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The Utilization of l -Methionine, dl -Methionine and Methionine Hydroxy Analogue by the Growing Chick1
The Utilization of L-Methionine, DL-Methionine and Methionine Hydroxy Analogue by the Growing Chick1 SMITH2
ROBERT E.
Canada Department of Agriculture, Research Branch, Nappan, Nova Scotia (Received for publication November 2, 1965)
M
1 Contribution No. 182. Department of Animal and Poultry Science. 2 Present address: Animal Research Institute, Central Experimental Farm, Ottawa, Ontario.
utilization of L-methionine, DL-methionine and methionine hydroxy analogue (Casalt) using both purified and semi-purified diets. The chief criterion of utilization was chick growth. In certain experiments plasma amino acid titres were determined. These values were compared with the growth results to see if this technique could be used to evaluate the different sources of methionine. EXPERIMENTAL Day-old male chicks (White Rock X Cornish) were fed a corn-soybean meal diet for a pretest period of 7 days. On the seventh day the chicks were selected for uniformity of weight. Chicks to be fed crystalline amino acid diets were subjected to the pre-experimental adaptation procedures of Green et al. (1962). The composition of the crystalline amino acid diet employed in Experiments 1, 2 and 5 is shown in Table 1. All amino acids, with the exception of methionine, were included in the diet in the L-form at levels capable of supporting maximum growth when sufficient methionine is supplied (Dean and Scott, 1962). Chicks were fed the experimental crystalline amino acid diets for a period of 6 days during which time daily weights and feed consumption were recorded. The composition of the intact protein diet employed in Experiments 3 and 4 is shown in Table 2. Chicks fed this diet were placed on the experimental diets immediately after selection at 7 days. Feed was offered on an ad libitum basis for the experimental period of 14 days. Chick weight and feed consumption were recorded at the
571
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ETHIONINE hydroxy analogue, because of its lower cost, is being used as a substitute for supplemental DLmethionine in commercial poultry rations. Nevertheless, its potency as a source of methionine activity for the chick remains a controversial question. Some workers (Gordon and Sizer, 1955; Machlin and Gordon, 1959; Bruggemann et al., 1962) have found it to be equivalent to DL-methionine, whereas others (Sullivan and Bird, 1957; Calet and Melot, 1961) report that it is less potent. All of these workers used diets containing intact protein ranging from 12% to optimal levels in the diet. There also appears to be a lack of agreement on the relative potencies of the L-, Dand DL-isomers of methionine. It has been reported that the D-isomer is as well utilized as the L- form when tested with purified diets in which the other amino acids are all in the L- form (Marrett et al., 1964) but not when the diet contains other amino acids in the D- form (Marrett and Sunde, 1965). However, Bruggemann et al. (1962) found D-methionine to be less potent than L-methionine added to a diet in which the other amino acids were supplied by protein, the amino acids being in the Lform. It would thus appear that other components of the diet may influence the relative utilization of the different isomers and analogues of methionine. In the following experiments, comparisons were made of the
572
R. E. SMITH TABLE 1.—Composition of crystalline amino acid diet
Ingredients Corn starch Amino acid mixture Corn oil, refined Mineral mix 1 Cellulose 2 NaHCOi Choline chloride Vitamins (2 gm./kg.) 1 Total
Percent 51.705 23.725 15.00 5.37 3.00 1.00 .20
+ ~—
100.00
Amino acid mixture
Total
1.33 (1.10)i .62 ( .50) 1.40(1.12) .63 .225 .68 .35
+
.85 1.20 .80 1.04 12.00 1.60 1.00 23.725
1 2
Klain el al., 1958. Solka Floe, a product of the Brown Company, Chicago 3, Illinois. 3 Free base.
end of the first and second weeks of the experiment. Supplemental amino acids were added at the expense of corn starch and glucose in the respective rations. The levels of supplemental methionine or its analogue were chosen so that maximum chick growth would not be achieved within at least the first three incremental levels. Chick gains were subjected to analysis of variance and when significant differences were obtained comparisons of means were conducted by either individual degree of
RESULTS AND DISCUSSION
Using a crystalline amino acid assay diet (Table 1) the L-isomer of methionine was found to support significantly (P < .01) greater gains than an equivalent amount of the racemized form (Tables 3 and 4). Maximum growth was achieved with less of the L- form of methionine than with the racemic isomer. This is indicated by significant quadratic and cubic effects in the former case but not in the latter (Table 4). Marrett and Sunde (1965) suggest that differences in utilization between D- and informs of methionine can only be demonstrated when the basal diet contains quantities of other n-amino acids. In the present work differences were demonstrated beTABLE 3.—Weight gain and feed utilization of chicks fed increasing dietary levels of L-methionine, DLmethionine and methionine hydroxy analogue in a crystalline amino acid diet Diet No.
TABLE 2.—Composition of intact protein basal diet Ingredient Glucose monohydrate Soybean meal (47.86% crude protein) 1 Mineral mix2 Corn oil, refined Choline chloride Vitamins (2 gm/kg.) 3 Penicillin (11 mg/kg.)
Percent 72.64 20.89 5.27 1.00 .20
+ + 100.00
1
Equivalent to a 10% crude protein diet. 2 Salts as a percent of the total diet. CaC03, 2.166; KH 2 P0 4 , 1.050; CaHP0 4 -2H 2 0, 0.940; NaCl, 0.800; MgS0 4 , 0.250; FeS0 4 -7H 2 0, 0.30; MnS0 4 •H 2 0, 0.020; ZnCOa, 0.010; CuS0 4 -5H 2 0, 0.002; KI, 0.001; Na 2 Mo0 4 -2H 2 0, 0.001. 'KlainrfaZ. (1958).
Supplement to basal diet 1
Gain/chick / d a y 2 (gm.)
