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THE EFFECTS ON THE WEAN'LING RAT OF THE
ADDITION OF WATER TO THREONINEDEFICIENT DIETS

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY

Rosemary .Louise Blyfh
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ABSTRACT
THE EFFECTS ON THE WEANLING RAT OF THE ADDITION OF
WATER TO THREONINE-DEFICIENT DIETS

by Rosemary Louise Blyth

Weanling rats of the Sprague-Dawley strain were fed
9% casein diets supplemented with 0.3% DL-methionine and
0.1%.DL-tryptophan, but not supplemented with threonine.
Varying levels of water were added as a 1%.agar solution to
the diet. The eXperimental period ranged from four days to
two weeks, food and.water consumption and weight gain
records were kept, and Protein Efficiency Ratios calculated.
The animals were sacrified, and livers removed for analysis
of moisture, fat and nitrogen content. The xanthine oxidase
activity of liver tissues was also measured.

Additions of 20, 50 or 80%.moisture to a 9% casein,
threonine-deficient diet cause a decrease in weight gain,
food and water consumption and Protein Efficiency Ratio in
weanling rats. Moreover, liver lipids are elevated in
animals fed the deficient diet plus 50%.water.

These effects are apparently not caused by chemical
interaction between water and the other diet components in

the food cup prior to ingestion. When the diet containing

 

Rosemary Louise Blyth
50%.moisture is allowed to remain at room temperature for 24
hours and then freeze-dried before feeding, liver lipids are
not elevated as observed when the diets are fed wet.

The addition of water to the threonine-deficient
diet increases the efficiency of utilization of the dietary
amino acids as measured by the xanthine oxidase activity of
the liver after four days of feeding the diet. This may
appear to contradict the data obtained in Part I, in which
the PER was decreased with increasing water in the diet.
However, in the imbalanced animals receiving the wet diet,
two different mechanisms are apparently in operation to
maintain homeostasis. Initially the efficiency of util—
ization of the amino acids is increased in the animals fed
the deficient diet wet as compared with those fed the same
diet dry, using xanthine oxidase activity of the liver as
the criterion. However, this initial increase in the
efficiency of protein utilization is followed by a decrease
in the food intake in response to the altered plasma amino
acid pattern (Yoshida et al., 1966). This pattern is more
rapidly distorted towards the "critical ratio" in the rats
fed the wet diet than is the case with those fed the dry
diet. Thus the increased availability of the amino acids

in the imbalanced diet induced by the addition of water

Rosemary Louise Blyth
serves only to aggravate the situation caused by the imbalance,
and over the two week experimental period results in a sig-

nificantly decreased food consumption, weight gain and PER.

THE EFFECTS ON THE WEANLING RAT OF THE ADDITION OF

WATER TO THREONINE-DEFICIENT DIETS

by

Rosemary Louise Blyth

A THESIS

submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Foods and Nutrition

1966

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ACKNOWLEDGEMENT

The author wishes to express her grateful appre-
ciation for the c00peration and guidance given by Dr.
Dorothy Arata during this course of study at Michigan

State University.

ii

ACKNOWLEDGEMENT .

TABLE OF CONTENTS

LIST OF TABLES .

TABLE OF CONTENTS

REVIEW OF LITERATURE

GENERAL METHODOLOGY .

PART I. THE EFFECT OF VARYING QUANTITIES OF WATER

ADDED TO LOW

DIETS .

INTRODUCTION .
METHODS . . .
RESULTS . . .
DISCUSSION . .
SUMMARY . . .

PART II. EFFECTS OF

INTRODUCTION .
METHODS . . .
RESULTS . . .
DISCUSSION . .
SUMMARY . . .

PART III. THE EFFECT

PROTEIN, THREONINE-DEFICIENT

FREEZE—DRYING ON THE RESPONSE OF
THE WEANLING RAT TO WET VERSUS DRY THREONINE-
DEF IC IENT DIETS o o o o o o o I o o o o o 0

OF ADDING 50%»WATER TO LOW

PROTEIN, THREONINE-DEFICIENT DIETS ON
THE LIVER XANTHINE OXIDASE ACTIVITY OF
WEANLING RATS . . . . . . . . . . . . . .

iii

Page
ii

iii

17

21

22
24
26
33
37

38

39
41
43
50
52

53

Page

INTRODUCTION . . . . . . . . . . . . . . . . . . . 53
METHODS . . . . . . . . . . . . . . . . . . . . . 55
RESULTS . . . . . . . . . . . . . . . . . . . . . 57
DISCUSSION . . . . . . . . . . . . . . . . . . . . 69
SUMMARY . . . . . . . . . . . . . . . . . . . . . 71

GENERAL DISCUSSION AND SUMMARY . . . . . . .

BIBLImR'APIN O O O O O O O O O O O O O O O O O O O O 74

iv

TABLE

LIST OF TABLES

PAGE
PART I

The effects of varying levels of water added
to low protein, threonine-deficient diets on
weight gain, food consumption, PER and water
consumption of weanling rats. . . . . . . 29

The effects of varying levels of water added
to low protein, threonine-deficient diets on
the liver composition of weanling rats . . . 30

The effect of pair feeding on weight gain,

food consumption, PER and water consumption

of weanling rats fed wet or dry low protein

diets deficient in threonine. . . . . . . 31

The effect of pair feeding on liver compo-
sition of weanling rats fed wet or dry low
protein diets deficient in threonine . . . . 32

PART II

A comparison of the effect of adding 50%

water to low protein, threonine-deficient

diets, and the same diets freeze-dried with

and without agar on the weight gain, food
consumption, PER and water consumption of

the weanling rat. . . . . . . . . . . 46

A comparison of the effect of adding 50%

water to low protein, threonine-deficient

diets, and the same diets freeze-dried with

and without agar on the liver composition

of the weanling rat. . . . . . . . . . 47

TABLE

3.

