly 2
(nmol/mL) Basal (B) B + .075% DL-Ethionine As 2 + .25% DL-methionine As 2 + .50% DL-methionine As 2 + .20% choline'Cl As 2 + .40% choline'Cl As 2 + .60% choline'Cl Pooled standard error of the mean
36 24 25 105 23 28 27 4.5
34 39 37 48 39 36 35 2.6
582 3,121 1,571 1,028 2,035 2,324 2,020 120
Significant (P<.05) quadratic increase due to methionine (treatments 2 to 4); no response to choline or ethionine (P>.05). 2 Significant (P<.001) increase due to ethionine and decrease (P-C001) due to choline (i.e., Treatment 5, 6, and 7 concentrations are less than Treatment 2); significant (P<.01) quadratic decrease due to methionine (Treatment 2 to 4).
glycine could have resulted from (ethionine-induced) enhanced flux in the serine transhydroxymethylase-catalyzed pathway of serine -• glycine or in the threonine aldolase pathway that results in threonine catabolism to glycine and acetaldehyde. Thus, serine and threonine were supplemented alone or in combination with methionine, the latter being the likely first-limiting amino acid in ethionine toxicity. Weight gain was again reduced due to ethionine toxicity, and methionine ameliorated the toxicity (Table 5). However, chicks did not respond to either serine or threonine supplementation, regardless of whether supplemental methionine was present or absent. DISCUSSION
Results of these experiments indicate that dietary ethionine drastically reduces weight gain of young chicks, which is congruent with the work of Christensen and Anderson (1980). The improvement in performance upon adding either methionine or methyl donors (choline or betaine), but not cystine, suggests that the toxic effect of ethionine is mediated through an effect on methionine rather than cysteine. That both extra methionine and choline (or betaine) were ameliorative, moreover, suggests that at least
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
ethionine-containing diet ameliorated the ethionine toxicity, whereas dietary cystine had no effect. Both choline and betaine partially alleviated the depression in weight gain and feed efficiency caused by ethionine. In Experiment 2, graded levels of methionine and choline were fed to determine if they could completely overcome the deleterious effects of ethionine. There was a linear response in both weight gain and efficiency of feed utilization when methionine was added to the ethioninecontaining diet (Table 3). Choline supplementation improved gain and feed conversion, and the lowest level of supplemental choline (.20%) was as efficacious as the higher levels. Dietary ethionine addition resulted in a 6-fold increase in plasma glycine concentration while having no effect on plasma methionine or cystine levels (Table 4). Chicks supplemented with .50% methionine exhibited elevated plasma methionine and cystine levels, and plasma glycine concentration was reduced markedly. Choline supplementation lowered plasma glycine concentration to a lesser extent, but it had no effect on plasma methionine or cystine concentration. Experiment 3 was designed to investigate possible metabolic effects of the elevated plasma glycine levels observed with ethionine toxicity. Because glycine is a product of both serine and threonine metabolism, the increased plasma
ETHIONINE TOXICITY IN CHICKS TABLE 5. Performance of chicks fed ethioninecontaining diets supplemented with methionine, serine, threonine, or various combinations (Experiment 3)1 Gain 2
Gain
Basal (B) B + .075% DL-Ethionine As 2 + .30% DL-methionine As 2 + .75% L-serine As 2 + .75% L-threonine As 3 + .75% L-serine As 3 + .75% L-threonine
(g) 254 62 203 57 56 191 183
.652 .419 .636 .380 .394 .621 .639
3
.013
Pooled standard error of the mean
1 Data represent means of triplicate groups of five male chicks fed experimental diets from 8 to 22 days posthatching; chicks weighed 71.5 g at Day 8.