Gain Feed
20% 25% 30% 35%
DL-methionine DL-methionine DL-methionine DL-methionine
5.22 9.33 13.61 16.00
.401 .522 .675 .-733
20% 25% 30% 35%
L-methionine L-methionine L-methionine L-methionine
6.28 10.56 15.33 15.78
.414 .558 .686 .707
15% 25% 35% 45%
DL-methionine DL-methionine DL-methionine DL-methionine
3.22 10.72 15.53 15.55
.286 .581 .713 .700
18% 30% 42% 54%
M. M. M. M.
2.22 6.33 13.22 15.34
.228 .434 .640 .709
H. H. H. H.
A." A. A. A.
i Table 1. 2 Average gain of 3 replicates of 3 chicks from 8-14 days of age.
3 Methionine hydroxy analogue calcium (Nutritional Biochemicals, Cleveland, Ohio). Equivalent ratio 8 3 % .
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L-arginine IICI L-histidine HC1 H 2 0 L-tysine HC1 L-tyrosine L-tryptophan L-phenylalanine L-cystine methionine L-threonine L-leucine L-isoleucine L-valine L-glutamic acid glycine L-proline
Percent
freedom analysis or by Duncan's multiple range test (1955). Plasma free amino acid titres were determined with chicks fed certain of the diets of Experiments 1 and 2. These studies involved the use of two sets of 50 chicks as per the method of Smith and Scott (1965).
573
METHIONINE AND ANALOGUE TABLE 4.—Analysis of variance of chick gains of Experiments 1 and 2 Experiment 1 Source Treatments DL- vs. L-methionine DL-methionine (linear) (quadratic) (cubic) L-methionine (linear) (quadratic) (cubic)
d.f.
M.S.
7 1
5,879 722**
1 1 1
20,823** 100 <1
1 1 1
18,000** 1,133** 376*
56
87
Source Treatments DL-methionine vs. M.H.A. DL-methionine (linear) (quadratic) (cubic) M.H.A. (linear) (quadratic) (cubic) Error
d.f.
M.S.
7 1
10,103 2,358**
1 1 1
27,801** 4,203** 32
1 1 1
35,084** 358* 929**
56
74
1
Analyzed on an individual chick basis. ** P < . 0 1 *P<.05
tween the L- form and the racemic mixture in spite of the absence of other D-amino acids. When the utilization of DL-methionine was compared with that of equimolar levels of methionine hydroxy analogue (Table 3), chick growth on the analogue was inferior to that with DL-methionine. Growth with DL-methionine approached maximum at the third level of supplementation as indicated by highly significant quadratic effects (Table 4). Growth on the methionine hydroxy analogue diets barely reached maximum level at the fourth level (Table 4). It would appear that as maximal growth is approached, differences in the efficiency of utilization of DL-methionine, L-methionine and methionine hydroxy analogue tend to disappear. Having demonstrated differences between DL- and L-methionine and between DL-methionine and methionine hydroxy analogue using crystalline diets, an attempt was made to see if similar results could be obtained when intact protein served as the source of amino acids in the assay diet (Experiment 3). The isomers of methionine and its analogue were compared in a lowprotein, soybean meal-glucose type diet
(Table 2). This diet was shown to be deficient in methionine (Table 5). Methionine hydroxy analogue appeared to be inferior to DL- and L-methionine but differences were not as marked as with the crystalline amino acid diets. Unlike Sullivan and Bird (1957) who demonstrated a response to urea or diammonium citrate, the addition of a source of non-specific nitrogen (glutamic acid) in this experiment did not have a beneficial effect upon chick gain or feed efficiency when fed along with methionine hydroxy analogue. A repeat of the first 7 treatments of Experiment 3 was conducted with the exception that methionine hydroxy analogue supplement was added on an equimolar basis. Once again a significant deficiency of methionine is indicated in the basal diet (Table 5). In this trial there were no differences in utilization of the three sources of methionine at any level in the two week trial. Why methionine hydroxy analogue is as well utilized on an equimolar basis as DLmethionine when assayed in intact protein diets but is obviously poorer in crystalline amino acid diets is not clear. Since most crystalline amino acid assays are costly
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Error 1
Experiment 2
574
R. E. SMITH TABLE 5.—Weight gain and feed utilization of chicks fed a low protein soybean diet supplemented with DL- and L-methionine and methionine hydroxy analogue
Diet No.
Supplement tci basal soy diet1
7-14 days 2,3
7-21 days 2 ' 3
Gain
Av. gain (gm.)
Av. gain (gm.)
Feed
Nil .05% DL-methionine . 10% DL-methionine .05% L-methionine . 10% L-methionine .05%M.H.A. . 10% M.H.A. .05% M.H.A.+ .05% L-glutamic acid
51= 63" 66» 62»b 66" 60»b 62"b 57b
98" 124b° 146" 120b° 140* 128b 109"
.325 .345 .398 .367 .395 .347 .371 .333
Experiment 4 1 2 3 4 5 6 7
Nil .05% DL-methionine . 10% DL-methionine . 05% L-methionine . 10% L-methionine .06% M.H.A. ( = . 0 5 % L-methionine) .12% M.H.A. ( = . 1 0 % L-methionine)
50"d 63°
92d 131" 146"b 134b° 150" 127° 145"b
.311 .366 .381 .366 .391 .357 .384
1 2 3
7 jab 65bcd
72" 61 d 6 gabc
117od
Table 2. Average gain of triplicate groups of 10 chicks each. Any 2 mean values within a column not having one letter in common differ significantly (P<0.05).
they are usually conducted for 1 week only. This may not allow sufficient time for .adaptation on the part of the chick. How-ever, comparison of the first and second week chick gains on the semi-purified diet (Table 5) does not support this hypothesis. In crystalline amino acid diets the entire source of methionine arises from one of the three test substances whereas in the semipurified diet the protein supplies about 75% of the methionine in the natural L-
form, thus leaving a very small proportion of the methionine requirement to be supplied by the test substance. This appears a more likely explanation of the disparity in results using purified versus semi-purified assay diets. To check this point a final experiment (Experiment 5) was conducted using the crystalline amino acid assay diet. In this diet L-methionine was incorporated at a level equal to that provided by the soybean
TABLE 6.—Weight gain and feed efficiency of chicks fed various dietary levels of L-methionine, DL-methionine and methionine hydroxy analogue in a crystalline amino acid diet Diet No. Experiment 5 1 2 3 4 5 6 7 8 1 1
Supplement to basal diet 1
. 25% L-methionine .20% L-methionine+.05% .20% L-methionine+ .06% .30% M.H.A. .30% L-methionine . 20% L-methionine-r-. 10% . 20% L-methionine + . 12% .36% M.H.A.