PAGE

A comparison of the effect of adding 50%

water to low protein, threonine-deficient

diets, and the same diets freeze—dried with

and without agar on the weight gain, food
consumption, PER and water consumption of

the weanling rat. . . . . . . . . . . 48

A comparison of the effect of adding 50%

water to low protein, threonine-deficient

diets, and the same diets freeze-dried with

and without agar on the liver composition

of the weanling rat. . . . . . . . . . 49

PART III

Food intake, water consumption and weight

gain of rats fed low protein, threonine—

deficient dietswith and without the addition

of 50% water . . . . . . . . . . . . 61

Food intake, water consumption and weight

gain of rats fed low protein, threonine-

deficient dietswith and without the addition

of 50%.water . . . . . . . . . . . . 62

Liver composition of rats fed low protein,
threonine-deficient diets with and without
the addition of 50% water. . . . . . . . 63

Xanthine oxidase activity in rats fed low
protein, threonine-deficient dietswith and
without the addition of 50% water . . . . . 64

Food intake, water consumption and weight

gain of rats fed low protein, threonine-
supplemented diets, and low protein, thre-
onine-deficient diets with and without the

addition of 50% water . . . . . . . . . 65

Food intake, water consumption and weight

gain of rats fed low protein, threonine-
supplemented diets, and low protein, thre-
onine-deficient diets with and without the

addition of 50% water . . . . . , . . . 66

vi

TABLE

PAGE

Liver composition of rats fed low protein,
threonine-supplemented diets, and low

protein, threonine-deficient diets with

and without the addition of 50%Iwater. . . 67

Xanthine oxidase activity in rats fed low
protein, threonine-supplemented diets,
and low protein, threonine-deficient diets

with and.without the addition of 50%»water . 68

vii

REVIEW OF LITERATURE

Harper (1959) has defined amino acid imbalance as
"a change in the prOportion of the amino acids of the diet
that results in a depression of food intake or growth rate
that can be completely prevented by a supplement of the amino
acid present in the least amount in the diet in relation to
the amount required for Optimal performance." The evolution
of this concept as defined by Harper has been long and
tedious, and complicated by a great deal of confusing data.

The first instance of the phenomenon, noted in 1946
by Krehl and coaworkers, was perplexing in that the addition
of protein to diets deficient in that nutrient caused a

growth retardation in rats. Krehl et a1. added gelatin to

 

diets deficient in tryptophan and niacin, and observed a
significantly lower growth rate as compared with that of
rats fed the unsupplemented diet. A similar retardation in
growth was seen with certain amino acid supplements to low
protein diets. When Hankes et al. (1949) added an amount
of threonine equivalent to that in 6%.casein to a 9% casein
diet supplemented with 0.2% cystine, a growth depression

1

2
occurred. This could be reversed by the addition of 1.5
mg% niacin or 5.0% tryptophan. These workers suggested
that the inhibition of growth occurring with the addition
of threonine could be due to some reaction taking place in
the intestinal tract or tissues between cystine, threonine
and tryptophan, which utilized the tryptophan and left in-
sufficient amounts for niacin synthesis. Thus these early
attempts to correct deficient diets by supplementing at
least one of the deficient amino acids resulted in a situa-
tion measurably worse than the original deficiency. The
effort to explain this phenomenon occupied many years.
First efforts centered around the documentation of various
kinds of growth depressions induced by amino acid supple-
mentation. These were followed by studies aimed at
unearthing the underlying physiological and biochemical

mechanisms.

Growth Depression Studies

 

Salmon in 1954 provided the first concrete data
implicating the balance of amino acids in the diet as the
critical factor. He found that 20 mg niacin/kg diet
reversed the decreased growth seen in rats When 10% gelatin

was added to a ration containing 20%.casein, 40% corn grits

E?"

 

 

 

3

and 0.3%>L-cystine. However, the same quantity of niacin
added to a 7% casein, 40% corn grits ration had no effect.
Only the addition of 0.2% DL-tryptophan increased growth.
He suggested that niacin corrected the growth depression
only when the addition was made to diets that contained a
limited surplus of tryptophan above that needed for use as
amino acid per se. This surplus would normally be converted
to niacin, but when the tryptophan deficient protein was
added, the conversion failed to occur. In this case, niacin
was the limiting factor for growth. However, in the 7%
casein diet, there was no surplus of tryptophan to begin
with, and tryptophan was growth limiting. Then the addition
of niacin had no effect.

Sauberlich and Salmon (1955) observed a slow rate
of growth in animals fed a 7% casein diet deficient in
niacin. Growth was further reduced with the addition of
gelatin, and restored with a supplement of 0.05%.DL-tryto-
phan. The addition of niacin had no effect. When 20%
oxidized casein (devoid of cystine, methionine and trypto-
phan) was added to a 10% casein basal diet, the growth of
animals fed this supplemented ration was depressed below
the basal diet controls. Growth was restored in this case

with 0.4% DL-tryptophan, and the authors concluded that the

14425":

 

4
tryptophan requirement of the rat was not constant, but
related to the diet employed and the protein level.

