1274 M. GRIFFITH AND R.
SCHEXNAILDER
ash and growth. The particles larger than
420 microns accumulated in the gizzard
while smaller sizes did not. In a third ex-
periment it was found that coarsely ground
agar particles caused little or no improve-
ment in phosphorus utilization, indicating
that the presence of a rough particle in the
food consumed was not sufficient to im-
prove utilization unless it remained in a hard
and rough state in the digestive tract.
REFERENCES
Association of Official Agricultural Chemists, 1960.
Official Methods of Analysis. Ninth edition,
Washington, D.C.
Carlson, C. W., H. C. Saxena, L. S. Jensen and
J. McGinnis, 1964. Rachitogenic activity of soy-
bean fractions. J. Nutrition, 82: 507-511.
Preferential Utilization of Methionine in Chicks
J. A. HEBERT, R. A. TEEKELL AND A. B. WATTS
Department of Poultry Science, Louisiana State University, Baton Rouge, Louisiana 70803
(Received for publication April 7, 1970)
M ETHIONINE is known to possess a
dual metabolic function for protein
synthesis and as a labile methyl group
for one-carbon metabolism. McKittrick
(1947) stated that choline and methionine
could replace each other in a diet but only
to the level of their essential increments.
Quillin et al. (1961) revealed improved
growth from choline additions on the lower
methionine supplementation and likewise
methionine supplementation resulted in
improved growth with low choline rations;
however, these rations were limiting in to-
tal methyl donors. Cardin (1967) at-
tempted to determine if a preferential utili-
zation of methionine for protein synthesis or
transmethylation existed at various dietary
levels of methionine using methyl-C-14-
methionine and methionine-2-C-l 4. He
found transmethylation was occurring rap-
idly at all supplemental levels of methio-
Griffith, M., R. J. Young and M. L. Scott, 1966.
Influence of soybean meal on growth and phos-
phorus availability in turkey poults. Poultry Sci.
45: 189-199.
Griffith, M., 1968a. Influence of soybean products
on the bone ash of chicks fed certain phosphorus-
deficient purified diets. Poultry Sci. 47: 765-
771.
Griffith, M., 1968b. Influence of type of diet on
phosphorus availability studies. Proceedings,
1968 Georgia Nutrition Conference. P. 83-90.
Griffith, M., 1969. Influence of dietary particle size
and composition on phosphorus availability.
Poultry Sci. 48: 1255-1261.
Scott, M. L., H. E. Butters and G. O. Ranit, 1962.
Studies on the requirements of young poults for
available phosphorus. J. Nutrition, 78: 223-230.
Steel, R. G. D., and J. H. Torrie, 1960. Principles
and Procedures of Statistics. McGraw-Hill Book
Co., New York.
nine. Due to the widespread distribution of
labeled methionine in the tissues, he could
not postulate a true preference of methio-
nine for protein synthesis or transmethyla-
tion.
Methionine has been shown to be toxic
when fed in excessive amounts to rats. Rus-
sell et al. (1952) added the same percent-
age excess of each of the essential amino
acids to a diet containing the recommended
levels of the rest of the essential amino
acids. Of the ten amino acids tested, only ly-
sine and methionine caused a definite
growth depression when fed in excess.
McKittrick (1947) produced a growth
depression with Single Comb White Leg-
horn chicks by feeding 0.5% methionine in
the presence of adequate choline. Tipton et
al. (1965) produced a growth depression in
chicks with 0.80% added DL-methionine to
a basal diet containing 0.24% methionine.
 METHIONINE UTILIZATION 1275

The following study was conducted to TABLE 2.—Rations used in feed trial
determine if methionine is preferentially
DL- Total
utilized for either protein synthesis or one- Ration Basal Methi- Sucrose Methi-
carbon metabolism at various dietary levels onine onine
of this amino acid. Percent
1 98.80 — 1.20 0.30
EXPERIMENTAL PROCEDURE 2 98.80 0.30 0.90 0.60
3 98.80 0.60 0.60 0.90
Day-old, unsexed, broiler-type chicks 4 98.80 0.90 0.30 1.20
were wingbanded, weighed and placed in
5 98.80 1.20 — 1.50