DL-methionine M.H.A. DL-methionine M.H.A.
Gain/chick /day 2 , 3 (gm.)
Gain Feed
12.651b11.67 9.97° 5.87* 15.38" 15.78" 14.98" 9.80°
.655 .615 .611 .448 .738 .752 .705 .583
Table 1. Average gain of 3 replicates of 3 chicks from 8-14 days of age. Any 2 mean values not having one letter in common differ significantly (P<0.05).
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Experiment 3 1 2 3 4 5 6 7 8
575
METHIONINE AND ANALOGUE TABLE 7. —Plasma
free amino acid titres of chicks fed DL-, L-methionine or methionine hydroxy analogue Experiment 7
Experiment 6 L--methionine
M . -methionine
.20
1 2 3
123.61 81.6 17.0 6.3 25.4 33.7 16.3 16.1 116.7 37.8 81.5 102.9 82.0 37.6 109.3 116.1
.25
.30
.20
.25
.30
120.6 68.3 19.3 6.8 25.5 24.6 17.7 14.8 104.2 44.2 60.7 104.4 67.6 47.1 129.4 186.7
119.2 60.2 21.6 13.0 12.4 14.3 14.7 12.1 85.4 32.0 53.6 107.8 55.7 42.8 113.7 182.8
199.8 77.2 17.0 8.0 34.5 32.2 17.2 17.0 95.7 34.7 91.2 114.0 77.0 40.9 129.2 154.5
208.9 66.9 19.6 6.7 23.6 21.9 15.9 15.7 85.2 32.0 64.3 108.7 68.8 53.5 119.9 199.1
204.6 63.1 18.7 10.6 23.2 17.1 13.1 13.3 105.2 32.8 57.8 112.1 63.9 37.1 116.9 199.0
.25 137.5 73.9 12.7 5.1 38.5 34.4 16.1 19.0 107.9 . 29.7 61.8 128.6 88.6 67.9 95.8 113.9
.35
.45
.30
.42
.54
154.5 65.6 19.5 7.6 24.7 22.1 20.5 15.1 99.3 37.8 60.6 84.5 73.8 58.1 85.9 130.9
137.8 58.7 22.1 11.1 22.1 9.1 13.8 8.5 66.4 29.4 38.2 81.8 45.7 52.1 102.8 175.8
155.7 69.2 13.1 5.3 26.6 31.9 14.4 17.5 102.9 31.8 69.2 161.9 90.2 60.0 122.9 133.5
116.9 76.1 19.2 6.5 28.5 26.7 24.5 17.7 120.8 43.3 77.9 77.0 65.6 54.5 119.1 151.0
98.1 67.2 19.6 8.7 23.5 21.4 19.9 14.9 87.9 35.4 54.7 77.8 62.7 50.0 104.4 178.7
Micrograms/ml. plasma. Corrected for methionine sulfoxides. Includes 3-methyl histidine.
in the semi-purified basal used previously. Thus the basal diets of Experiments 4 and 5 were comparable with respect to methionine except that in the latter situation the methionine was provided in crystalline as opposed to protein form. The test materials were then added at the same level as in Experiment 4. Here again methionine hydroxy analogue appeared to be less well utilized, but there was the same lack of sensitivity as with the semi-purified diet (Table 6). The lack of sensitivity when approximately 75% of the dietary methionine is provided in the L- form is not surprising. Under these circumstances the tests are being conducted at a relatively high point on the growth curve and using rather low levels of the test materials. There appears to be no essential difference whether the non-sulfur amino acids are supplied in protein or crystalline form. Plasma amino acid titres were obtained to see if they could be used as a measure of the potency of the test substances. In general, as the dietary level of methionine sources was increased, the plasma level of the other, essential amino acids decreased (Table 7). The plasma level of the sulfur-
amino acids remained constant with increasing supplementation until dietary levels presumably exceeding the chicks' requirement were reached. At this point a sharp increase in plasma titre occurred. Specifically, plasma amino acid titres responded to increasing levels of L- and DLmethionine supplementation in much the same way. However, when methionine hydroxy analogue was the supplement, plasma titres of the essential amino acids did not decrease to the same extent as when DLmethionine was used and the rise in the sulfur amino acids was less marked. It is a logical premise that a diet deficient in an amino acid will cause an accumulation of the other essential amino acids in the plasma. As the dietary level of the limiting amino acid increases, blood titres of the other amino acids decrease, a reflection of increased utilization for protein synthesis. At the same time the limiting amino acid will remain at a constant level until dietary levels exceed requirement (Zimmerman and Scott, 1965). The data in this study strongly support this premise. If an amino acid source is not well uti-
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Threonine Valine Cystine Methionine 2 Isoleucine Leucine Tyrosine Phenylalanine Lysine His ti dine 5 Arginine Serine Proline Glutamic acid Glycine Alanine
Methionine hydroxy analogue
DL -methionine
576
R. E. SMITH
SUMMARY Three sources of methionine were compared in purified and semi-purified basal diets fed to growing chicks. Rate of gain, feed efficiency and in certain cases blood amino acid titres were used as the criteria of comparison. L-methionine was more efficiently utilized than the DL- form when tested in crystalline amino acid diets. DL-methionine in turn was superior to equimolar amounts of methionine hydroxy analogue. When they were compared using a semipurified basal diet, or a crystalline amino acid diet simulating the former, methionine hydroxy analogue again appeared to be inferior to the other forms of methionine, but the assay was not sufficiently sensitive to prove this conclusively. The lack of sensitivity was considered due to the high proportion of natural (L)-methionine in the semi-purified basal diet. This resulted in