'Treatment 2 mean is significantly less (P<.001) than that of Treatment 1 or 3 (P<.001); Treatment means 4 and 5 are not significantly (P>.05) different from that of Treatment 2; Treatment means 6 and 7 are not significantly (P>.05) different from that of Treatment 3.
part of the toxicity may reside in decreased synthesis of S-adenosylmethionine, the obligatory methyl donor in choline biosynthesis. Thus, the ethyl analog of S-adenosylmethionine may be synthesized instead, as others have suggested. The linear improvement in gain observed with methionine supplementation, together with the lesser improvement in gain with choline, indicates that the primary effect of ethionine may be to interfere with methionine utilization per se, which may in turn cause a secondary methyl group deficiency. Shull et al. (1966) observed an increase in S-adenosylethionine in livers of rats administered ethionine. If ethionine competes with methionine for the enzyme (i.e., methionine adenosyl transferase), less methionine would be converted to S-adenosylmethionine, and reactions further down the pathway might be inhibited as well. Although other investigators have suggested that the toxic effects of ethionine are due to its catabolism by a transaminative pathway, our results may differ because we administered a lower dose of ethionine than these investigators (Steele and Benevenga, 1979; Steele, 1982). Steele (1982) supplemented his rat diet with .80% DLethionine, a level over 10 times greater than our
.075% addition. Thus, ethionine toxicity may act in two different ways: at low levels it may act on transsulfuration and transmethylation while at high levels it may saturate the enzymes of the transsulfuration pathway such that ethionine may have to be catabolized by an alternate transaminative pathway. The primary effect of ethionine on plasma amino acids appears to be on glycine. This amino acid increased dramatically in the plasma of ethionine-intoxicated chicks. This is in contrast to the work of Wu and Bollman (1954) wherein only a slight increase in plasma glycine was observed along with concomitant increases in other plasma amino acids when rats were injected with ethionine. The difference in response may be due to either species specificity or mode of administration of the ethionine, as the response observed by Wu and Bollman (1954) appeared to be a general effect of ethionine on protein catabolism, resulting in increased plasma amino acids while in our study ethionine appeared to primarily affect glycine metabolism. The increase in plasma glycine concentration could have resulted from changes in the activity of threonine aldolase or serine-hydroxymethylase, enzymes that catalyze the conversion of threonine and serine, respectively, to glycine, thus creating the possibility of a secondary deficiency of either threonine or serine. Although we did not measure the activity of these enzymes, the results of Experiment 3 suggested that neither serine nor threonine became deficient as a result of ethionine administration. Moreover, neither threonine nor serine were decreased in the plasma as a result of feeding ethionine. Addition of extra methionine to ethionine-intoxicated birds resulted in normalization of plasma glycine levels, but the mechanism of this response is not clear. It is possible that ethionine may somehow stimulate translocation of glycine from splanchnic tissues to plasma. It is also possible, however, that glycine uptake by renal tissue may be inhibited by ethionine or one of its metabolities. Still another possibility is that ethionine may inhibit glycine utilization for uric acid or bile acid biosynthesis. REFERENCES Benevenga, N. J., and R. D. Steele, 1984. Adverse effects of excessive consumption of amino acids. Annu. Rev. Nutr. 4:157-181. Christensen, A. C , and J. O. Anderson, 1980. Factors affecting efficacy of methionine hydroxy analogue for
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
Treatment
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1032
LOWRY AND BAKER
chicks fed practical diets. Poultry Sci. 59:2485-2491. Friedman, M. A., D. E. Berry, and R. P. Elzay, 1977. Acute lethality of D- and L-ethionine in Swiss mice. Cancer Lett. 3:71-76. Hsu, J. M., P. J. Buchanan, J. Anilane, and W. L. Anthony, 1968. Hepatic glutathionine concentrations linked to ethionine toxicity in rats. Biochem. J. 106:639-643. Shull, K. H., J. McConomy, M. Vogt, A. Castillo, and E. Farber, 1966. On the mechanism of induction of hepatic adenosine triphosphate deficiency by ethionine. J. Biol. Chem. 241:5060-5070. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics, McGraw-Hill Book Co., New
York, NY. Steele, R. D., 1982. Role of 3-ethylthiopropionate in ethionine metabolism and toxicity in rats. J. Nutr. 112:118-125. Steele, R. D., and N. J. Benevenga, 1979. Identification of a transaminative pathway for ethionine catabolism. Cancer Res. 39:3935-3941. Wu, C , and J. L. Bollman, 1954. Effect of ethionine on the free amino acids in the rat. J. Biol. Chem. 210:673680. Yamada, M., and J. Takahashi, 1977. Reversal of ethionine intoxication in the domestic fowl with methionine and adenine sulphate. Br. Poult. Sci. 18:567-571.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
1987 Poultry Science 66:1028-1032 INTRODUCTION
The toxic effects of ethionine, the ethyl analog of methionine, have been demonstrated in rats, mice and poultry (Hsu et al., 1968; Friedman et al., 1977; Christensen and Anderson, 1980). However, neither the specific mechanism by which ethionine exerts its toxic effects nor the best means of ameliorating the toxicity have been clearly defined. Some investigators have suggested that the toxic effect of ethionine results from metabolites produced in a transaminative pathway of ethionine catabolism (Steele and Benevenga, 1979; Steele, 1982; Benevenga and Steele, 1984) but other workers have suggested that the primary effect of ethionine is to react with ATP and thereby trap adenosine as S-adenosylethionine (Shull et al., 1966). Thus, both methionine and adenine administration have been found to ameliorate ethionine toxicity in mice (Friedman et al., 1977) whereas only methionine was found to ameliorate ethionine toxicity in poultry, although adenine enhanced the response due to methionine supplementation (Yamada and Takahashi, 1977). If the mechanism by which ethionine exerts its toxic effects can be elucidated, it may be possible to use ethionine as a
'To whom reprint requests should be addressed.
