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THE EFFECTS OF SUPPLEMENTARY THREONINE AND/OR CHOLINE
ON ENZYME ACTIVITY AND FAT DEPOSITION IN LIVERS OF
ALBINO RATS FED 9% CASEIN DIETS

by

Betty Bondurant Brown

AN ABSTRACT
Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and Anolied Science

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition
College of Home Economics

1959

222% 0222:—

ABSTRACT

Threonine and choline deficiencies in a low protein
diet are known to induce different tynes of fatty livers. The
histological difference in these livers indicates that fat is
deposited by different routes. In this experiment the amount
of fat deposited and enzyme activity were studied to determine
if they, too, differed in these two types of fatty livers.

Forty weanling, male, albino rats were divided into
four groups and fed a 9% casein diet containing suoolements
of threonine and/or choline or neither. Food and water were
allowed ad libitum throughout the three week experimental
period.

The animals were sacrificed by decaoitation. Livers
were analyzed for fat, nitrogen and water content; and for
DPN cytochrome-c reductase and fatty acid oxidase activity.

There was little or no difference in the nitrogen
content, amount of fat deposited, or the activity of the DPN
cytochrome—c reductase system in the livers of the choline
deficient rats as comoared with the threonine deficient rats.
When the threonine deficiency was superimposed on the choline
deficiency there was likewise no difference in fat and nitrogen
content of these livers, but the activity of the DPN cytochrome-c
reductase system was significantly deoressed in this group.
Fatty acid oxidase activity was depressed by a choline defi-

ciency whereas the threonine deficient group and the doubly

deficient group maintained fatty acid oxidase activity

equivalent to that of the threonine and choline supplemented
group.

It was suggested that the chief difference between
choline induced and threonine induced fatty livers might be
the rate at which adaptation takes place. However, further

analysis must be made in order to test this hypothesis.

THE EFFECTS OF SUPPLEMENTARY THREONINE AND/OR CHOLINE
ON ENZYME ACTIVITY AND FAT DEPOSITION IN LIVERS OF
ALBINO RATS FED 9% CASEIN DIETS

by

Betty Bondurant Brown

A THESIS

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and.Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition
College of Home Economics

1959

ACKNOWLEDGEMENTS

The author wishes to express her sincere thanks
to Dr. Dorothy Arata for her encouragement, guidance and
help throughout the study; and to Miss Catherine Carroll
and Miss Marjorie Mastic for their assistance in checking
data.

She is also greatly indebted to her husband, Mr.
Wayne Brown, and her father, Mr. C. O. Bondurant, for their

constant and untiring patience and encouragement.

INTRODUCTION .

REVIEW OF LITERATURE .
EXPERIMENTAL PROCEDURE
RESULTS

DISCUSSION . . .
SUMMARY . .
LITERATURE CITED
APPENDIX .

TABLE

II

III

IV

VI

LIST OF TABLES

Composition of Diets

Growth Rate and Liver Fat of Rats Fed
9% Casein Diets

Liver Nitrogen and Water Content of
Rats Fed 9% Casein Diets

Activities of Two Enzyme Systems in
Livers of Rats Fed 9% Casein Diets

APPENDIX

Liver Nitrogen of Rats Fed 9% Casein
Diets Calculated on Basis of Dry
Weight of Livers

Activities of Two Enzyme Systems
Calculated on Basis of NitrOgen
Content of Livers

 

19

20

ii

INTRODUCTION

INTRODUCTION

In normal animals the lipid content of the liver
is approximately five percent of the w9t weight of tissue.
Under certain conditions, however, the fat content of the
liver may rise considerably to a point where the organ is
characterized as being a "fatty liver". Starvation, high fat
diet, feeding of cholesterol, carbon tetrachloride or phos—
phorus poisoning, choline and methionine deficiency, and
threonine deficiency on a low protein diet are a few of the
causes of fatty liver (Fruton and Simmonds 1958). Thus,
fatty infiltration of the liver can have a number of apparent—
ly unrelated causes, and prevention and cure of the abnormality
probably involves a variety of biochemical processes.

In recent years two types of fatty livers have
received much attention and subsequent study. It is believed
that the role of choline in lipid metabolism is to facilitate
fat transport by its effect on phospholipid synthesis and
turnover (Fruton and Simmonds 1958). Although threonine has
been established as a lipotropic factor on a low protein diet,
its role in lipid metabolism is not clear. Several enzyme
systems have been studied and various theories advanced as
to the mode of action of threonine as a lipotropic agent.

It is known that the lipotropic action of threonine
is not observed unless choline or methionine is present in

the diet in adequate amounts. Thus, some correlation between

the two lipotropic factors has been sought. This relation—
ship is complex for one very obvious reason -- each substance
is involved in other roles besides lipotrOpism and it is
difficult to ascertain which roles take precedence and which
are secondary to the lipotropic action, especially when there
is a limited supply of one or both of the factors.

Further investigation was stimulated by the fact
that fatty livers, induced by the absence of each of these
lipotropic factors individually, were histologically different;
and, hence, the route of fat deposition was probably different.

Some work has been done in comparing the effect of
a threonine deficiency z§_a choline deficiency on fat dep—
osition and enzyme activity in the livers of these animals.