the assays being conducted high on the growth curve, using low levels of the test materials. There was no evidence that the presence of protein as such in the basal diet has any influence on the utilization of the three sources of methionine tested. The positive relationship between plasma amino acid titres and growth as criteria for measuring the potency of methionine source materials is discussed. REFERENCES Briiggemann, J., K. Drepper and H. Zucker, 1962. Quantitative Bestimmung der Verwertung von D-, L-, DL-Methionine and Ca-ra,-2-Hydroxy-4methylthiobutyrat durch das Hiihnerkiiken. Die Naturwissenschaften, 49: 334. Calet, C , and M. Melot, 1961. Comparative efficacy of methionine and the calcium salt of hydroxy-methyl-thiobutyric acid (M.H.A.) for chicken growth. Ann. Zootech. .10: 205-213. Dean, W. F., and H. M. Scott, 1962. The development of an amino acid standard for the early growth of chicks. Poultry Sci. 4 1 : 1640. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Gordon, R. S., and I. W. Sizer, 1955. The biological equivalence of methionine hydroxy analogue. Poultry Sci. 34: 1198. Green, D. E., H. M. Scott and B. C. Johnson, 1962. The role of proline and certain non-essential amino acids in chick nutrition. Poultry Sci. 4 1 : 116-120. Klain, G. J., H. M. Scott and B. C. Johnson, 1958. The amino acid requirements of the growing chick fed crystalline amino acids. Poultry Sci. 37: 976-977. Machlin, L. J., and R. S. Gordon, 1959. Equivalence of methionine hydroxy analogue and methionine for chickens fed low protein diets. Poultry Sci. 38:650-652. Marrett, L., H. R. Bird and M. L. Sunde, 1964. The effects of different isomers of methionine on growth of chicks fed amino acid diets. Poultry Sci. 4 3 : 1113-1118. Marrett, L. E., and M. L. Sunde, 1965. The effect of other D amino acids on the utilization of the isomers of methionine and its hydroxy analogue. Poultry Sci. 44: 957-964. Smith, R. E., and H. M. Scott, 1965. The use of free amino acid concentrations in blood plasma
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lized, one would expect the decrease in the other essential amino acids to be more gradual and the point of abrupt increase in the titre of the deficient amino acid to occur at a higher dietary level. There is some suggestion that this has been the case with methionine hydroxy analogue as compared with DL-methionine. Maximum growth occurred at the second level of supplementation with DL-methionine, but not until the third level with methionine hydroxy analogue (Table 3). Similarly the amino acid titres for all essential amino acids were comparable at the second and third levels of supplementation of DL-methionine and methionine hydroxy analogue respectively (Table 7—Expt. 7). Thus, there appears to be some agreement between the interpretation of plasma amino acid titres and growth rate as indicators of the potency of the methionine sources studied.
METHIONINE AND ANALOGUE in evaluating the amino acid adequacy of intact proteins for chick growth. I. Free amino acid patterns of blood plasma of chicks fed unheated and heated fish meal proteins. J. Nutrition, 86: 37^t4. Sullivan, T. W., and H. R. Bird, 1957. Effect of quantity and source of dietary nitrogen on the utilization of the hydroxy analogues of methi-
577
onine and glycine by chicks. J. Nutrition, 62: 143-150. Zimmerman, R. A., and H. M. Scott, 1965. Interrelationship of plasma amino acid levels and weight gain in the chick as influenced by suboptimal and superoptimal dietary concentrations of single amino acids. J. Nutrition, 87 : 13-18.
Isolation and Tentative Identification of the Carotenoids Present in Chicken Skin and Egg Yolks1
(Received for Publication November 4, 1965)
groups of investigators have SEVERAL studied the characteristics of the carotenoids deposited in the skin and egg yolk of chickens (Palmer, 1915; Brockman and Volker, 1934; Zechmeister and Tuzson, 1934; Peterson et al, 1939; Schrenk et al., 1944; Thompson et al., 1946; Ganguly et al., 1953; Marusich et al, 1960; Anjaneyalu, 1962; and Williams et al., 1963). Much of this earlier work has been reviewed by Goodwin (1954). Generally, in the work cited, identification of the carotenoids present has depended upon phasic separation between different solvent systems. Though in the work of Schrenk et al. (1944), Thompson et al. (1946) and Anjaneyalu (1962) adsorption chromatography was used in identifying the pigments present in egg yolks. The identity of the carotenoids deposited in the egg yolk and skin of chickens in the studies reported by Marusich et al. (1960) and Williams et al. (1963) was presumptive since they fed isolated carotenoids and the resulting pig1 Presented in part before the meeting of the American Institute of Nutrition, April, 1963 (Federation Proa, 22: 201, 1963, abstract). 2 Agricultural and Veterinary Research Division.
ments deposited were assumed to be the same as the original pigments fed. The purposes of the studies reported here were: 1) to measure the relative amounts of carotenoids deposited in the skin and egg yolks obtained from chickens fed different dietary sources of carotenoids, and 2) to obtain further information on the identity of the carotenoids deposited, especially those deposited in the skin. EXPERIMENTAL PROCEDURE Feeding—Broilers and laying hens were supplied dietary carotenoids from 1) a combination of yellow corn and alfalfa meal in either a standard broiler or layer diet; or 2) dried algae meal {Chlorella pyrenoidosa) in a standard broiler or layer diet where the alfalfa was omitted and yellow corn was replaced with milo. The main hydroxy carotenoids found in these different sources are presented in Table 1. Chemical—The skin, taken from the shank area, or egg yolks were extracted repeatedly with acetone until essentially all of the pigment was removed. The acetone extracts were concentrated under a stream of nitrogen, transferred into diethyl ether, reconcentrated, and transferred into redis-
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I. D. SMITH AND H. S. PERDUE Abbott Laboratories, North Chicago, Illinois2
ROBERT E.