In 1953, Henderson, Koeppe and Zimmerman suggested
that niacin deficiency was really a result of amino acid
imbalance, and substantiated this with diets composed of
free amino acids. They produced a niacin deficiency in
rats fed 10% acid4hydrolyzed casein rations supplemented
with 0.2% L—cystine, and 0.1%.DL-tryptophan. When the
threonine content of the diet was increased from 0.33 to
0.30% of the L-isomer, growth was depressed. This was re-
versed with the addition of 2.5 mg% niacin. They suggested
that the addition of other amino acids to diets containing
a limited surplus of tryptophan for niacin synthesis caused
this tryptophan to be used preferentially for protein syn-
thesis, and conversion to niacin did not occur.

Sauberlich (1956) suggested that it was the amino
acid array in the diet that caused growth depression, and
not a Specific effect of one added amino acid or protein,
or the vitamin interrelationship that might be involved,
as was the case with niacin. By adding 20%.oxidized casein
to a 35% peanut meal ration supplemented with 0.2% L-lysine
and 0.3%.L-cystine and Vitamin B12, he made methionine the

most limiting amino acid. Growth was depressed with the

gs“:

 

 

 

 

5

addition of 0.2% DL-tryptophan and the depression reversed
by adding 0.5% DL—methionine. He also created an imbalance
with the addition of 15%»hemoglobin to 75% corn diets.
Corn is deficient in tryptophan, isoleucine, lysine, thre-
onine and valine, and 75% corn diets When supplemented with
these amino acids supported growth. The isoleucine content
of the supplemented diet was barely adequate however, and
when hemoglobin with its low isoleucine level was added, a
severe inhibition of growth occurred. This was reversed
by a supplement of 0.55% isoleucine to the diet. The fact
that he caused an imbalance of an amino acid (isoleucine)
without a known relationship to a vitamin suggested that
the imbalance is a general phenomenon associated with amino
acids.

That an amino acid imbalance is caused by the
addition of the second most-limiting amino acid in the
diet was established by Morrison and Harper (1960). They
used 8 or 9% casein diets supplemented with 0.3% DLamethion—
ine, and added separately a series of amino acids in
attempts to produce a growth retardation. Only those
animals fed diets supplemented with threonine, the second
most—limiting amino acid, showed depressed growth.

Deshpande (1958fl created an imbalance in rats fed

THESIS

 

 

 

'1'.

 

 

6

6% fibrin diets. He found that four amino acids, leucine,
isoleucine, valine and histidine were all about equal and
first-limiting for growth, and methionine and phenylalanine
about equally second most-limiting. The addition of 0.4%
methionine and 0.6%7phenylalanine to 6% fibrin diets caused
a growth retardation that was reversed with the addition of
all four of the most limiting amino acids.

The level of protein in the diet was found to be
important in producing an imbalance. Harper (1959b) fed
rats various levels of casein supplemented with 0.3%.me-~
thionine. He added an amino acid mixture equivalent to the
L isomers of 6% casein, but lacking in threonine to diets
containing 4, 6, 8, 10, and 15% casein. The growth depres-
sion was greatest with the 6% casein diets. It was less
at levels higher or lower than 6%, and very little depres-
sion of growth occurred when the amino acid mixture was
added to the 15% casein diets. Rats fed 4, 7 and 10% casein
diets supplemented with 0.3%.methionine and the amino acid
mixture lacking threonine suffered different magnitudes of
a growth depression, the most severe being with the 7%
casein ration, but all the depressions were reversed by
the same amount of threonine, 0.1%.of the DL isomer. Har-

per hypothesized that adding the same quantity of an amino

 

 

[SIQ

7423 “fit“

 

7
acid mixture which increased the requirement of the animal
by 0.1%.would always call for an equal supplement of the
amino acid, even though the growth depression caused by
the imbalance is less severe when it is produced with higher
quality proteins.

The problem of amino acid imbalance has been com-
plicated by the appearance of fatty livers in rats fed 9%
casein diets supplemented with choline and niacin. Harper
(1954a) found an increased deposition of fat in the livers
of animals fed a 9% casein diet supplemented with methionine
or cystine. When the sulphur-containing amino acids were
not added, growth was retarded, but there were no fatty in-
filtration of the liver. Harper added 0.1%»DLamethionine
to 9% casein diets containing choline and 0.1%.DL-trypto—
phan, and found an increase in liver fat on a dry weight
basis from 13.0% to 24.6%» The inclusion of 0.36%.DL-thre-
onine reduced the liver lipid to 13.1%. Increments of
methionine up to 0.6%Iof the DL isomer caused no further
increase in liver fat. When rats receiving 9% casein diets
containing choline and 0.3% DLamethionine were pair fed
against the same diet with no methionine supplement, there
was no difference found in the percent liver fat of either

group, nor did the addition of methionine cause as great

Wests

 

.52

 

 

 

8
an increase in growth as when the diet was fed ad libitum.
Harper suggested that a 9% casein diet is primarily defic-
ient in the sulphur-containing amino acids, and when
methionine was added, the secondary deficiencies of threonine
and tryptophan became evident. When tryptophan was added,
the threonine deficiency appeared, and fat accumulated in
the liver. When the intake of the diet containing methionine
was limited to that of the unsupplemented group, threonine
did not become sufficiently limiting to cause the deposition
of fat.

Deshpande and Harper (1958b) suggested that the
preportions of amino acids required for growth might differ
from those required for physiological processes, such as fat
metabolism. When the growth of amino acid imbalanced rats
is stimulated by adding the most limiting amino acid, more
of the other amino acids are used for protein synthesis
and less for control of fat deposition. This agreed with
Singal et al. (1953a) who fed rats an amino acid mixture
containing all the amino acids provided by a 9% casein diet
except for threonine. Consumption of this threonine-devoid
diet caused a growth depression, but the animals had normal
liver lipid levels. When 0.7% DL—threonine was added,

bringing the level to that in 9% casein, growth improved

E5“:

 

 

 

9
markedly, but there was an increased deposition of fat in
the liver. Not until 1.1% DL-threonine was added and
growth was Optimum was a surplus of threonine available,
and liver fat levels reduced to normal.