The purpose of this study was to further investigate and
compare those two types of fatty livers re: the fat content

and two enzyme systems of the liver -— fatty acid oxidase

and DPN cytochrome-c reductase.

REVIEW OF LITERATURE

REVIEW OF LITERATURE

The fact that choline is a potent lipotropic factor
in the liver was established by Best and co—workers in 1932.

Subsequent studies indicated there were other
factors besides choline involved in this lipotropic action.
Tucker and Eckstein (1937) and Channon, Manifold and Platt
(1938) were the first to report that the deposition of lipids
in livers of rats fed low protein rations deficient in choline
could be reversed by feeding not only choline but also methio—
nine and cystine. The lipotropic action of methionine was
shown to be dependent on the ability of this compound to
furnish methyl groups for the synthesis of choline (Tucker
and Eckstein 1937).

Several groups of workers (Channon, g§_§1 1938,
l9AO; Best and Ridout 1940; Tucker and Eckstein 1938) have
suggested that some proteins are more effective in reducing
the liver lipids which accumulate during choline deficiencies
than would be expected from their methionine and cystine
content. This led to many attempts to demonstrate a lipo-
tropic action for other amino acids and protein, and conse-
quently, much controversy. Tucker, Treadwell and Eckstein
(1940) maintained that the lipotropic effects of protein could
be accounted for solely by the cystine: methionine balance.

In support of this theory Beeston and Platt (1939), Setton
and Grail (1942), Channon, Mills and Platt (1943) and Eckstein

A.
(1952) failed to produce evidence that specific amino acids
other than methionine and cystine influence the deposition
of fat in the livers of animals receiving diets deficient in
choline.

Other workers (Dick 23 El 1952) suggested that some
fatty livers may reflect a nonspecific effect of protein
deficiency rather than a specific lack of any one essential
amino acid. .According to this theory, inadequate dietary
protein might lead to a faulty production of protein in the
cells of the liver or to faulty formation of the lipotropic
complexes characteristic of this tissue.

Evidence began to accumulate which indicated that
fatty livers could be produced by a deficiency of an amino
acid other than methionine or cystine. Singal 23.51 (1953a)
and Harper gthgl (195Aa) agreed that the secondary lipotropic
effect of protein (the sparing of choline by methionine being
the primary) is not a choline sparing action, but results
from the provision of certain amino acids; i.e. threonine, a
deficiency of which causes fat to accumulate in the liver.
This effect was observed only when the diet contained either
choline or methionine in amounts approaching what is considered
to be the requirement.

Harper 23,31 (1954b) reported that the amount of
fat deposited in the livers of young rats receiving choline
was dependent on the interaction of several factors. They

considered threonine to be of major importance in low casein

5.

diets but warned that when proteins of different amino acid
composition were fed the proper balance of other amino acids
must be kept in mind.

This observation confirmed that made by Singal
§t_gl (1949) while studying niacin-tryptophan relationships.
in rats fed a 9% casein ration containing choline. They
found that under these conditions liver lipids developed to
the extent of 16.0% (dry wt.) whereas the addition of threonine
to the diet reduced the liver lipids to 5.1% (dry wt.).
However, the addition of threonine also created an amino
acid imbalance which markedly decreased growth. To correct
this imbalance, niacin or tryptophan was added to the diet
and the rats grew at a more rapid rate. Under these con-
ditions liver lipids were 14.4% in the absence of threonine
and 5.9% when threonine was supplemented to the diet.

Since the amount of fat deposited in the livers of
rats receiving choline decreased slightly as the amount of
dietary fat increased, Harper §§_§1'(1954b) concluded that
the liver fat arose from the conversion of carbohydrate or
protein to fat. Further evidence supporting this conclusion
was obtained from the observation that high fat deposits
occurred in animals on a fat free diet (Harper 2313; 1954b).

The specific lipotropic effect of threonine was
conclusively established by several workers. By increasing
the choline content of the basal ration (9% casein) threefold

and raising the methionine to 0.3%, Harper and co—workers

(1953) observed some reduction of liver fat, but not to the
extent which was obtained with the addition of Just 0.18%

DL threonine. They were also able to demonstrate a reversable
effect of threonine by withdrawing it from the diet after two
weeks of supplementation and observing a subsequent increase
in liver fat. Inclusion of threonine for two weeks after the
rats were fed the deficient diet two weeks reduced the accum—
ulation of liver fat.

The effects of choline, methionine and additional
protein on fat deposition in the livers of rats fed low pro-
tein diets were studied by Harper §t_gl_(l954a). They, too,
found that neither choline nor methionine in amounts sufficient
to meet the stated requirement of the rat for growth and pre—
vention of fat infiltration prevented some excess of fat from
accumulating in the liver. Only when either the protein or
threonine content of the diet was increased was the fat
content of the liver reduced to what is considered the normal
range. These results were in agreement with those obtained
by Singal gt §1_(1953b) and Nine-Herrera and co-workers (1954).

That the lipotropic action of threonine was not
the result of increased food intake was demonstrated in a
paired feeding experiment by Singal £3 E; (1949). The liver
lipids of rats pair—fed a casein ration supplemented with
tryptophan was 14.4% compared to 5.9% when threonine was
included. They also found that the livers of rats on an

amino acid diet simulating the 9% casein ration supplemented

7.

with tryptophan contained 17.0% lipids, and supplementary
threonine reduced the lipids to 5.3%. When a diet completely
free of threonine was fed, the rats lost weight and the liver
(lipids were only slightly above normal.