Canada Department of Agriculture, Research Branch, Nappan, Nova Scotia (Received for publication November 2, 1965)
M
1 Contribution No. 182. Department of Animal and Poultry Science. 2 Present address: Animal Research Institute, Central Experimental Farm, Ottawa, Ontario.
utilization of L-methionine, DL-methionine and methionine hydroxy analogue (Casalt) using both purified and semi-purified diets. The chief criterion of utilization was chick growth. In certain experiments plasma amino acid titres were determined. These values were compared with the growth results to see if this technique could be used to evaluate the different sources of methionine. EXPERIMENTAL Day-old male chicks (White Rock X Cornish) were fed a corn-soybean meal diet for a pretest period of 7 days. On the seventh day the chicks were selected for uniformity of weight. Chicks to be fed crystalline amino acid diets were subjected to the pre-experimental adaptation procedures of Green et al. (1962). The composition of the crystalline amino acid diet employed in Experiments 1, 2 and 5 is shown in Table 1. All amino acids, with the exception of methionine, were included in the diet in the L-form at levels capable of supporting maximum growth when sufficient methionine is supplied (Dean and Scott, 1962). Chicks were fed the experimental crystalline amino acid diets for a period of 6 days during which time daily weights and feed consumption were recorded. The composition of the intact protein diet employed in Experiments 3 and 4 is shown in Table 2. Chicks fed this diet were placed on the experimental diets immediately after selection at 7 days. Feed was offered on an ad libitum basis for the experimental period of 14 days. Chick weight and feed consumption were recorded at the
571
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ETHIONINE hydroxy analogue, because of its lower cost, is being used as a substitute for supplemental DLmethionine in commercial poultry rations. Nevertheless, its potency as a source of methionine activity for the chick remains a controversial question. Some workers (Gordon and Sizer, 1955; Machlin and Gordon, 1959; Bruggemann et al., 1962) have found it to be equivalent to DL-methionine, whereas others (Sullivan and Bird, 1957; Calet and Melot, 1961) report that it is less potent. All of these workers used diets containing intact protein ranging from 12% to optimal levels in the diet. There also appears to be a lack of agreement on the relative potencies of the L-, Dand DL-isomers of methionine. It has been reported that the D-isomer is as well utilized as the L- form when tested with purified diets in which the other amino acids are all in the L- form (Marrett et al., 1964) but not when the diet contains other amino acids in the D- form (Marrett and Sunde, 1965). However, Bruggemann et al. (1962) found D-methionine to be less potent than L-methionine added to a diet in which the other amino acids were supplied by protein, the amino acids being in the Lform. It would thus appear that other components of the diet may influence the relative utilization of the different isomers and analogues of methionine. In the following experiments, comparisons were made of the
572
R. E. SMITH TABLE 1.—Composition of crystalline amino acid diet
Ingredients Corn starch Amino acid mixture Corn oil, refined Mineral mix 1 Cellulose 2 NaHCOi Choline chloride Vitamins (2 gm./kg.) 1 Total
Percent 51.705 23.725 15.00 5.37 3.00 1.00 .20
+ ~—
100.00
Amino acid mixture
Total
1.33 (1.10)i .62 ( .50) 1.40(1.12) .63 .225 .68 .35
+
.85 1.20 .80 1.04 12.00 1.60 1.00 23.725
1 2
Klain el al., 1958. Solka Floe, a product of the Brown Company, Chicago 3, Illinois. 3 Free base.
end of the first and second weeks of the experiment. Supplemental amino acids were added at the expense of corn starch and glucose in the respective rations. The levels of supplemental methionine or its analogue were chosen so that maximum chick growth would not be achieved within at least the first three incremental levels. Chick gains were subjected to analysis of variance and when significant differences were obtained comparisons of means were conducted by either individual degree of
RESULTS AND DISCUSSION
Using a crystalline amino acid assay diet (Table 1) the L-isomer of methionine was found to support significantly (P < .01) greater gains than an equivalent amount of the racemized form (Tables 3 and 4). Maximum growth was achieved with less of the L- form of methionine than with the racemic isomer. This is indicated by significant quadratic and cubic effects in the former case but not in the latter (Table 4). Marrett and Sunde (1965) suggest that differences in utilization between D- and informs of methionine can only be demonstrated when the basal diet contains quantities of other n-amino acids. In the present work differences were demonstrated beTABLE 3.—Weight gain and feed utilization of chicks fed increasing dietary levels of L-methionine, DLmethionine and methionine hydroxy analogue in a crystalline amino acid diet Diet No.
TABLE 2.—Composition of intact protein basal diet Ingredient Glucose monohydrate Soybean meal (47.86% crude protein) 1 Mineral mix2 Corn oil, refined Choline chloride Vitamins (2 gm/kg.) 3 Penicillin (11 mg/kg.)
Percent 72.64 20.89 5.27 1.00 .20
+ + 100.00
1
Equivalent to a 10% crude protein diet. 2 Salts as a percent of the total diet. CaC03, 2.166; KH 2 P0 4 , 1.050; CaHP0 4 -2H 2 0, 0.940; NaCl, 0.800; MgS0 4 , 0.250; FeS0 4 -7H 2 0, 0.30; MnS0 4 •H 2 0, 0.020; ZnCOa, 0.010; CuS0 4 -5H 2 0, 0.002; KI, 0.001; Na 2 Mo0 4 -2H 2 0, 0.001. 'KlainrfaZ. (1958).
Supplement to basal diet 1
Gain/chick / d a y 2 (gm.)