By this time, a significant number of amino acid
imbalances had been identified, and attention in this field
was turned toward an effort to identify the physiological
and biochemical basis for the occurence of amino acid im-

balances.

Physiological and Biochemical Studies

Deshpande (1958b) attempted to study the fateof
the amino acids that could not be used for protein syn-
thesis when one amino acid is limiting. Salmon in 1954
had suggested that the surplus of amino acids arising from
an imbalance might stimulate amino acid catabolism, and
result in some additional loss of the amino acid that is
already in shortest supply. Deshpande (1958b) found some
support for this theory. He fed 6% fibrin diets imbalanced
with respect to leucine, isoleucine, valine and histidine
by the addition of 0.4%»methionine and 0.6% phenylalanine.
He found that the imbalance led to a decrease in food in-

take, total nitrogen retained, and percent absorbed nitrogen

mm:

 

 

10
retained. Force feeding the diet in an attempt to elevate
the food intake resulted in the death of the animals in 2
or 3days.. He hypothesized that the rapid fall in food in—
take and percentage of absorbed nitrogen retained might
indicate a reduced ability of the rat to utilize the nitro-
gen present in the diet. However, Kumpta et al. in 1958
found the percent nitrogen retained by animals fed a 6%
fibrin diet imbalanced in leucine, isoleucine, valine and
histidine was not significantly different from that of
animals fed the control 6% fibrin diets pair fed to the
same low food intake as the imbalancedanimals. Thus it
was very hard to separate the effects of the imbalance from
the effects of the decreased food Consumption.

These workers found the food intake fell within 24
hours after feeding the imbalanced diet. However, in rats
trained to eat a single daily feeding in a one or two
hour period, the foOd intake did not fall until the second
day, indicating that there was some sort of systemic
effect in Operation, and not merely a difference in pala-
tability (Kumpta, 1958). They suggested that the plasma
amino acid patterns might provide some answer. As early
as 1955, Sauberlich and Salmon noted an altered plasma

amino acid pattern in rats fed tryptophan-deficient diets.

"3!qu

 

 

 

11
They added 20% oxidized casein to 10% casein diets supple-
mented with L-cystine, and found a lowered plasma trypto-
phan and two to three times as much tryptophan in the urine
of rats fed the imbalanced diets compared to those fed
control diets to which no oxidized casein had been added,
or corrected diets that were supplemented with 0.4%.DL—
tryptophan.

Kumpta and Harper (1962) studied the plasma amino
acid pattern of rats fed an imbalanced diet of 6% fibrin
and an amino acid mixture lacking histidine. Despite the
fact that the animals had been pair fed so that both groups
consumed exactly the same amount of histidine, the plasma
histidine levels of the imbalanced rats fell far below
those of the controls within an hour.. The other amino
acids in plasma from imbalanced rats reflected the dietary
pattern.

Sanahuja and Harper (1962) investigated the problem
of the decreased food intake of rats fed imbalanced diets
in the light of these data. They pr0posed a homeostatic
mechanism that resPonds to changes in the plasma amino acid
pattern, and explored this possibility by means of experi-
ments that allowed rats to choose between imbalanced and

protein-free diets. When rats depleted in protein for a

l2
seven day period were offered a choice between an imbalanced
and a protein-free diet, they chose the latter within three
days (Sanahuja and Harper, 1962). Correction of the imbal-
anced diet with the addition of histidine caused a reversal
of the preference for the protein-free diet. Although rats
that had not been protein depleted began by consuming more
of the imbalanced diet, by the end of 24 hours, almost all
of the animals were consuming the protein-free diet.

The difference in behavior of the depleted rats was
only in the time at which the response occurred, and.was
ascribed to the plasma amino acid pattern. As soon as the
non-depleted rats consumed the imbalanced diet, plasma
levels of the limiting amino acid would fall rapidly, and
the ratio between the level of this amino acid and all the
othenswould become very great. 0n the other hand, this
ratio would ggt_be so great in the depleted rats because
although the histidine level would be very low, the levels
of the other amino acids would not reach as high a level
as quickly as they would in the non-depleted animals.
According to this theory, the homeostatic mechanism would
be activated by the "critical ratio" of the limiting amino
acid to the others, and would prompt the animals to begin

consuming a protein-free diet that would promote a more

13
normal plasma amino acid pattern.

These experiments on the amino acid pattern of the
blood prompted a more thorough examination of the immediate
fate of the most limiting amino acid in the diet. Yoshida
et al. (1966) created amino acid imbalances of threonine and
histidine using casein diets. Six percent of casein was im-
balanced with respect to threonine by the addition of an
amino acid mixture lacking in threonine, and a histidine
imbalance created with the addition of an amino acid.mixture
minus histidine to a 5% casein diet. Additions of threonine
-Cl4 and histidine-C14 to each diet as tracers were of
quantities too small to affect growth.