After threonine was proven to be a lipotropic factor
in low protein diets, much interest was directed toward
determining the mode of action of threonine. The accumulation
of abnormal amounts of liver lipids in choline deficiency is
usually explained as a failure of fat transport. In the
threonine deficient animal in which the level of dietary fat
is low, the accumulation of abnormal amounts of liver lipids
is difficult to explain.

A study of the biochemical changes in the livers
by Sidransky and Farber (1958b) revealed that the liver
protein content of animals on a threonine free diet was the
same as —- or slightly greater than —- that of the control
animals. Protein synthesis in the liver as measured by radio-
active amino acid incorporation into liver protein was also
not decreased in the rats on the threonine free diet, and in
three experiments increased amino acid incorporation was
'observed. They point out that the failure to find a general
inhibition of protein synthesis with a threonine free diet
does not rule out selective inhibition of the synthesis or
turnover of one or more liver enzymes in these animals.

Dick 2E.§l (1952) and Singal gpygi (1953a,b) also indicated
the possibility that the liver fat was-due to reduced enzyme

function.

Singal g; g; (1953b) found that a threonine de-
ficiency depresses the rate of synthesis of phospholipids
and nucleoprotein phosphorus fractions of the livers, while
supplementary threonine stimulated the turnover to near
normal values. They stated that this could be effected
through a reduction in the amount of enzymes due to an in—
adequate supply of amino acids to be synthesized into enzymes,
or an impairment of the system by which these enzymes are
elaborated. In regard to the latter, Spiegelman and Kamen
(1946) have suggested that nucleoprotein may be "specific
energy donators“ which make possible reactions leading to
protein and enzyme synthesis.

A study of the effects of feeding a protein free
diet on the activities of oxidative enzymes was reported by
Whinio gt gl,(l953). They found that DPN cytochrome-c
reductase decreased in unit activity and total activity when
the depleted.anima1s and their pair-fed controls were compared.
A general decrease in activity per unit of nitrogen indicated
that the enzyme proteins were lost more rapidly than was
total protein.

Pilsum, Speyer and Samuels (1957) while studying
the effects of protein and amino acid deficiencies on several
enzyme systems concluded that the enzymes studied seemed to
divide into two groups: those which change with the protein

of the organ irrespective of the cause of protein alteration,

and those which decrease with the absence of a given amino
acid in the diet irrespective of the total protein of the
organ. Their results indicated (a) the missing amino acids
do not play a role in the structure of the first group while
they are vital to the formation of the second; (b) the turn—
over of the second group is more rapid and, therefore,
interference with synthesis would appear earlier; or (c) the
system synthesizing the first group have priority over the
second in the utilization of the limited amount of the de—
ficient amino acid in circulation. The effects of the amino
acid deficiency appear to depend upon the relative “priority”
of the particular protein for the available amino acid supply
as a whole.

Arata (1959) observed profound disturbances in the
metabolism of adenosinetriphosphate (ATP) and diphosphopyridine
nucleotide (DPN) with a concommitant disruption of the ATP -—
and DPN -- dependent enzyme systems in fatty livers induced
by a threonine deficiency. She states that the fat accumulation
is probably due to a loss in the ability of that organ to
oxidize fatty acids, eSpecially since ATP is necessary for
activation of fatty acid oxidation and DPN is an obligatory
hydrogen acceptor in the fatty acid cycle (Van Baalen and
Gurin, 1953).

A similar observation was made by Dianzani (1955,
1957) who studied the ATP and DPN content of fatty livers

induced by 0014 poisoning and choline deficiency.

10.

As a result of the many theories involving enzyme
function as affected by threonine deficiency, much research
has been undertaken in that area. Harper 33 El (1953)
observed that the addition of threonine to a 9% casein diet
resulted in a reduced deposition of fat in the liver and a
concommitant increase in the activities of certain soluble
liver enzymes and in endogenous oxidation and decarboxylation.
A decrease in the activity of certain mitochondrial enzymes
in the liver was also observed.

Arata and co—wprkers (1954) observed that the in-
clusion of 0.36% threonine to a basal diet (9% casein
supplemented with choline, tryptophan and methionine) caused
a significant increase in endogenous oxidation and decar—
boxylation. In their experiments, there was no significant
effect of threonine on the activities of the mitochondrial
enzymes (succinic, choline and pyruvic oxidases). These
latter findings were not consistant with those of Harper 33 §_
(1953). It wasalso observed that the accumulation of fat in
the liver does not, in itself, cause the depression of the
endogenous respiration and the two soluble enzymes observed.

In a later study (Arata pt 31 1956) of the pyridine
nucleotide-linked enzyme systems, they concluded that both a
defect in DPN production and the improper metabolism of
endogenous DPN in the liver are major factors in liver fat
accumulation in a partial threonine deficiency.