Gain Feed
20% 25% 30% 35%
DL-methionine DL-methionine DL-methionine DL-methionine
5.22 9.33 13.61 16.00
.401 .522 .675 .-733
20% 25% 30% 35%
L-methionine L-methionine L-methionine L-methionine
6.28 10.56 15.33 15.78
.414 .558 .686 .707
15% 25% 35% 45%
DL-methionine DL-methionine DL-methionine DL-methionine
3.22 10.72 15.53 15.55
.286 .581 .713 .700
18% 30% 42% 54%
M. M. M. M.
2.22 6.33 13.22 15.34
.228 .434 .640 .709
H. H. H. H.
A." A. A. A.
i Table 1. 2 Average gain of 3 replicates of 3 chicks from 8-14 days of age.
3 Methionine hydroxy analogue calcium (Nutritional Biochemicals, Cleveland, Ohio). Equivalent ratio 8 3 % .
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L-arginine IICI L-histidine HC1 H 2 0 L-tysine HC1 L-tyrosine L-tryptophan L-phenylalanine L-cystine methionine L-threonine L-leucine L-isoleucine L-valine L-glutamic acid glycine L-proline
Percent
freedom analysis or by Duncan's multiple range test (1955). Plasma free amino acid titres were determined with chicks fed certain of the diets of Experiments 1 and 2. These studies involved the use of two sets of 50 chicks as per the method of Smith and Scott (1965).
573
METHIONINE AND ANALOGUE TABLE 4.—Analysis of variance of chick gains of Experiments 1 and 2 Experiment 1 Source Treatments DL- vs. L-methionine DL-methionine (linear) (quadratic) (cubic) L-methionine (linear) (quadratic) (cubic)
d.f.
M.S.
7 1
5,879 722**
1 1 1
20,823** 100 <1
1 1 1
18,000** 1,133** 376*
56
87
Source Treatments DL-methionine vs. M.H.A. DL-methionine (linear) (quadratic) (cubic) M.H.A. (linear) (quadratic) (cubic) Error
d.f.
M.S.
7 1
10,103 2,358**
1 1 1
27,801** 4,203** 32
1 1 1
35,084** 358* 929**
56
74
1
Analyzed on an individual chick basis. ** P < . 0 1 *P<.05
tween the L- form and the racemic mixture in spite of the absence of other D-amino acids. When the utilization of DL-methionine was compared with that of equimolar levels of methionine hydroxy analogue (Table 3), chick growth on the analogue was inferior to that with DL-methionine. Growth with DL-methionine approached maximum at the third level of supplementation as indicated by highly significant quadratic effects (Table 4). Growth on the methionine hydroxy analogue diets barely reached maximum level at the fourth level (Table 4). It would appear that as maximal growth is approached, differences in the efficiency of utilization of DL-methionine, L-methionine and methionine hydroxy analogue tend to disappear. Having demonstrated differences between DL- and L-methionine and between DL-methionine and methionine hydroxy analogue using crystalline diets, an attempt was made to see if similar results could be obtained when intact protein served as the source of amino acids in the assay diet (Experiment 3). The isomers of methionine and its analogue were compared in a lowprotein, soybean meal-glucose type diet
(Table 2). This diet was shown to be deficient in methionine (Table 5). Methionine hydroxy analogue appeared to be inferior to DL- and L-methionine but differences were not as marked as with the crystalline amino acid diets. Unlike Sullivan and Bird (1957) who demonstrated a response to urea or diammonium citrate, the addition of a source of non-specific nitrogen (glutamic acid) in this experiment did not have a beneficial effect upon chick gain or feed efficiency when fed along with methionine hydroxy analogue. A repeat of the first 7 treatments of Experiment 3 was conducted with the exception that methionine hydroxy analogue supplement was added on an equimolar basis. Once again a significant deficiency of methionine is indicated in the basal diet (Table 5). In this trial there were no differences in utilization of the three sources of methionine at any level in the two week trial. Why methionine hydroxy analogue is as well utilized on an equimolar basis as DLmethionine when assayed in intact protein diets but is obviously poorer in crystalline amino acid diets is not clear. Since most crystalline amino acid assays are costly
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Error 1
Experiment 2
574
R. E. SMITH TABLE 5.—Weight gain and feed utilization of chicks fed a low protein soybean diet supplemented with DL- and L-methionine and methionine hydroxy analogue
Diet No.
Supplement tci basal soy diet1
7-14 days 2,3
7-21 days 2 ' 3
Gain
Av. gain (gm.)
Av. gain (gm.)
Feed
Nil .05% DL-methionine . 10% DL-methionine .05% L-methionine . 10% L-methionine .05%M.H.A. . 10% M.H.A. .05% M.H.A.+ .05% L-glutamic acid
51= 63" 66» 62»b 66" 60»b 62"b 57b
98" 124b° 146" 120b° 140* 128b 109"
.325 .345 .398 .367 .395 .347 .371 .333
Experiment 4 1 2 3 4 5 6 7
Nil .05% DL-methionine . 10% DL-methionine . 05% L-methionine . 10% L-methionine .06% M.H.A. ( = . 0 5 % L-methionine) .12% M.H.A. ( = . 1 0 % L-methionine)
50"d 63°
92d 131" 146"b 134b° 150" 127° 145"b
.311 .366 .381 .366 .391 .357 .384
1 2 3
7 jab 65bcd
72" 61 d 6 gabc
117od
Table 2. Average gain of triplicate groups of 10 chicks each. Any 2 mean values within a column not having one letter in common differ significantly (P<0.05).
they are usually conducted for 1 week only. This may not allow sufficient time for .adaptation on the part of the chick. How-ever, comparison of the first and second week chick gains on the semi-purified diet (Table 5) does not support this hypothesis. In crystalline amino acid diets the entire source of methionine arises from one of the three test substances whereas in the semipurified diet the protein supplies about 75% of the methionine in the natural L-
form, thus leaving a very small proportion of the methionine requirement to be supplied by the test substance. This appears a more likely explanation of the disparity in results using purified versus semi-purified assay diets. To check this point a final experiment (Experiment 5) was conducted using the crystalline amino acid assay diet. In this diet L-methionine was incorporated at a level equal to that provided by the soybean
TABLE 6.—Weight gain and feed efficiency of chicks fed various dietary levels of L-methionine, DL-methionine and methionine hydroxy analogue in a crystalline amino acid diet Diet No. Experiment 5 1 2 3 4 5 6 7 8 1 1
Supplement to basal diet 1
. 25% L-methionine .20% L-methionine+.05% .20% L-methionine+ .06% .30% M.H.A. .30% L-methionine . 20% L-methionine-r-. 10% . 20% L-methionine + . 12% .36% M.H.A.