model to study sulfur amino acid metabolism, methyl donor activity and methionine bioavailability in practical corn-soy diets without fear that ethionine is acting elsewhere to confound the results. To examine the effects of ethionine and means of ameliorating its toxicity, a series of experiments were conducted. The objectives of the experiments were to determine if ethionine toxicity could be completely alleviated in chickens by the administration of either methionine or choline and to examine the effect of ethionine toxicity on plasma free amino acids. A third experiment was conducted to determine if the changes observed in plasma amino acid levels were due to secondary amino acid deficiencies caused by ethionine toxicity. MATERIALS AND METHODS
New Hampshire x Columbian chicks were used in each of the three experiments. Female chicks were used in Experiment 1 and male chicks were used in Experiments 2 and 3. The chicks were fed a corn-soybean meal starter diet during the first 7 days posthatching. On the 8th day posthatching, following an overnight fast, chicks were weighed and allotted to experimental groups so that all groups had similar mean initial weights and weight distributions. Triplicate groups of five chicks were placed on the experimental diets at 8 days posthatching. The
1028
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
ABSTRACT Several chick bioassays were conducted to evaluate means of ameliorating ethionine toxicity. Supplementing a corn-soy diet marginally deficient in sulfur amino acids (methionine + cystine) with .075% D,L-ethionine reduced weight gain in 8-day-old chicks by 70% compared to gains of unsupplemented controls. Dietary addition of .50% DL-methionine prevented reduction in weight gain and feed intake resulting from ethionine supplementation whereas feeding supplemental L-cystine was without effect. Supplementation of the ethionine-containing diet with either choline or betaine ameliorated the growth depression, although neither compound was able to completely overcome the toxic effects of ethionine. Dietary ethionine did not affect plasma levels of free methionine or cystine but did increase plasma free glycine 6-fold. Dietary addition of .50% DL-methionine caused normalization of plasma glycine levels whereas it elevated plasma methionine concentration. Although results suggested the possibility of ethionine-induced serine or threonine deficiency, dietary additions of .75% L-serine or .75% L-threonine failed to improve chick weight gain. These studies suggest that ethionine, in addition to affecting transsulfuration and transmethylation activity, may exert specific effects on certain amino acids in tissue pools. (Key words: ethionine, methionine, choline, betaine, plasma glycine)
ETHIONINE TOXICITY IN CHICKS TABLE 1. Composition of the basal diet Ingredient
1
52.13 37.00 2.00 1.00 4.00 2.20 1.00 .40 .05 .01 .10 .10 .01
Crude protein.
2
Vitamin premix provided per kilogram of diet: vitamin A, 4,400 IU; vitamin D 3 1,000 ICU; vitamin E, 11 IU; vitamin B , 2 , .01 mg; riboflavin, 4.41 mg; d-pantothenic acid, 10.0 mg; niacin, 22.0 mg; menadione sodium bisulfite, 2.3 3 mg.