Few reports are included in the literature of a

ll.

comparison of fatty livers induced by a threonine deficiency
with those caused by a deficiency of choline. Arata and
co—workers (1954) observed, when choline was omitted from
the basal ration and threonine added, a significant increase
in endOgenous oxidation and decarboxylation and in the
activities of two soluble enzyme systems (tyrosine oxidase
and xanthine oxidase). However, the fat content of the
liver was not lowered.when threonine was supplemented. Singal
and co-workers (1953a) also observed that when choline was
omitted from the basal diet, supplementation with threonine
caused no reduction in the level of fat in the liver.

A comparison of the type of fat deposition in
threonine zgicholine deficient diets was made by Sidransky
and Farber (1958a). While studying the morphologic changes
in immature rats fed a threonine free diet, they found that
the lobular distribution of the liver lipid differs with the
different types of fatty liver. In choline deficiency, the
fat appears about the central vein and then progressively
involves the remainder of the lobule. In contrast to this,
when fatty livers are induced by protein and amino acid
deficiencies, a periportal lipid distribution is found in
the liver.

This investigation was undertaken to study further
the similarities and dissimilarities between the choline in—
duced Kg threonine induced fatty livers. Two enzyme systems ——
fatty acid oxidase and DPN cytochrome-c reductase —— were

chosen for study and the results are presented in this paper.

EDCPERIl-EI~ITAL PROCEDURE

EXPERIMENTAL PROCEDURE

Forty male weanling rats of the Sprague—Dawley
strain weighing from 41 to 52 grams were divided into four
groups of ten animals each and were maintained in individual
screen-bompm cages in an air conditioned room. A nine percent
casein diet supplemented with 0.15% choline and/or 0.36% DL
threonine was fed ES libitum throughout the experimental
period of three weeks. The composition of these diets is
given in Table I. Weights of the animals were taken every
four or five days and prior to sacrificing them.

At the termination of the experimental period, the
rats were stunned by a sharp blow on the head, decapitated,
and the livers excised for the determination of fatty acid
oxidase, DPN cytochrome-c reductase, fat and nitrOgen.
Immediately after removal, the livers were chilled by immer-
sion in ice and then blotted free of moisture. Weighed
portions (2 to 3 gms.) of each liver were homegenized with
two volumes of ice cold 0.0393 sodium potassium phosphate
buffer (pH 7.3) using a Potter-Elvehjem type homogenizer.
The chilled liver homogenates were pipetted into previously
prepared, chilled Warburg flasks. In the determination of
both enzyme systems, carbon dioxide was adsorbed on pieces
of filter paper saturated with 0.2 ml. 10% KOH in the center
well of each flask. The flasks were mounted on manometers
and immersed in a constant temperature water bath maintained

at 25°C. A five minute temperature equilibration period was

13.

allowed for all flasks before the manometers were adjusted
to 15 and'the systems closed. Thereafter, readings were
taken at five minute intervals for thirty minutes. Calcula-
tions were based on 15 minutes for fatty acid oxidase and 20
minutes for DPN cytochrome—c reductase; the activity of the
respective enzymes decreased after this interval of time.
Results are expressed as ul OZ/hr/unit weight fresh liver
tissue and u1 OZ/hr/unit weight of nitrogen.

The substrate for DPN cytochrome-c reductase
(0.2 ml.of 0.5% DPN) was tipped into the main compartment
of the flask from the side arm following temperatureequili-
bration. In the fatty acid oxidase system, the octonoate
was pipetted directly into the main compartment.

The fatty acid oxidase activity of the liver
homogenates was determined by the method of Colowick and
Kaplan (1955) with 0.6 ml. sodium potassium phOSphate buffer
(pH 7.3), 0.3 ml. sodium octanoate and distilled water to
make 3.2 ml. A control flask contained the same components
except that the substrate was replaced by water.

DPN Cytochrome-c reductase activity of the liver
homogenates was determined by the method of Potter (Umbreit
33 E1 1951) with 0.4 ml. of 3 x 10-4 g cytochrome—c and
buffer to make a total volume of 3.2 ml. Crude malic
dehydrogenase was isolated from fresh pig liver according
to the method of Potter (1946). The control flask was

treated the same except 0.2 m1. of 0.5% DPN was replaced

 

14.

with distilled water.

The remaining portions of the livers were weighed,
homOgenized.with water and dried for 12 hours at 90°C. The
dried liver homogenates were cooled, weighed and ground to a
powder. Fat content was determined by ether extraction of
weighed samples (appx. 1 gm.) in a Goldfisch fat extractor.
The fat content of these samples was expressed as percent of
dry weight of tissue.

Nitrogen was determined on the dried fat free liver
by the macro Kjeldahl method. KJeldahls were run in duplicate
on 0.350 gm. samples; copper sulfate was used as a catalyst.
Percent nitrogen was calculated on the basis of dry and fresh

weight of the sample.

 

15.

TABLE I.

COMPOSITION OF DIETS

INGREDIENTS DIET

I II III IV

(gm) (em) (gm) (gm)
Sucrose 80.80 81.20 81.00 81.40
Casein 9.00 9.00 9.00 9.00
DL Methionine 0.30 0.30 0.30 0.30
DL TryptOphan 0.10 0.10 0.10 0.10
Corn 0111 5.00 5.00 5.00 5.00
Salts w2 4.00 4.00 4.00 4.00
Vitamin mix3 0.25 0.25 0.25 0.25
Choline 0.15 0.15 —___ _-__
DL Threonine 0.36 -——- 0.36 ————

1. Containing 7.5 mg. tocopherol, 0.38 mg. menadione.

2. Obtained from National Biochemicals Corporation.

3. Containing in milligrams per 100 grams ration: thiamin
hydrochloride 0.5, riboflavin 0.5, niacin 1.0, calcium
pantothenate 0.25, pyridoxine hydrochloride 0.25, biotin
0.01, folic acid 0.02, vitamin B12 0.002, inositol 10.0,

vitamin A 10.0, vitamin D 0.18, and sucrose to make 0.25

gm. of mix.