DL-methionine M.H.A. DL-methionine M.H.A.
Gain/chick /day 2 , 3 (gm.)
Gain Feed
12.651b11.67 9.97° 5.87* 15.38" 15.78" 14.98" 9.80°
.655 .615 .611 .448 .738 .752 .705 .583
Table 1. Average gain of 3 replicates of 3 chicks from 8-14 days of age. Any 2 mean values not having one letter in common differ significantly (P<0.05).
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Experiment 3 1 2 3 4 5 6 7 8
575
METHIONINE AND ANALOGUE TABLE 7. —Plasma
free amino acid titres of chicks fed DL-, L-methionine or methionine hydroxy analogue Experiment 7
Experiment 6 L--methionine
M . -methionine
.20
1 2 3
123.61 81.6 17.0 6.3 25.4 33.7 16.3 16.1 116.7 37.8 81.5 102.9 82.0 37.6 109.3 116.1
.25
.30
.20
.25
.30
120.6 68.3 19.3 6.8 25.5 24.6 17.7 14.8 104.2 44.2 60.7 104.4 67.6 47.1 129.4 186.7
119.2 60.2 21.6 13.0 12.4 14.3 14.7 12.1 85.4 32.0 53.6 107.8 55.7 42.8 113.7 182.8
199.8 77.2 17.0 8.0 34.5 32.2 17.2 17.0 95.7 34.7 91.2 114.0 77.0 40.9 129.2 154.5
208.9 66.9 19.6 6.7 23.6 21.9 15.9 15.7 85.2 32.0 64.3 108.7 68.8 53.5 119.9 199.1
204.6 63.1 18.7 10.6 23.2 17.1 13.1 13.3 105.2 32.8 57.8 112.1 63.9 37.1 116.9 199.0
.25 137.5 73.9 12.7 5.1 38.5 34.4 16.1 19.0 107.9 . 29.7 61.8 128.6 88.6 67.9 95.8 113.9
.35
.45
.30
.42
.54
154.5 65.6 19.5 7.6 24.7 22.1 20.5 15.1 99.3 37.8 60.6 84.5 73.8 58.1 85.9 130.9
137.8 58.7 22.1 11.1 22.1 9.1 13.8 8.5 66.4 29.4 38.2 81.8 45.7 52.1 102.8 175.8
155.7 69.2 13.1 5.3 26.6 31.9 14.4 17.5 102.9 31.8 69.2 161.9 90.2 60.0 122.9 133.5
116.9 76.1 19.2 6.5 28.5 26.7 24.5 17.7 120.8 43.3 77.9 77.0 65.6 54.5 119.1 151.0
98.1 67.2 19.6 8.7 23.5 21.4 19.9 14.9 87.9 35.4 54.7 77.8 62.7 50.0 104.4 178.7
Micrograms/ml. plasma. Corrected for methionine sulfoxides. Includes 3-methyl histidine.
in the semi-purified basal used previously. Thus the basal diets of Experiments 4 and 5 were comparable with respect to methionine except that in the latter situation the methionine was provided in crystalline as opposed to protein form. The test materials were then added at the same level as in Experiment 4. Here again methionine hydroxy analogue appeared to be less well utilized, but there was the same lack of sensitivity as with the semi-purified diet (Table 6). The lack of sensitivity when approximately 75% of the dietary methionine is provided in the L- form is not surprising. Under these circumstances the tests are being conducted at a relatively high point on the growth curve and using rather low levels of the test materials. There appears to be no essential difference whether the non-sulfur amino acids are supplied in protein or crystalline form. Plasma amino acid titres were obtained to see if they could be used as a measure of the potency of the test substances. In general, as the dietary level of methionine sources was increased, the plasma level of the other, essential amino acids decreased (Table 7). The plasma level of the sulfur-
amino acids remained constant with increasing supplementation until dietary levels presumably exceeding the chicks' requirement were reached. At this point a sharp increase in plasma titre occurred. Specifically, plasma amino acid titres responded to increasing levels of L- and DLmethionine supplementation in much the same way. However, when methionine hydroxy analogue was the supplement, plasma titres of the essential amino acids did not decrease to the same extent as when DLmethionine was used and the rise in the sulfur amino acids was less marked. It is a logical premise that a diet deficient in an amino acid will cause an accumulation of the other essential amino acids in the plasma. As the dietary level of the limiting amino acid increases, blood titres of the other amino acids decrease, a reflection of increased utilization for protein synthesis. At the same time the limiting amino acid will remain at a constant level until dietary levels exceed requirement (Zimmerman and Scott, 1965). The data in this study strongly support this premise. If an amino acid source is not well uti-
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Threonine Valine Cystine Methionine 2 Isoleucine Leucine Tyrosine Phenylalanine Lysine His ti dine 5 Arginine Serine Proline Glutamic acid Glycine Alanine
Methionine hydroxy analogue
DL -methionine
576
R. E. SMITH
SUMMARY Three sources of methionine were compared in purified and semi-purified basal diets fed to growing chicks. Rate of gain, feed efficiency and in certain cases blood amino acid titres were used as the criteria of comparison. L-methionine was more efficiently utilized than the DL- form when tested in crystalline amino acid diets. DL-methionine in turn was superior to equimolar amounts of methionine hydroxy analogue. When they were compared using a semipurified basal diet, or a crystalline amino acid diet simulating the former, methionine hydroxy analogue again appeared to be inferior to the other forms of methionine, but the assay was not sufficiently sensitive to prove this conclusively. The lack of sensitivity was considered due to the high proportion of natural (L)-methionine in the semi-purified basal diet. This resulted in