chicks were housed in heated, thermostatically controlled wire-floor starter batteries, and a 24-h constant light schedule was maintained. Feed and water were provided ad libitum throughout the 14-day assay periods, and weight gain and feed intake were monitored at regular intervals. The composition of the basal 23% protein diet used in all three experiments is presented in Table 1. The basal diet was not supplemented with methionine in order to make it marginally deficient in sulfur amino acids (SAA). Experiment 1 was designed to examine the efficacy of SAA and methyl donors in ameliorating ethionine toxicity. Thus, isosulfurous dietary additions of DL-methionine (.25%) and L-cystine (.203%), as well as .20% choline-Cl and .20% betaine-HCl, were studied in terms of their capacity to overcome the growth depression caused by .075% DL-ethionine supplementation. In Experiment 2, the capacity of methionine and choline to completely alleviate the ethionine-induced growth depression was evaluated by adding graded levels of DLmethionine (.25% or .50%) or choline-Cl (.20%, .40% or .60%) to the ethionine-containing diet. At the termination of Experiment 2, the heaviest and lightest birds were culled from each replicate group and the three remaining chicks were bled by heart puncture using a heparinized syringe. The blood was pooled by replicate and plasma then prepared by centrifugation at 1300 x g for
15 min. The three pooled plasma samples for each dietary treatment were subsequently deproteinized with 3.5% sulfosalicylic acid, after which amino acid concentrations were quantified by ion-exchange chromatography using an automated amino analyzer (Model 6300, Beckman Instruments, Palo Alto, CA). Experiment 3 was conducted to determine if the elevated plasma glycine levels observed in Experiment 2 were indicative of an ethionine-induced deficiency of metabolic glycine precursors, i.e., serine or threonine. Thus, .75% additions of L-serine or L-threonine were evaluated in terms of their capacity to ameliorate ethionine toxicity in both the presence and absence of .30% supplemental DL-methionine (to assure that methionine deficiency would not limit the response to a second- or third-limiting amino acid). Pen means data for performance and plasma analyses were analyzed by analysis of variance procedures for a completely randomized design. Meaningful nonorthogonal single degree-offreedom comparisons were made to assess treatment effects (Steel and Torrie, 1980). RESULTS
The results of Experiment 1 are summarized in Table 2. Both weight gain and feed utilization were depressed severely by ethionine supplementation. Addition of methionine to the
TABLE 2. Performance of chicks fed ethioninecontaining diets supplemented with sulfur amino acids or methyl donors (Experiment l)1 Treatment
Gain2
Gain:feed 2
Basal (B) B + .075% DL-Ethionine As 2 + .25% DL-methionine As 2 + .203% L-cystine As 2 + .20% choline-Cl As 2 + .20% betaine«HCl
(g) 203 72 174 68 118 117
.579 .417 .615 .408 .547 .538
5
.011
Pooled standard error of the mean 1
Data represent means of triplicate groups of five female chicks fed experimental diets from 8 to 22 days posthatching; chicks weighed 66.9 g at Day 8. 2 Treatment means 1, 3, 5, and 6 are all significantly greater (P<.001) than that in Treatment 2; Treatment 4 mean is not significantly different (P>.05) from that of treatment 2.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
Ground corn Dehulled soybean meal (48% CP 1 ) Fishmeal, Menhaden (60% CP) Alfalfa meal, dehydrated (17% CP) Corn oil Dicalcium phosphate Ground limestone Iodized salt Manganese sulfate Zinc carbonate Choline chloride (60%) Vitamin premix 2 Lincomycin premix (4.4%)
Percent
1029
LOWRY AND BAKER
1030
TABLE 3. Performance of ethionine-intoxicated chicks fed graded levels of DL-methionine or choline (Experiment 2)1 Treatment
Gain 2
Gain:feed 2
(g) Basal (B) B + .075% DL-Ethionine As 2 + .25% DL-methionine As 2 + .50% DL-methionine As 2 + .20% choline°Cl As 2 + .40% choline-Cl As 2 + .60% choline'Cl Pooled standard error of the mean
230 64 167 231 138 148 144
.618 .417 .609 .679 .586 .585 .574
5
.011
1 Data represent means of triplicate groups of five male chicks fed experimental diets from 8 to 22 days posthatching; chicks weighed 67.6 at Day 8. 2 Significant (P<.001) linear effect due to methionine (Treatments 2 to 4); Treatment means 1, 3, 4, 5, 6, and 7 are all greater (P<.001) than that of Treatment 2.