RESULTS

RESULTS

The results from this study are summarized in
Tables II, III and IV.

The average weight gain in grams per week and the
average percent liver fat (dry weight) for the four groups
of animals are presented in Table II. The animals fed a 9%
casein diet supplemented with threonine (Group III) or
threonine and choline (Group I) showed significantly higher
growth rates than did the animals fed diets which did not
contain additional threonine (Groups II and IV). The in-
clusion of choline in the diet,had no effect on the growth
rate regardless of whether or not threonine was added.
Group I (+ threonine and.+ choline) was not significantly
different from Group III (4 threonine) nor was Group II
(+ choline) significantly different from Group IV (no
supplement), according to the standard error of the mean.

When neither threonine nor choline were supplemented
to the 9% casein diet (Group IV), an abnormally high quantity
of fat was deposited in the livers of the rats. The addition
of either choline (Group II) or threonine (Group III) to this
diet was ineffective in reducing the amount of liver fat.
However, when both of these compounds were added to the 9%
casein ration (Group I), the liVer fat was significantly
depressed. Although the standard error of the mean indicated

a slight difference in the extent to which threonine was

170

effective in reducing liver fat in Group III as compared with
Groups II and IV, in view of the variation and overlapping of
the values within the grOups, the author did not consider
this difference significant.

The nitrogen content of the liver was calculated on
the basis of wet weight of tissue. These data are summarized
in Table III. Data for the percent nitrogen on the basis of
dry weight are recorded in the Appendix (Table V). When the
unsupplemented diet (Group IV) was fed to the animals, the
nitrogen content of the liVers was slightly less than when
choline and threonine were supplemented (Group I). This diffs
erence indicated by the standard error of the mean was so
slight it was not considered significant by the author upon
considering the differences within the groups. There were no
significant differences in liver nitrogen between Groups II, I
III and IV.

Although the nitrogen content of the liVers remained
fairly constant from one group to another, the amount of moisture
in these tissues varied to a measurable degree (Table III). In
fact, a comparison of the moisture content with the percent fat
in the livers (Table II) revealed an inverse relationship; i.e.,
the higher percent fat, the lower the percent water, etc.

Enzyme activity data are reported as u1.02/ hr./
unit weight of fresh liver tissue in Table IV. Enzyme
activity expressed as ul.02/ hr./ unit weight of nitrogen
may be found in the Appendix (Table VI). The former unit of

measure is used in this report because fluctuations in the

18.

nitrogen content of the livers did not follow fluctuations
in enzyme activity.

DPN cytochrome—c reductase activity was greatly
depressed in the livers of animals receiving no supplementary
threonine or choline (Group IV) as compared with the supple-
mented group (I). The addition of either choline (Group II)
or threonine (Group III) to the 9% casein diet resulted in
an increase in the activity of this enzyme to equal that of
the supplemented control (Group I). The removal of threonine
or choline from the diet did not alter the DPN cytochrome-c
reductase activity; only when both threonine and choline were
omitted was the enzyme activity reduced to a significant
degree.

Fatty acid oxidase activity in the livers of
animals fed a diet containing no supplementary choline
(Group III) was depressed to approximately 30% of the activ-
ity of the remaining three groups of animals. When threonine
was not added to the diet, no significant effect on the
activity of this enzyme was observed regardless of whether

or not choline was present in the diet (Groups II and IV).

19.

TABLE II .

Growth Rate and Liver Fat of Rats Fed 9% Casein Diet

GROUP SUPPLEMEN WEIGHT GAIN FAT sm./100 gm. LIVER
(gm./wk.) (dry weight)
I 0.15% choline 32.3 i 1.3* 11.4 i 0.6*
0.36% DL threonine
II 0.15% choline 20.7 f_1.1 22.3 i 1.7
III 0.36% DL threonine 30.4 i 1.4 18.2 :_l.3
IV no supplement 20.9 i.l.5 24w? 1 1.7

*Standard error of mean

TABLE III

Nitrogen and Water Content of Livers From RataFed 9% Casein Diets

GROUP SUPPLEMENT N/100 gm. LIVER H20 gn./100 gm. LIVER
(wet weight)
I 0.15% choline 2.7 i 0.2* 74.9 f 0.7*
0.36% DL threonine
II 0.15% choline 2.2 i 0.1 71.3 i 0.6
III 0.36% DL threonine 2.3 i 0.1 73.1 i 0.7
IV no supplement 2.1 i 0.1 70.3 i.1.1

“Standard error of mean

TABLE IV.