the assays being conducted high on the growth curve, using low levels of the test materials. There was no evidence that the presence of protein as such in the basal diet has any influence on the utilization of the three sources of methionine tested. The positive relationship between plasma amino acid titres and growth as criteria for measuring the potency of methionine source materials is discussed. REFERENCES Briiggemann, J., K. Drepper and H. Zucker, 1962. Quantitative Bestimmung der Verwertung von D-, L-, DL-Methionine and Ca-ra,-2-Hydroxy-4methylthiobutyrat durch das Hiihnerkiiken. Die Naturwissenschaften, 49: 334. Calet, C , and M. Melot, 1961. Comparative efficacy of methionine and the calcium salt of hydroxy-methyl-thiobutyric acid (M.H.A.) for chicken growth. Ann. Zootech. .10: 205-213. Dean, W. F., and H. M. Scott, 1962. The development of an amino acid standard for the early growth of chicks. Poultry Sci. 4 1 : 1640. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Gordon, R. S., and I. W. Sizer, 1955. The biological equivalence of methionine hydroxy analogue. Poultry Sci. 34: 1198. Green, D. E., H. M. Scott and B. C. Johnson, 1962. The role of proline and certain non-essential amino acids in chick nutrition. Poultry Sci. 4 1 : 116-120. Klain, G. J., H. M. Scott and B. C. Johnson, 1958. The amino acid requirements of the growing chick fed crystalline amino acids. Poultry Sci. 37: 976-977. Machlin, L. J., and R. S. Gordon, 1959. Equivalence of methionine hydroxy analogue and methionine for chickens fed low protein diets. Poultry Sci. 38:650-652. Marrett, L., H. R. Bird and M. L. Sunde, 1964. The effects of different isomers of methionine on growth of chicks fed amino acid diets. Poultry Sci. 4 3 : 1113-1118. Marrett, L. E., and M. L. Sunde, 1965. The effect of other D amino acids on the utilization of the isomers of methionine and its hydroxy analogue. Poultry Sci. 44: 957-964. Smith, R. E., and H. M. Scott, 1965. The use of free amino acid concentrations in blood plasma
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lized, one would expect the decrease in the other essential amino acids to be more gradual and the point of abrupt increase in the titre of the deficient amino acid to occur at a higher dietary level. There is some suggestion that this has been the case with methionine hydroxy analogue as compared with DL-methionine. Maximum growth occurred at the second level of supplementation with DL-methionine, but not until the third level with methionine hydroxy analogue (Table 3). Similarly the amino acid titres for all essential amino acids were comparable at the second and third levels of supplementation of DL-methionine and methionine hydroxy analogue respectively (Table 7—Expt. 7). Thus, there appears to be some agreement between the interpretation of plasma amino acid titres and growth rate as indicators of the potency of the methionine sources studied.
METHIONINE AND ANALOGUE in evaluating the amino acid adequacy of intact proteins for chick growth. I. Free amino acid patterns of blood plasma of chicks fed unheated and heated fish meal proteins. J. Nutrition, 86: 37^t4. Sullivan, T. W., and H. R. Bird, 1957. Effect of quantity and source of dietary nitrogen on the utilization of the hydroxy analogues of methi-
577
onine and glycine by chicks. J. Nutrition, 62: 143-150. Zimmerman, R. A., and H. M. Scott, 1965. Interrelationship of plasma amino acid levels and weight gain in the chick as influenced by suboptimal and superoptimal dietary concentrations of single amino acids. J. Nutrition, 87 : 13-18.
Isolation and Tentative Identification of the Carotenoids Present in Chicken Skin and Egg Yolks1
(Received for Publication November 4, 1965)
groups of investigators have SEVERAL studied the characteristics of the carotenoids deposited in the skin and egg yolk of chickens (Palmer, 1915; Brockman and Volker, 1934; Zechmeister and Tuzson, 1934; Peterson et al, 1939; Schrenk et al., 1944; Thompson et al., 1946; Ganguly et al., 1953; Marusich et al, 1960; Anjaneyalu, 1962; and Williams et al., 1963). Much of this earlier work has been reviewed by Goodwin (1954). Generally, in the work cited, identification of the carotenoids present has depended upon phasic separation between different solvent systems. Though in the work of Schrenk et al. (1944), Thompson et al. (1946) and Anjaneyalu (1962) adsorption chromatography was used in identifying the pigments present in egg yolks. The identity of the carotenoids deposited in the egg yolk and skin of chickens in the studies reported by Marusich et al. (1960) and Williams et al. (1963) was presumptive since they fed isolated carotenoids and the resulting pig1 Presented in part before the meeting of the American Institute of Nutrition, April, 1963 (Federation Proa, 22: 201, 1963, abstract). 2 Agricultural and Veterinary Research Division.
ments deposited were assumed to be the same as the original pigments fed. The purposes of the studies reported here were: 1) to measure the relative amounts of carotenoids deposited in the skin and egg yolks obtained from chickens fed different dietary sources of carotenoids, and 2) to obtain further information on the identity of the carotenoids deposited, especially those deposited in the skin. EXPERIMENTAL PROCEDURE Feeding—Broilers and laying hens were supplied dietary carotenoids from 1) a combination of yellow corn and alfalfa meal in either a standard broiler or layer diet; or 2) dried algae meal {Chlorella pyrenoidosa) in a standard broiler or layer diet where the alfalfa was omitted and yellow corn was replaced with milo. The main hydroxy carotenoids found in these different sources are presented in Table 1. Chemical—The skin, taken from the shank area, or egg yolks were extracted repeatedly with acetone until essentially all of the pigment was removed. The acetone extracts were concentrated under a stream of nitrogen, transferred into diethyl ether, reconcentrated, and transferred into redis-
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I. D. SMITH AND H. S. PERDUE Abbott Laboratories, North Chicago, Illinois2