There was no difference in the excretion of the
labelled amino acids in C02, urine or feces between the
balanced and the imbalanced rats. However, rats fed the
imbalanced diets retained more radioactivity in the liver
and carcass. There were no observed differences between
the radioactivities of the gastrointestinal contents of the
groups, so the imbalance did not affect absorption. The
blood plasma showed significantly less threonine-C14 and
histidine-C14 in rats fed the imbalanced diets than those
fed the control. They hypothesized the following succession

of events. The imbalanced meal was digested and absorbed

14
normally, causing a flow of all but one of the essential
amino acids to the liver, where protein synthesis was stimu-
lated. In the imbalanced animals, more of the amino acid
in shortest supply was incorporated into protein so that
the amount in the peripheral system.was reduced, and food
intake depressed. Sidransky and Farber (1958) have shown
that pathological lesions developed when severely deficient
diets were force-fed to the rat. This depression in food
intake is apparently a protective response on the part of
the animal.

These data effectively eliminated Salmon's theory
of increased catabolism of the limiting amino acid, but
supported Henderson, who had earlier (1953) suggested an
increased protein synthesis accounted for the lack of try-
ptophan conversion to niacin when tryptophan was not the
limiting amino acid. Sidransky and Farber (1958) found
that force-feeding an amino acid diet devoid of threonine
caused an increase in the RNA content of the liver, and
that the incorporation of amino acids into liver protein
was increased.

Yoshida et a1. did not find conclusive evidence of
enhanced protein synthesis in organs other than the liver,

but there was no depression of labelled amino acid incor-

15
poration into any other compartment, and the same altera—
tions would occur in the plasma amino acid pattern if the
synthesis were only accelerated in a few organs, and pro-
ceded at a normal rate in others. Of course, protein
synthesis over a longer period of time would be decreased
once the initial rapid rate had resulted in the decreased
food intake and depressed growth.

Kumpta and Harper (1961) showed that When the food
intake of rats fed a 6% fibrin diet, imbalanced.with respect
to leucine, isoleucine, valine and histidine by the addition
of methionine and phenylalanine, was increased by the in-
jection of insulin, the animals grew well, and showed no
adverse effects. Thus, provided that the food intake can
be maintained, the limiting amino acid would be used as
efficiently, if not more so, than the same amino acid in
a control diet (Yoshida et al., 1966). The resPonse of the
animal, the depressed food intake, is a response to a plasma
amino acid pattern that looks like that of a much more
severely deficient diet than the imbalanced diet actually
is, and if the food intake can be stimulated by some method
such as insulin injection, the animals should grow well and
show no ill effects.

Since the problem of amino acid imbalance has there-

WES“:

 

 

 

 

 

16
fore been suggested by Harper (1964) as being primarily one
of overcoming the depressed food intake, any method that
increases the availability of the amino acids should accom-
plish the same end. In 1962, Keane and Denton found that
they could increase the food consumption, weight gain and
efficiency of utilization of protein of rats fed low protein
diets by the addition of water. It was therefore decided to
attempt the correction of the threonine-imbalanced, low pro-
tein diets used in this laboratory with the addition of

varying levels of water.

THESIS

 

GENERAL METHODOLOGY

18

Male weanling rats of the Sprague-Dawley strain
were used in all exPeriments. The initial weight ranged
from 43 to 52 grams, and the animals were divided into
groups so that the mean weights of the groups did not
differ more than i 0.5 grams. They were individually
housed in screen bottom cages, in a temperature controlled
room, and weighed twice weekly over the experimental
period. weight gain was reported as grams gain per week.

The basal diet was of the following percent compo-
sition: sucrose, 81.2; casein, 9.0; salts W,1 4.0; fat
(corn oil),2 5.0; vitamin mix,3 0.25; choline chloride,
0.15; DLémethionine, 0.3; DL—tryptophan, 0.10. Alphacel
was added to the diet to the level of 10% to retard diar-
rhea. In those experiments where threonine was supple-
mented to this basal diet, 0.36%.of the DL isomer replaced

an equal weight of sucrose.

The addition of water to the basal diet was accom-

 

1Wesson modification of Osborne and Mendel salt
mixture. L. G. Wesson, Sci., 25, 339 (1932).

2Containing 75 mgtx-tocopherol per kilogram diet.

3For composition of vitamin mix see L. L. Rikans,
D. Arata and D. C. Cederquist, J. Nutri., 83, 83 (1964).

WES":

 

 

 

l9
plished with a 1%agar4 solution. The agar was disolved
in water with heat, and allowed to cool before being added
by weight to the desired percentage of the diet solids.
The mixture formed a thick gel when set, which prevented
the separation of the solid and liquid components of the
diet, thus ensuring the consumption of the prOper prOportion
of solids to water. The wet diets were mixed fresh daily,
while the basal dry diet was replenished only when neces-
sary. Food consumption was recorded as grams of diet
solids consumed, and the animals were allowed to eat ad
libitum, except where the pair feeding technique was used.
Individual water consumption was not measured. The animals
were allowed to drink ad libitum, but the bottles were
filled from a weighed reservoir for the group. The dif-
ference between this amount and the total remaining in the
bottles at the end of one week was recorded as the total
weekly consumption of the group. This figure divided by
the number of animals gave the average weekly consumption
per rat. These data were totalled for the experimental
period, and the average consumption per week per rat com-

puted.

 

4Bacteriology grade, General Biochemicals, Chagrin
Falls, Ohio. (Lot #54395)

20

At the end of the two week experimental period, the
animals were killed by decapitation and exsanguinated. The
livers were excised,weighed and homogenized in distilled
water in a Potter—Elvehjem homogenizer.5 They were placed
in a forced air drying oven at 850C for 12 hours, and then
removed to a vacuum oven at 50°C where they were allowed to
come to constant weight. The moisture content of the liver
was calculated, and the dried tissue ground in a Wiley mill.
Fat content of the liver was determined by continuous ex—
traction with ether of the dried ground tissue for three
hours on a Goldfisch apparatus. The data were calculated
as the percent of the dry weight of the tissue. Nitrogen
was determined on 0.10 gram samples of the fat extracted
tissue, and calculated as the percent of fresh tissue.
Standard errors of the means were calculated for all data
except water consumption where there were no individual
values, and the Student's t test used to measure signifi-
cance. Unless otherwise stated, a probability of 0.01 or

less was used as a criterion of significance.