Activities of Two Enzyme Systems in Liver Tissue
of Rats Fed 9% Casein Diets

GROUP SUPPLEMENT DPN Cytochrome-c Fatty Acid Oxidase
Reductase u1.02/nr./gm. LIVER
u1.02/hr./10 mg. LIVER

I 015% choline 177 i_6 253 i 64
0.36% DL threonine

II 0.15% choline 167 1,8 374 f,85
III 0.36% DL threonine 161 i,lu 103 f_39
IV no supplement 115 i 9 328 i 52

, DISCUSSION

DISCUSSION

In this experiment, threonine was the limiting
factor for growth. Maximal growth rates (approx. 30 gm./
week) were attained only when threonine was present in the
ration; the presence or absence of choline had no effect on
the growth rate. Some correlation was noted between the rate
at which the animals grew and the amount of nitrogen present
in 100 gm. of dry weight of liver tissue. Livers from rats
growing at a less rapid rate contained less nitrogen than
did those from rats growing more rapidly, as would be exp
pected. This correlation was not observed when nitrogen was
calculated on the basis of fresh weight of tissue.

The fact that the addition of choline alone or
threonine alone to a 9% casein diet was insufficient to
reduce liver fat has also been observed by other workers
(Singal 22.El 1953b, Harper 33 al.1954b, and Nino Herrera
23.§l 195u). Only when both of these factors are present in
adequate quantities is liver fat reduced to a more normal
value. It has been shown that the threonine induced fatty
liver is histologically different from the choline induced
fatty liver (Sidransky and Farber 1958a). Thus, in these
two types of fatty livers, the fat must be deposited Egg
different routes. However, under the conditions of this
experiment, the two types were not additive since the fat

deposited in the liver was no greater in quantity when both

choline and threonine were omitted from the diet than it was
when either threonine or choline was omitted.

It was somewhat puzzling to observe that either
choline or threonine was equally effective in increasing the
activity of DPN cytochrome-c reductase to a level comparable
with the positive control group. These data are in conflict
with those of Arata gt a; (1959) who observed a significant
depression in the activity of this enzyme system when thre—
onine was not added to a 9% casein ration containing choline.
A possible explanation for this disagreement is the fact that,
in this study, the activity of DPN cytochrome—c reductase
was determined at 2500, while the optimum temperature for
this enzyme system is 37°C (Umbriet 23.2i 1951). The lower
temperature was chosen in this study to make possible the
determination of DPN cytochrome-c reductase and fatty acid
oxidase simultaneously. It was found that the DPN cytochrome-c
reductase system was active at 25°C, however, the activity
was not maximal. As a result, small differences which might
have existed between experimental groups might have been
obscured. In any case, the activity of DPN cytochrome—c
reductase did not parallel liver fat levels.

The activity of the fatty acid oxidase system
appeared to be independent of the quantity of threonine avail—
able to the animals. There was no significant difference in
the uptake of 02 whether threonine was supplemented to the ration

or not. These data do not agree with those of Arata £3 £1

23.

(1959) who observed that fatty acid oxidase activity in the
livers of rats fed a threonine deficient ration was signif-
icantly lower than that in the livers of animals receiving
threonine supplements.

However, Arata g§_gl observed a tendency of this
enzyme system to adapt to the deficiency of threonine, although
their study was not of sufficient duration to measure the
extent of this adaptation. In a previous paper, Carroll gj_§l
(1959) observed varying degrees of adaptation depending upon
the enzyme system studied. If fatty acid oxidase is one of
the systems which undergoes complete adaptation (as is the '
case with malic dehydrOgenase), it is entirely possible that
this system had already adapted in the study reported here.

This possibility illustrates the hazard involved in
selecting a single time interval for analytical determinations
in a syndrome which changes with time. Since both the liver
fat and the enzyme systems tend to describe hyperbolic curves
when plotted against time, the time of maximum differences is
difficult to predict for a single analysis.

Though no effect on fatty acid oxidase activity was
observed when threonine was removed from the supplemented
control ration, a marked effect was noted when choline was
removed from this diet. If one assumes that the fatty acid
oxidase system has already adapted in the animals fed the
threonine-deficient diet, then the inhibition observed in

this system in animals fed a choline-deficient diet suggests

2%.

another interesting possibility. It is possible that the
deficiency of choline places a greater stress on the rat
than does a deficiency of threonine —- at least with respect
to the adaptation of fatty acid oxidase. It may be that the
albino rat cannot adapt to a choline deficiency, or that if
such adaptation can take place its appearance is much more
prolonged than it is when the diet is deficient in threonine.

The observation that the inhibition of the fatty acid
oxidase system does not persist when a threonine deficiency
is superimposed on a choline deficiency (Group IV) is puzzling.
However, the rats fed the doubly—deficient diet (Group IV)
grew less rapidly than those fed the choline—deficient diet
(Group III). Therefore, it is possible that two factors were
operating: (1) the slower rate of growth created less stress
in the unsupplemented rats, allowing them to adapt more easily
and (2) the slower growth rate in this group (IV) and the
concommitant lower requirement for choline for purposes of
growth would allow more of this compound to be used to
maintain the activity of the fatty acid oxidase system.

The only significant differences in the two types of
fatty livers produced in this experiment were refleCted in the
growth rate and the fatty acid oxidase activity of the livers.
The threonine deficiency affected the growth rate adversly,
but had no effect on the activity of either enzyme system
studied, as compared to the control group (I). The rats

fed the choline deficient diet (Group III) grew at a rate

25.

comparable to the control group (I), but a significant
depression in fatty acid oxidase activity was observed.