TABLE 4. Plasma amino acid concentrations of ethionine-intoxicated chicks fed graded levels of DL-methionine or choline (Experiment 2) Plasma amino acid concentration Met 1
Treatment
Cys 1
Gly 2
(nmol/mL) Basal (B) B + .075% DL-Ethionine As 2 + .25% DL-methionine As 2 + .50% DL-methionine As 2 + .20% choline'Cl As 2 + .40% choline'Cl As 2 + .60% choline'Cl Pooled standard error of the mean
36 24 25 105 23 28 27 4.5
34 39 37 48 39 36 35 2.6
582 3,121 1,571 1,028 2,035 2,324 2,020 120
Significant (P<.05) quadratic increase due to methionine (treatments 2 to 4); no response to choline or ethionine (P>.05). 2 Significant (P<.001) increase due to ethionine and decrease (P-C001) due to choline (i.e., Treatment 5, 6, and 7 concentrations are less than Treatment 2); significant (P<.01) quadratic decrease due to methionine (Treatment 2 to 4).
glycine could have resulted from (ethionine-induced) enhanced flux in the serine transhydroxymethylase-catalyzed pathway of serine -• glycine or in the threonine aldolase pathway that results in threonine catabolism to glycine and acetaldehyde. Thus, serine and threonine were supplemented alone or in combination with methionine, the latter being the likely first-limiting amino acid in ethionine toxicity. Weight gain was again reduced due to ethionine toxicity, and methionine ameliorated the toxicity (Table 5). However, chicks did not respond to either serine or threonine supplementation, regardless of whether supplemental methionine was present or absent. DISCUSSION
Results of these experiments indicate that dietary ethionine drastically reduces weight gain of young chicks, which is congruent with the work of Christensen and Anderson (1980). The improvement in performance upon adding either methionine or methyl donors (choline or betaine), but not cystine, suggests that the toxic effect of ethionine is mediated through an effect on methionine rather than cysteine. That both extra methionine and choline (or betaine) were ameliorative, moreover, suggests that at least
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 27, 2015
ethionine-containing diet ameliorated the ethionine toxicity, whereas dietary cystine had no effect. Both choline and betaine partially alleviated the depression in weight gain and feed efficiency caused by ethionine. In Experiment 2, graded levels of methionine and choline were fed to determine if they could completely overcome the deleterious effects of ethionine. There was a linear response in both weight gain and efficiency of feed utilization when methionine was added to the ethioninecontaining diet (Table 3). Choline supplementation improved gain and feed conversion, and the lowest level of supplemental choline (.20%) was as efficacious as the higher levels. Dietary ethionine addition resulted in a 6-fold increase in plasma glycine concentration while having no effect on plasma methionine or cystine levels (Table 4). Chicks supplemented with .50% methionine exhibited elevated plasma methionine and cystine levels, and plasma glycine concentration was reduced markedly. Choline supplementation lowered plasma glycine concentration to a lesser extent, but it had no effect on plasma methionine or cystine concentration. Experiment 3 was designed to investigate possible metabolic effects of the elevated plasma glycine levels observed with ethionine toxicity. Because glycine is a product of both serine and threonine metabolism, the increased plasma
ETHIONINE TOXICITY IN CHICKS TABLE 5. Performance of chicks fed ethioninecontaining diets supplemented with methionine, serine, threonine, or various combinations (Experiment 3)1 Gain 2
Gain
Basal (B) B + .075% DL-Ethionine As 2 + .30% DL-methionine As 2 + .75% L-serine As 2 + .75% L-threonine As 3 + .75% L-serine As 3 + .75% L-threonine
(g) 254 62 203 57 56 191 183
.652 .419 .636 .380 .394 .621 .639
3
.013
Pooled standard error of the mean
1 Data represent means of triplicate groups of five male chicks fed experimental diets from 8 to 22 days posthatching; chicks weighed 71.5 g at Day 8.