 

5
V. R. Potter and C. A. Elvehjem, J. Biol. Chem.,

114, 499 (1936).

mtg:

 

 

 

 

PART I

THE EFFECT OF VARYING QUANTITIES OF WATER ADDED
TO LOW PROTEIN, THREONINE-DEFICIENT DIETS

may:

 

 

 

 

INTRODUCTI ON

Keane, Smutko, Krieger and Denton in 1962 studied
the effects of the addition of water to low protein diets.
They fed male weanling rats 9% casein diets containing
choline and added varying amounts of water. The water was
added alone or in combination with such diet thickeners as
agar and guar gum. In the absence of any thickener, the
addition of water at the 20% level produced a highly
significant increase in PER compared with the dry control
diet. In the presence of agar, the addition of water at
the 20 and 50% levels also produced a highly significant
(P < 0.01) increase in PER over control values, but compar-
able to water alone. An increased food consumption was
noted, but when animals were pair fed a diet containing
20% water to animals receiving a 9% casein diet dry the
PERs of the "wet" group were still significantly increased,
despite the same protein consumption (Keane et al., 1963).
The effects of the addition of different levels of water
to various levels of protein in the diet were also studied
(Keane et al., 1962). With 6 and 9% casein diets,

22

23
PERs were significantly increased (P < 0.01) with additions
of 20% water. There was no increase in PER with 18% casein
diets. The authors suggested that the osmotic pressure in
the gastrointestinal tract prompted the increased utiliza-
tion of protein in the low protein diets.

Since Keane and Denton worked with a low protein
diet similar in composition to the 9% casein diet used to
produce a threonine imbalance in this laboratory, it was
suggested that the addition of water to this low protein,
threonine-deficient diet might increase the food consump-
tion, weight gain and efficiency of utilization of protein
in rats fed these diets. Harper (1964) suggested that the
main problem in overcoming an amino acid imbalance is in
counteracting the depressed food intake. Increasing the
level of water in the diet might increase the efficiency
of utilization of the amino acids in the deficient diet
and achieve the same end as improving the food intake.
ExPeriments studying these possibilities are described in

the following sections.

METHODS

Five groups of ten animals each were fed the basal
diet plus five different levels of added water. Data from
rqiicate groups were combined at the end of the study.

The groups were as follows:

Group I - basal diet,no added water.

Group II - basal diet, 10%»water.

Group III - basal diet, 20%Iwater.

Group IV — basal diet, 50% water.

Group V - basal diet, 80% water.

Weight gain, food consumption and water consumption
were recorded and the Protein Efficiency Ratio calculated
(weight gained in grams per week per gram of protein con—
sumed) for each group. Livers were analysed for moisture,
fat and nitrogen.

In the first experiment conducted in this series,
animals were given these diets ad libitum. Rats fed the
wet diets ate significantly less on a solids basis than
did those fed the dry diet. In order to eliminate this
complicating factor, the pair feeding technique was used

24

25
in succeeding experiments. Since the greatest effect on
liver lipid from the addition of moisture was seen in the
50%.wet group, it was decided to pair feed to this group
on a solids basis. Three groups of ten animals each were
used in the study, and data from replicate groups combined.
Group I was fed the basal diet with no added moisture.
Group IV received the basal diet containing 50%.moisture,
and Group VI received the basal diet with no added moisture,
pair fed to the solids consumption of Group IV (50%»wet).
The analytical procedures were the same as for the ad

libitum studies.

RESULTS

Weight gain, food consumption, PER and water con—
sumption data for the ad libitum studies are presented in
Table l. A significant growth depression from the control
(P < 0.01) is obtained with the addition of 20, 50 and 80%
water. Food consumption is significantly reduced from the
control at each of these levels. The depression of the
food intake is of equal magnitude in Groups III and IV,
with a more severe decrease as the water content of the
diet is increased from 50 to 80%. As would be expected,
the water consumption of rats in each group varies in—
versely with the water content of the diet. When the
total water consumed, including that provided by the diet,
is calculated, there still remains a difference between
the amount consumed by the animals fed the basal dry diet,
and those fed the wet. Group .V (80%.moisture) is an
exception, the total consumption being very nearly the
same as that of Group I. The PERs are significantly re-
duced with the addition of 20 and 80%.moisture, and since
there are no significant differences between these two

26

27
groups and Group IV (50%Hwet), the reduced PER with 50%
moisture must also be significant. The addition of 10%
moisture causes no significant decrease in PER.

Liver composition data are presented in Table 2.
Livers taken from rats in Group IV (50% wet) have signifi-
cantly less moisture than do those from control animals,
but there are no significant differences among the other
groups. The addition of 50%.moisture (Group IV) to the
basal diet (Group I) causes a significant elevation in
liver lipid from 25.7 to 30.1%. However, a further in-
crease in moisture content of the basal diet to 80%.(Group
V) does not elevate the lipid content of liver tissues
above the control value. There are no significant differ-
ences in the percent liver nitrogen between groups.