Thus, few biochemical differences were observed
between these two types of fatty livers. Moreover, the
differences which were noted might have been more qualitative
than quantitative. It is possible that the primary differ-
ence between choline induced and threonine induced fatty
livers is the rate at which adaptation takes place. This

study should be repeated with more frequent analyses in

 

order to test this hypothesis. . i

SUM-IARY

 

SUMMARY

Forty, male, weanling albino rats were fed a 9%
casein diet supplemented with choline and/or threonine.

The control animals were fed the 9% casein ration plus
choline and plus threonine. Food and water were allowed
‘gd libitum for a period of three weeks. Records of weight
gain were kept.

At the end of the experimental feeding period, the
rats were killed and the livers were analyzed for fat, nitrogen,
moisture, DPN cytochrome—c reductase and fatty acid oxidase
activity.

Supplementary threonine was found to have signif-
icantly increased the growth rate, regardless of the presence
or absence of the choline supplement. When threonine was
omitted from the diet, the growth rate was only 65% of the
threonine supplemented animals.

A deficiency of threonine, or choline, or both,
resulted in an abnormally high deposition of fat in the
livers of rats. When both threonine and choline were added
to the diet, liver fat levels were reduced to normal. This
effect was not observed when threonine or choline alone was
supplemented. These data are in agreement with several
published reports.

The nitrogen content of the livers remained fairly

constant between groups, but the amount of moisture present

27.

varied inversely with the fat content (dry weight).

When neither threonine nor choline were added to
the diet, the activity of DPN cytochrome-c reductase was
decreased. The addition of either threonine or choline was
equally effective in increasing the activity of DPN cytochrome-c
reductase to the level of the control group.

The presence or absence of threonine appeared to
have no effect on the fatty acid oxidase activity in the
liver. The removal of choline from the supplemented control
ration, however, resulted in a marked decrease in the activity
of the enzyme system; while the removal of both choline and
threonine had no effect. The possibility of enzyme adaptation
and the possible effect of the growth rate on these data are
explained and discussed at length.

Under the conditions of this experiment, the two
types of fatty livers produced were comparable in fat content,
nitrogen content and DPN cytochrome—c reductase activity.

The growth rate of the animals deficient in choline was near
that of the supplemented control animals, but the fatty acid
oxidase activity was greatly depressed on this diet. On the
other hand, a deficiency of threonine had the reverse effect
on these two factors. A double deficiency (threonine and
choline) resulted in decreased growth rate and depressed

DPN cytochrome-c reductase activity. In this group, the
fatty acid oxidase activity was comparable to that of the

control group.

LITERATURE CITED

 

'l wi'l‘.‘

LITERATURE CITED

Arata, D., Harper, A. E., Svenneby, G., Williams, J. N. Jr.,
and Elvehjem, C. A. 1954. Some effects of dietary
threonine, tryptophan and choline on liver enzymes and
fat. Proceedings Society for Experimental Biology and
Medicine. Ԥ7:544.

Arata, D., Svenneby, G., Williams, J. N. Jr., and Elvehjem,
C. A. 1956. Metabolic factors and the development of
fatty livers in partial threonine deficiency. Journal
of Biological Chemistry. 212:327-33.

Arata, D., Carroll, 0., and Cederquist, D. 1959. Manuscript
in preparation.

Beeston, A. W., and Platt, A. P. 1939. The effect of
supplements of tyrosine on the dietary fatty liver.
Journal Society of Chemists in Industry. 5§z557.

Best, C. H., Hershey, J. M., and Huntsman, M. E. 1932. The
control of the deposition of liver fat. American Journal
of Physiology. lglg7.

Best, C. H. and Huntsman, M. E. 1932. The effect of the
components of lecithine upon deposition of fat in the
liver. Journal of Physiology. 15:405.

Best, C. H. and Ridout, J. H. 1940. The lipotropic action
of methionine. Journal of Physiology. 213489.

Carroll, 0., Arata, D., Cederquist, D. 1959. Adaptation to
a threonine deficiency in rats. In press.

Shannon, H. J., Manifold, M. C. and Platt, A. P. 1938. The
action of cystine and methionine on liver fat deposition.
Biochemical Journal. 32:969.

Channon, H. J., Manifold, M. C. and Platt, A. P. 1938. The
action of sulfur containing amino acids and proteins
on liver fat depositions. Ibld., 33:866.

Channon, H.-J., Mills, G. T., and Platt, A. P. 1943. The
action of amino acids and proteins on liver fat deposition.
Ibidi, 31:483

Colowick, S. P. and Kaplan, N. O. 1953. Methods in Enzymology,
Volume I. Academic Press, Inc., New York. pp. 16—17,
545—8.

29.

Dianzani, M. U., 1955. Content and distribution of pyridine
nucleotides in fatty livers. Piochimica et Biophysica.
Acta 12:391.

Dianzani, M. U. 1957. The content of adenosine polyphosphates
in fatty livers. Biochemical Journal. 65:116—24.

Dick, F., Hall, W. K., Sydenstricker, V. P., McCollum, W.,
and Bowles, L. L. 1952. Accumulation of fat in the
liver with deficiency of threonine and of lysine.
Archives of Pathology. 52:154.