three electrically heated battery-type

brooders, each containing five decks or the expense of sucrose (Table 2). Feed
pens. Feed and water were provided ad li- consumption and weight gain were ob-
bitum and strip fluorescent lighting was tained weekly for four weeks.
provided twenty-four hours per day. Ten The experimental design was a random-
chicks were placed in each pen. ized block. Blocks consisted of three bat-
The chicks were fed a semi-purified diet tery brooders into which the various methi-
consisting of the basal ration (Table 1) onine levels (treatments) were randomly
plus various levels of methionine added at assigned. The data were analyzed by an
analysis of variance with appropriate or-
TABLE 1.—Composition of basal ration thogonal comparisons illustrated by Sne-
decor (1956).
Ingredients %
Four birds from each dietary methionine
Sucrose 48.59 level were each orally dosed with 50 micro-
Isolated soybean protein" 15.00
Gelatin 15.00 curies of methionine-2-C-14. Two of these
Corn oil 12.00 birds were sacrificed thirty minutes after
Ground cellulose 1.00
Vitamin A mix (30,000 I.U./gm.) 0.03 dosing. The remaining two were sacrificed
Vitamin D 3 mix (7,500 I.C.U./gm.) 0.02 ninety minutes after dosing. The same pro-
Butylated hydroxy toluene 0.01
L-tryptophan 0.15 cedure was repeated on another four birds
L-phenylalanine 0.20 from each dietary methionine level using 50
Choline chloride (70%) 0.30
Vitamin premix b 0.50 microcuries of methyl-C-14-methionine.
Mineral mix0 6.00 Blood samples were taken by heart punc-
Total 98.80 ture and the total liver and kidneys were
Calculated analysis:
removed from each bird, placed in tared
Crude protein (%) 26.48 sample jars and refrigerated.
Energy (M.E. kcal./Kg.) 3996.28 The liver plus two volumes of 0.25 mo-
C/P ratio 68.44
Methionine (%) 0.30 lar (M) sucrose were homogenized in an
Cystine (%) 0.19 Omni mixer (Ivon Sorvall Model). Four
Lysine (%) 1.54
Choline chloride (mg./Kg.) 1995.00 ml. of this homogenate plus 18 ml. of 0.25
M sucrose were homogenized in a Potter
" Purina RP-100 Isolated Soybean Protein.
b
Furnished in mg. per Kg. of finished ration: Elvehjem type mixer.
vitamin Bi 2 , 0.03; biotin, 0.30; menadione, 1.0; The latter liver homogenate was sepa-
pyridoxine-HCl, 8.0; folic acid, 4.0; riboflavin, 16.0;
nicotinic acid, 100.0; calcium pantothenate, 20.0; rated into four subcellular fractions by a
thiamin-HCl, 24.0; vitamin E (I.U.), 15.0. method adapted from that of Hogebroom
0
Furnished in gm./Kg. of ration: CaC03, 15;
K 2 HP0 4 , 9; NasHPOi, 7.3; Ca3(PO<)2, 14; NaCl, (1955). This differential centrifugation was
8.8; MgS0 4 -7H 2 0, 5; ferric citrate, 0.4; MnSO< accomplished using a Sorvall refrigerated
•4H 2 0, 0.42; KI, 0.04; ZnCOs, 0.02; CuSCv5H 2 0,
0.02. centrifuge model RC-2 with angle rotor
1276 J. A. HEBERT, R. A. TEEKELL AND A. B. WATTS