The results from the pair feeding trials are re-
ported in Tables 3 and 4. Increasing the moisture content
of the basal diet to 50%,results in a significant decrease
in food consumption and PER (Table 3). These results agree
inith those from previous experiments (see Table 1). When
'the basal diet is pair fed with Group IV, an identical decrease
in.growth, food consumption and PER is observed, and the
imater consumption rises, as would be exPected, since the

diet is fed dry.

nests

 

28

The animals fed the 50% wet diet ad libitum (Group
IV) have livers significantly lower in moisture and higher
in fat compared to the basal dry controls. These results
corroborate data from the previous studies (see Table 2).
However, animals consuming the same amount of dry diet as
those in Group IV (50% wet) show liver moisture levels
comparable to controls, and liver fat reduced even below
control levels. No significant differences are found

between any of the three groups‘in percent liver nitrogen.

29

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DISCUSS ION

The addition of 10%.moisture to a low protein, threo-
nine-deficient diet has no significant effect on the para—
meters measured in this study. However, at all levels above
10%, there is a significant decrease in food consumption,
weight gain and PER. There is no significant change in the
liver composition until the moisture content of the diet
reaches 50%, at which point moisture is decreased and fat
elevated. The low lipid levels in livers from rats in
Group V (80%wwater) may result from the greatly depressed
food intake. The threonine deficiency may have been too
severe to allow the appearance of fatty livers. Singal et
al. (1953a) fed weanling rats an amino acid diet to simulate
9.0% casein. It contained 0.7% DL-threonine, and produced
liver fat levels of 13.9% of the fresh tissue weight. When
he reduced the amount of threonine to 0.55%, the liver
lipid was 11.7%, and at 0.4%.threonine, it fell to 8.1%.

The addition of 1.1% DL—threonine brought the liver fat to
5.9%, and the complete omission of threonine produced levels
of 5.7%. There was a range between the 0.7% level of thre—

33

may:

 

 

34
onine and the complete lack of the amino acid when growth
was retarded to a greater degree, and less fat deposited
in the liver than was observed when higher levels of the
amino acid were fed. In this experiment, the animals fed
the diet with 80%.moisture added consumed only 73.5% as
much threonine as the control animals, and it is possible
that this reduction brings the threonine intake within the
range where fatty livers do not appear.

The results from the pair feeding studies indicate
that the addition of moisture, rather than the reduction
in food intake is responsible for the increase in liver
fat. Livers from animals consuming the same quantity of
the dry ration as those fed the 50%.wet diet (on a solids
basis) contain significantly less fat than those fed the
wet diet.

The PER data from the ad libitum studies indicate
that the addition of water to 20% or more of the diet has
an adverse effect on the absorption and/or utilization of
the diet. However, in the pair feeding trials the PERs
of the animals fed the wet diet are very nearly identical
to those of the animals fed the same quantity of dry solids,
and are significantly lower than controls. It is difficult

to define which are the effects of the diet and which the

35

decreased food intake. These data are contrary to those
reported by Keane and Denton (1962). They observed in-
creased growth and higher food efficiency resulting from
increasing the water content of the diet. They hypothe-
sized that the difference in osmotic pressure of the gastric
contents of the animals fed the wet diets caused the
increase in weight gain, food consumption and PER. There
is in fact evidence that altered tonicity of the gastric
contents affects gastric motility. Hunt (1959) states

that there are receptors in the duodenum that are sensitive
to the osmotic pressure of the chyme. Thomas (1934)
suggests that the mechanism is a vagal reflex that lowers
the tone of the stomach muscle and the strength of the
peristaltic contractions, rather than shutting down the
pylorus to prevent the entrance of a hyper- or hypotonic
chyme. If this mechanism is operative, the rate of flow
through the gastrointestinal tract would be slowed, and
there would be time for more thorough absorption. This
would produce a higher efficiency of utilization of the
diet solids, and account for the increased weight gain and
PER found in the exPeriments of Keane and Denton. The de-
creased weight gain, food consumption and PER found in this

experiment are not in agreement with their work, but these

36
diets are low in protein and imbalanced with respect to
threonine, while Keane and Denton's eXperiments were not
complicated by the presence of an amino acid imbalance.
Furthermore, PER values are relatively gross measurements,
compounding the error from two parameters, and this combined
with an eXperimental period of only two weeks may make the
PER a somewhat less than ideal method for measuring protein
'utilization under these conditions.

These grounds do not seem sufficient to eliminate
entirely osmotic pressure as a consideration in the studies
under discussion, and it may Operate in conjunction with
some other factor. The effect of added moisture to the
diet may be reduced to two possibilities; either the effect
is a chemical one, resulting from the interaction of the
water with the diet solids prior to ingestion by the animal,
or a physical one yi§_the water's presence in the gastro-
intestinal tract. ExPeriments exploring both of these

possibilities are discussed in the following sections.

SUMMARY

When 20, 50 or 80%.water are added to low protein,
threonine-deficient diets (9% casein supplemented with 0.3%
DL- methionine and 0.1%.DL- tryptophan) a significant de-
crease (P < 0.01) is observed in the weight gain, food
consumption and PER of animals fed these diets. Alteration
in the liver composition does not occur until 50% water is
added, when moisture is decreased and fat elevated. In the
animals receiving 80%»moisture in the diet, fatty livers do
not appear, but this may be due to the low food and.hence
threonine intake. The effect of the water may be mediated
through either chemical alterations in the diet solids
caused by their contact with the water over a 24 hour period
prior to ingestion, or through the physical presence of the
water in the animal's gastrointestinal tract. Experiments

exPloring these possibilities will be described.