Eckstein, H. C. 1952. Dietary essential amino acids and the
liver lipid content of young white rats. Journal of
BiolOgical Chemistry. 195:167.

Fruton , J. S., and Simmonds, S. 1958. General Biochemistry.
2nd edition. John Wiley and Sons, Inc., New York.

pp. 537—9.

Harper, A. E., Benton, D. A., Winje, M. E. and Elvehjem, C. A.
1954a. On the lipotropic action of protein. Journal
of Biological Chemistry. 209:171.

 

Harper, A. E., Monson, W. J., Benton, D. A., Winje, M. E.,
Elvehjem, C. A. 1954b. Factors other than choline
which affect the deposition of liver fat. Ibid.
226:151.

Harper, A. E., Monson, W. J., Benton, D. A., and Elvehjem, C. A.
1953. The influence of protein and certain amino acids,
particularly threonine, on the deposition of fat in the
liver of the rat. Journal of Nutrition. 59:383.

Nino-Herrera, H., Harper, A. E., Elvehjem, C. A. 1954.
Histological differentiation of fatty livers produced
by threonine or choline deficiency. Ibid. 52:469.

Pilsum, J. F., Speyer, J. F., and Samuels, L. T. 1957.
Essential amino acid deficiency and enzyme activity.
Archives of Biochemistry and Biophysics. 6§;42—53.

Potter, V. R. 1946. The assay of animal tissues for res-
piratory enzymes. Journal of BIOIOgical Chemistry.

.léjz311.

Setton, D. Jr., and Grail, F. G. 1942. Effect of dietary
choline, ethanol-amine, serine, cystine, homocysteine
and guanido-acetic acid on the liver lipids of rats.
Ibidi l3£:l75.

Sidransky, H. and Farber, E. 1958c. Chemical pathology of
acute amino acid deficiencies I. Morphologic changes
in immature rats fed threonine -, methionine -, or
histidine - devoid diets. American Medical Association
Archives of Pathology. 665119.

Sidransky, H. and Farber, E. 1958b. II Biochemical changes
in rats fed threonine - or methionine - devoid diets.
Ibid. 66:135.

Singal, S. A., Hazan, S. J., Sydenstricker, V. P. and ¢;i_
Littlejohn, J. M. 1953a. The production of fatty -
livers in rats on threonine and lysine deficient diets.
Journal of Biological Chemistry. .gggz867.

Singal, S. A., Hazan, S. J., Sydenstricker, V. P. and
Littlejohn, J. M. 1953b. The effect of threonine
deficiency on the synthesis of some phosphorus fractions r”
in the rat. Ibid. 222:375- J

 

Singal, S. A., Sydenstricker, V. P. and Littlejohn, J. M.
1949. The lipotropic action of threonine. Federation
Proceedings. 6:251.

Spiegelman, S. and Kamen, M. D. 1946. Genes and nucleo—
proteins in the synthesis of enzymes. Science. 104:581.

Tucker, H. F. and Eckstein, H. C. 1937. The effect of
supplementary methionine and cystine on the production
of fatty livers by diet. Journal of B1010gical Chemistry.
121:479.

Tucker, H. F. and Eckstein, H. C. 1938. The effect of
supplementary lysine, methionine and cystine on the
production of fatty livers by high fat diets containing
cystine. Ibid. 1§63117.

Tucker, H. F., Treadwell, C. R. and Eckstein, H. C. 1940.
The effect of supplementary cystine and methionine on
the production of fatty livers by rats on high fat diets
containing casein or edestin. Ibid. l36:85.

Umbreit, W. H., Burris, R. H. and Stauffer, J. E. 1951.
Manometric Techniques and Tissue Metabolism. Burgess
Publishing 00., Minneapolis 15, Minn. pp. 140.

Van Baalen, J. and Gurin, S. 1953. Cofactor requirements for
lipogenesis. Journal of Biological Chemistry. 205:303.

wainio, W. H., Eichel, B., Eichel, H. J., Person, P., Estes,
F. L. and.Allison, J. B. 1953. Oxidative enzymes of
theuéivers in protein depletion. Journal of Nutrition.
49: 5.

APPENDIX

TABLE V.

Liver NitrOgen of Rats fed 9% Casein Diets
Calculated on Basis of Dry Weight of Liver

GROUP SUPPLEMENT N gm./100 gm. LIVER
(dry weight)
I 0.15% choline 10.8 i_0.3*
0.36% DL threonine
II 0.15% choline 7.7 1.0.3
III 0.36% DL threonine 8.7 :_0.2
IV No supplement 7.4 i 0.4

*Standard error of the mean

11

TABLE VI.

Activities of Two Enzyme Systems in Livers of Rats Fed
9% Casein Diets, Calculated on Basis of Nitrogen Content

GROUP SUPPLEMENT DPN Cytochrome-c Fatty Acid
reductase Oxidase
u1.02/hr./mg. N u1.02/hr./10 mg. N
I 0.15% Choline 656 i 27* 73 i 20*
0.36% DL threonine
II 0.15% choline 767 i 42 177 i 40
III 0.36% DL threonine 708 i 68 61 i 21
IV No supplement 539 i 42 160 i 30

“Standard error of the mean.

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