TABLE 3.—Four week methionine utilization with ration considered a random effect and
both label and time being fixed effects.
Total Total Feed Feed
Methi- Weight Consump- Conver-
onine Gain tion sion1" RESULTS AND DISCUSSION
(percent) (grams) (grams) (grams)
The criteria employed in this feed trial
0.30 98 + 20.0" 238+ 9.6 2.58 + 0.38 were weight gain, feed consumption and
0.60 259+24.5 453 + 11.5 1.72 + 0.18
0.90 344+ 5.8 457 + 28.0 1.33 + 0.10 feed conversion. The results obtained for
1.20 305+11.9 432+ 7.2 1.42 + 0.07 weight gain are shown in Table 3. The low-
1.50 294+ 6.5 435 + 15.7 1.48 + 0.07
est weight gain was obtained with 0.30%
" Mean and standard deviation. total methionine. The highest weight gain
b
Grams feed consumed per gram gained. was obtained with 0.90% total methionine.
Table 4 gives the analysis of variance of
SS-34. Liver was separated into nuclear, this data. There were real differences be-
mitochondrial, microsomal and soluble pro- tween rations but none between batteries.
tein fractions. The orthogonal comparisons between ra-
The amount of radioactivity present in tions revealed that the 0.30 and 0.60% to-
whole blood, liver, kidney and liver frac- tal methionine levels resulted in signifi-
tions was determined. The procedure of cantly lower weight gains than did the
Smith et al. (1964) was used to combust other levels. A true, statistically significant
samples and collect the carbon-14 as car- growth depression was not shown with the
bon dioxide. The samples were counted in a higher levels of methionine.
Beckman Model LS-250 liquid scintillation The feed consumption data is shown in
counter. All data was expressed as percent Table 3. The lowest feed consumption was
recovery of the administered dose. obtained with chicks fed the lowest level of
An analysis of variance was used to sta- methionine while the birds fed the 0.90%
tistically analyze the parameters measured total methionine level consumed the most
by isotope incorporation. A S X 2 X 2 fac- feed. The analysis of variance of feed con-
torial arrangement of treatments was used sumption shown in Table 4 revealed a
with five rations, two isotope labels and highly significant difference from ration
two time periods. A mixed model was used variation. The calculated feed conversion is

TABLE 4.—Analysis of variance and orthogonal comparisons of methionine utilization

Weight Gain Feed Consumption Feed Conversion

Source of D.F.
Variation M.S. F M.S. F M.S. F

Total 14
Blocks 2 35.5 0.04 1556 2.65 0.010 0.01
Rations 4 27401.2 30.27** 25795 43.94** 0.785 0.69
Comparisons
1 vs. 2, 3, 4, 5 1 98577 108.90** 101764 173.36** 2.797 2.48
2 vs. 3, 4, 5 1 6844 7.67* 330 0.56 0.311 0.28
3 vs. 4, 5 1 3901 4.31 1073 1.83 0.029 0.03
4: VS. 5 1 182 0.02 14 0.02 0.005 0.004

Error 8 905.2 — 587 — 1.128 —

* Significant at the 0.05 level of probability.
** Significant at the 0.01 level of probability.
 METHIONINE UTILIZATION 1277

TABLE 5.—Analysis of variance of isotope incorporation from the blood, kidney and liver

Blood Kidney Liver

Source of
D.F.
Variation M.S. F M.S. F M.S. F

Total 39 — — —. —.
Ration (R) 4 0.062 6.20** 0.352 32.00** 129.2 16.35**
Label (L) 1 0.127 12.70** 1.156 3.41 41.4 1.75
Time (T) 1 0.032 3.20 0.176 1.98 84.6 3.22
RXL 4 0.012 1.20 0.339 30.82** 23.6 2.99*
RXT 4 0.011 1.10 0.089 8.09** 26.3 3.33*
LXT 1 0.025 2.50 0.327 3.99 119.3 15.10**
RXLXT 4 0.012 1.20 0.082 7.46* 5.6 0.71
Error 20 0.010 — 0.011 •— 7.9 —
Significant at the 0.05 level of probability.
* Significant at the 0.01 level of probability.

shown in Table 3. The analysis of variance
revealed no significant differences with feed
conversion.
The percent recovery of radioactivity
from labeled methionine from whole blood,
kidney, liver and liver cell fractions was de-
termined. It was believed that these analy-
ses could provide information to determine
if there was a preferential utilization of me-
thionine for protein synthesis or transmeth-
ylation at various dietary levels of the
amino acid.
The blood data revealed a highly signifi-
cant label effect shown in Table 5. The
mean percent recovery of methionine-2-C-
14 (0.134%) was higher in whole blood than
the methyl-C-14-methionine (0.022%).
These findings would indicate rapid
transmethylation assuming that methio-
nine-2-C-14 and methyl-C-14-methionine
are equally absorbed from the gastrointes-
tinal tract. This observation was noted by
Cardin (1967) who concluded that trans-
methylation was very rapid in all of the
various dietary levels of methionine.
The analysis of variance of isotope incor-
poration into kidney tissue revealed a
highly significant difference between ra-
tions. However, no evidence of a preferen-
tial utilization of methionine was noted by
the kidney data.
The percent recovery of isotope from to-
tal liver was higher than any other tissue
tested. Recovery of labeled methionine
from total liver is shown in Table 6 and the
analysis of variance of this data is shown in
Table 5. There was a higher incorporation
of carbon-14 from methyl-C-14-methionine
at the 1.20 percent and the 1.50 percent
dietary methionine levels. A preferential util-
ization of methyl groups at high dietary
methionine levels could explain this observa-
tion.
The liver cells were separated into vari-
ous components to determine where in the
cell that the incorporation of isotope was
occurring. This could presumably isolate
the metabolic activity by a knowledge of
what activity is occurring in certain frac-
tions.
There was no significant difference in
any source of variation tested from the nu-
clear fraction. It was concluded that a low,
steady-state of isotope was being incorpo-
rated into this fraction. It was observed
that isotope incorporation into the mito-
chondria was higher at the ninety minute
time period than at the thirty minute time
period. There was no evidence of a prefer-
ential mitochondrial incorporation from
methionine at any particular dietary level.
The conclusion is made that methionine, or
1278 J. A. HEBERT, R. A. TEEKELL AND A. B. WATTS