'Treatment 2 mean is significantly less (P<.001) than that of Treatment 1 or 3 (P<.001); Treatment means 4 and 5 are not significantly (P>.05) different from that of Treatment 2; Treatment means 6 and 7 are not significantly (P>.05) different from that of Treatment 3.
part of the toxicity may reside in decreased synthesis of S-adenosylmethionine, the obligatory methyl donor in choline biosynthesis. Thus, the ethyl analog of S-adenosylmethionine may be synthesized instead, as others have suggested. The linear improvement in gain observed with methionine supplementation, together with the lesser improvement in gain with choline, indicates that the primary effect of ethionine may be to interfere with methionine utilization per se, which may in turn cause a secondary methyl group deficiency. Shull et al. (1966) observed an increase in S-adenosylethionine in livers of rats administered ethionine. If ethionine competes with methionine for the enzyme (i.e., methionine adenosyl transferase), less methionine would be converted to S-adenosylmethionine, and reactions further down the pathway might be inhibited as well. Although other investigators have suggested that the toxic effects of ethionine are due to its catabolism by a transaminative pathway, our results may differ because we administered a lower dose of ethionine than these investigators (Steele and Benevenga, 1979; Steele, 1982). Steele (1982) supplemented his rat diet with .80% DLethionine, a level over 10 times greater than our
.075% addition. Thus, ethionine toxicity may act in two different ways: at low levels it may act on transsulfuration and transmethylation while at high levels it may saturate the enzymes of the transsulfuration pathway such that ethionine may have to be catabolized by an alternate transaminative pathway. The primary effect of ethionine on plasma amino acids appears to be on glycine. This amino acid increased dramatically in the plasma of ethionine-intoxicated chicks. This is in contrast to the work of Wu and Bollman (1954) wherein only a slight increase in plasma glycine was observed along with concomitant increases in other plasma amino acids when rats were injected with ethionine. The difference in response may be due to either species specificity or mode of administration of the ethionine, as the response observed by Wu and Bollman (1954) appeared to be a general effect of ethionine on protein catabolism, resulting in increased plasma amino acids while in our study ethionine appeared to primarily affect glycine metabolism. The increase in plasma glycine concentration could have resulted from changes in the activity of threonine aldolase or serine-hydroxymethylase, enzymes that catalyze the conversion of threonine and serine, respectively, to glycine, thus creating the possibility of a secondary deficiency of either threonine or serine. Although we did not measure the activity of these enzymes, the results of Experiment 3 suggested that neither serine nor threonine became deficient as a result of ethionine administration. Moreover, neither threonine nor serine were decreased in the plasma as a result of feeding ethionine. Addition of extra methionine to ethionine-intoxicated birds resulted in normalization of plasma glycine levels, but the mechanism of this response is not clear. It is possible that ethionine may somehow stimulate translocation of glycine from splanchnic tissues to plasma. It is also possible, however, that glycine uptake by renal tissue may be inhibited by ethionine or one of its metabolities. Still another possibility is that ethionine may inhibit glycine utilization for uric acid or bile acid biosynthesis. REFERENCES Benevenga, N. J., and R. D. Steele, 1984. Adverse effects of excessive consumption of amino acids. Annu. Rev. Nutr. 4:157-181. Christensen, A. C , and J. O. Anderson, 1980. Factors affecting efficacy of methionine hydroxy analogue for
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chicks fed practical diets. Poultry Sci. 59:2485-2491. Friedman, M. A., D. E. Berry, and R. P. Elzay, 1977. Acute lethality of D- and L-ethionine in Swiss mice. Cancer Lett. 3:71-76. Hsu, J. M., P. J. Buchanan, J. Anilane, and W. L. Anthony, 1968. Hepatic glutathionine concentrations linked to ethionine toxicity in rats. Biochem. J. 106:639-643. Shull, K. H., J. McConomy, M. Vogt, A. Castillo, and E. Farber, 1966. On the mechanism of induction of hepatic adenosine triphosphate deficiency by ethionine. J. Biol. Chem. 241:5060-5070. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics, McGraw-Hill Book Co., New
York, NY. Steele, R. D., 1982. Role of 3-ethylthiopropionate in ethionine metabolism and toxicity in rats. J. Nutr. 112:118-125. Steele, R. D., and N. J. Benevenga, 1979. Identification of a transaminative pathway for ethionine catabolism. Cancer Res. 39:3935-3941. Wu, C , and J. L. Bollman, 1954. Effect of ethionine on the free amino acids in the rat. J. Biol. Chem. 210:673680. Yamada, M., and J. Takahashi, 1977. Reversal of ethionine intoxication in the domestic fowl with methionine and adenine sulphate. Br. Poult. Sci. 18:567-571.
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