TABLE 6.—Percent recovery of labeled methionine more specifically some metabolic product
from total liver and the microsomal fraction*
of this amino acid is being oxidized by the
Level Methy l-C-14- mitochondria and that this oxidation is oc-
of Methionine-2-C-14 Meth ionine curring within a ninety minute time period.
Methi-
onine1" 30 Min. 90 Min. 30 Min. 90 Min. The soluble protein fraction revealed a
larger recovery of isotope than the other
Liver
liver fractions but did not reveal a prefer-
0.30 7.030 9.981 3.512 4.672 ential utilization. The analysis of variance
7.205 12.590 3.270 4.715
of isotope incorporation from the liver cell
0.60 15.980 19.392 12.205 7.329 fractions is shown in Table 7.
16.700 13.125 16.830 12.208
The microsomal fraction is a centrifugal
0.90 8.418 12.365 6.515 6.382 term denoting the precipitate obtained by
5.692 14.266 7.280 3.941
centrifugation at 39,000 X g for ninety
1.20 10.680 20.111 16.215 24.620 minutes. It is assumed to consist primarily
9.450 22.580 12.984 12.228
of ribosomes. If we assume the ribosomes
1.50 5.310 16.050 12.577 9.276 to be the major site of protein synthesis in
2.485 12.121 11.760 12.320
the cell, then we can conclude that activity
Microsomal Fraction found in this fraction is associated with
0.30 0.339 3.459 0.126 0.246 protein synthesis.
0.295 5.515 0.114 0.255 The percent recovery of labeled methio-
0.60 0.263 0.755 0.216 0.495 nine from the microsomal fraction is shown
0.302 0.987 0.994 0.473 in Table 6 and the analysis of variance
0.90 0.238 0.302 0.228 0.202 shown in Table 7. The analysis of the mi-
0.252 0.493 0.293 0.194 crosomal fraction revealed a high level of
1.20 0.387 0.715 1.209 0.491 incorporation of carbon-14 from methio-
0.327 3.810 0.259 0.503 nine-2-C-14 at the low dietary methionine
1.50 0.163 0.796 0.712 0.242 levels and a high level of incorporation of
0.163 0.454 1.986 0.314 carbon-14 from methyl-C-14-methionine at
a
Data expressed as percent recovery of radio- the high levels of dietary methionine. This
activity on a total liver basis.
b
is revealed by an orthogonal comparison of
Percent of total methionine in the ration.
the ration by label interaction which is

TABLE 7.—Analysis of variance of isotope incorporation from the liver cell fractions

Nuclear Mitochondrial Microsomes Sol.;protein

Source of
D.F.
Variation M.S. F M.S. F M.S. F M.S. F

Total 39 — — — — .—
Ration (R) 4 0.699 1.46 0.143 3.04* 1.256 2.97* 2.934 3.13*
Label (L) 1 0.019 0.04 0.979 20.83** 2.773 1.33 0.031 0.03
Time (T) 1 0.146 0.30 0.570 12.13** 3.543 1.92 0.021 0.02
RXL 4 0.197 0.41 0.085 1.81 2.088 4 94** 0.132 0.14
RXT 4 0.182 0.38 0.102 2.17 1.842 4.36* 0.102 0.11
LXT 1 0.071 0.15 0.140 2.98 7.370 17.42** 0.043 0.04
RXLXT 4 0.632 1.32 0.014 0.30 1.116 2.64 1.476 1.57
Error 20 0.480 — 0.047 — 0.423 •— 0.937 —
* Significant at the 0.05 level of probability.
** Significant at the 0.01 level of probability.
 METHIONINE UTILIZATION
given in Table 8. These observations pre-
sent evidence for a preferential utilization of
methionine for protein synthesis at low di-
etary methionine levels and a preferential
utilization of methionine for transmethyla-
tion at high dietary methionine levels. The
data also revealed that carbon-14 from me-
thionine-2-C-14 was more highly incorpo-
rated at the ninety minute time period
while carbon-14 from methyl-C-14-methi-
onine was more highly incorporated at the
thirty minute time period. This again pre-
sents evidence for rapid transmethylation.
SUMMARY
The performance of chicks fed diets dif-
fering in methionine levels was best with a
dietary methionine level of 0.90% as mea-
sured by weight gain and feed conversion.
Levels of 1.20 and 1.50 percent total methi-
onine gave lower weight gains than did the
0.90% level. The latter were not signifi-
cantly different from the 0.90% level and
therefore could not be termed a significant
growth depression or a toxic effect of excess
methionine.
The percent recovery of orally adminis-
tered methionine-2-C-14 and methyl-C-14-
methionine at two time periods and at five
dietary methionine levels was obtained in
the blood, kidney, liver and liver cell frac-
tions. Transmethylation was shown to be
occurring rapidly by a higher recovery of
carbon-14 from methyl-C-14-methionine at
thirty minutes than at ninety minutes in
most of the tissues analyzed. No evidence
was obtained for a preferential utilization
of methionine at any dietary methionine
level in either the nuclear, mitochondrial or
soluble protein fraction.
A preferential utilization of methionine
for protein synthesis at low dietary methio-
nine levels was noted in the microsomal
fraction of the liver cell. This was shown
by the high incorporation of carbon-14
1279
TABLE 8.—Orthogonal comparisons of the RY.L
interaction of the microsomal fraction of the liver
Comparisons 8 D.F. M.S. F
0.90^.0.30,0.60,1.20,1.50 1 0.49 1.16
0 . 3 0 , 0 . 6 0 M. 1.20,1.50 1 1.90 4.49*
0.30 vs. 0.60 1 4.77 11.28**
1.20OT. 1.50 1 1.19 2.81
Error 20 0.423 —
a
Comparisons are levels of methionine at each
label averaged over time.
* Significant at the 0.05 level of probability.
** Significant at the 0.01 level of probability.
from methionine-2-C-14 at the low dietary
levels of methionine. A preferential utiliza-
tion of methionine for transmethylation at
high dietary methionine levels was shown
by the high incorporation of methyl-C-14-
methionine at high dietary methionine lev-
els.
REFERENCES
Cardin, D. W., 1967. Metabolism of methionine
by certain tissues of the intact chick. A disserta-
tion: Louisiana State University.
Hogebroom, G. H., 1955. Fractionation of cell com-
ponents of animal tissues. I n : Methods in En-
zymology. Vol. 1. Ed. S. P. Colowick and N. O.
Kaplan, Academic Press, Inc., New York.
McKittrick, D. S., 1947. The interrelations of
choline and methionine in growth and the ac-
tion of betaine in replacing them. Arch. Bio-
chem. IS: 133-155.
Quillin, E. C , G. F. Combs, R. D. Creek and
G. L. Romsier, 1961. Effect of choline on the
methionine requirement of broiler chickens.
Poultry Sci. 40: 639-645.
Russell, W. C , M. W. Taylor and J. M. Hogan,
1952. Effects of excess essential amino acids on
growth of the white rat. Arch. Biochem. Biophys.
39: 249-253.
Smith, G. N., P. D. Ludwig, D. C. Wright and
W. R. Bauriedel, 1964. Pesticide residue method-
ology; simple apparatus for combustion of sam-
ples containing C-14-labeled pesticides for resi-
due analysis. J. Agri. Food Chem. 12: 1721.
Snedecor, G. W., 1956. Statistical Methods. The
Iowa State University Press. Ames, Iowa.
Tipton, H. C , B. C. Dilworth and E. J. Day, 1965.
The relative biological value of DL-methionine
and methionine hydroxy analogue in chick diets.
Poultry Sci. 44: 987-992.