37

'I-‘l-! ‘

 

 

PART II

EFFECTS OF FREEZE-DRYING ON THE RESPONSE
OF THE WEANLING RAT TO WET VERSUS
DRY THREONINE-DEFICIENT DIETS

INTRODUCTION

Adding water to dry, low protein, threonine-
deficient diets may cause some chemical change in the diet
solids that would produce the effects described in Part I
when fed to the animal. The wet diets remain in the cages
at room temperature throughout a 24 hour period, and under
these conditions microbial action, for instance, might
alter one or more of the dietary constituents.

This experiment was designed to preserve in the
diet solids any changes that might have occurred during
this time period. If the diets are allowed to stand at
room temperature for 24 hours, and then freeze-dried, any
changes should remain in the solid components, as the
process only removes the water with no effect on the rest
of the diet mixture. In order to eliminate the possibility
that the addition of agar as a vehicle for the water has
an effect on its own, wet diets were prepared with the
usual agar solution, and also with water alone.

If the added water has a chemical effect on the
diet solids prior to ingestion by the animal, the data

39

THESIS

.‘HJ-m

hub.

“"

 

40
from the freeze-dried groups should compare favourably

with those of the animals fed the wet diets.

METHODS

Four groups of ten animals each were used in the
experiment. Group I received the basal diet dry,1 and
Group IV the basal diet with 50%.moisture added. The
ration for Group VII (wet diet, freeze-dried with agar)
was prepared exactly as for Group IV (50% moisture) except
that the quantity calculated to last for the entire experi-
mental period was prepared at once. The mixture was
allowed to stand for 24 hours at room temperature, and
then frozen at -20°C, and freeze-dried in a Stokes freeze—
drier (Model Number 2003F—2, lot p6569, serial number
p65753) for 72 hours. The internal pressure fell to a
minimum of 100 microns.

Rats in Group VIII were given a diet prepared by
the same procedure, with the exception that the 50%:mois—
ture was added as water alone, not as a 1%.agar solution.
The mixture was shaken in a mechanical shaker over the 24

hour period, in order to keep the water in constant contact

lFor composition of basal diet, see p. 18.

41

mes“

 

42

with the diet solids. The mixture was frozen and freeze—
dried as above for 48 hours.

The animals in Groups I and IV were permitted to
eat ad libitum, but the animals in Groups VII and VIII
(the freeze-dried diets) were pair fed to the solids con-
sumption of the animals in Group IV (50%.moisture).

At the end of the experimental period, the animals
were sacrificed, and the livers removed for analysis. The
entire experiment was repeated with a second batch of rats,

and the results are reported separately.

RESULTS

Experiment I

The results from this first study are presented in
Tables 1 and 2. There are differences in some biological
compartments at the 1% level. The weight gains of the
rats in Group VII (freeze-dried with agar) are signifi-
cantly greater than those of Group VIII (freeze-dried
without agar). The rats fed the 50%.wet diet consume
significantly more diet solids than controls, and the PER
values for the rats in Group VII are significantly in—
creased over both the controls and those of Group IV (50%
moisture).

The liver analysis data are presented in Table 2.
The peicent moistures of the livers from rats in Group I
(basal diet dry) and Group IV (basal diet wet) are signifi-
cantly lower than those from Groups VII and VIII (freeze-
dried with and without agar). No significant differences
are seen among groups in percent fat or nitrogen.

Since liver weights vary more than usual between

groups in this experiment (those of Group IV significantly

43

44
heavier (P < 0.01) than those of Groups I and VIII) total
values for moisture, fat and nitrogen have also been calcu-
lated and tabulated. No significant differences are
observed either in total moisture or total nitrogen between
groups, but Group IV (50%.wet) has a significantly higher
level of total fat than either Group VII or VIII (freeze—
dried with and without agar). Since there are no differ—
ences among Groups I, VII and VIII with respect to liver
fat, liver lipids in Group IV’must be significantly

greater than those in Group I as well.

Experiment 2

The data from the replicate exPeriment varies
sufficiently from the initial that it is reported sepa—
rately in Tables 3 and 4. Although the same trends may
be observed in food consumption, weight gain and PER
(Table 3), the significant differences found in the pre-
vious study are lost.

The liver composition data (Table 4) also show
trends comparable to the initial experiment. Significant
differences are seen between groups in percent nitrogen
that do not appear previously. The animals in Group VII

have percent nitrogen values significantly higher than

114:6“:

 

 

45
Groups I and IV, and those in Group VIII have values sig-
nificantly increased over Group IV.

The liver weights in this experiment show the same
variation seen previously. This time a significant in-
crease in liver weight is found in the animals of Group IV
(50%.wet) over all other groups, and those of rats in
Group VIII are significantly reduced from control weights.
The same trends in total liver values are seen - signifi—
cant differences in total moisture are retained, and the
total fat levels of the animals in Group IV are increased
(P < 0.01) over the levels observed with Groups VII and
VIII. Since there is no significant difference between
Groups I, VII and VIII in this parameter, Group IV must be

increased significantly over Group I as well.

46

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DISCUSSION

The data from the initial and the replicate experi-
ment have been tabulated separately because of the differ-
ences observed in some of the biological compartments
measured. Differences that are significant at the 1% level
in the initial experiment are no longer so in the replicate,
but the same trends are observed. Absolute values in these
two exPeriments are slightly different as well from the
data in the previous studies, but again, the same trends
may be seen. Although the food consumption, weight gain
and PERS of the group fed the 50%.wet diet are much higher
than previously observed, the liver composition is not very
different when the total values are considered. The in-
crease in deposition of fat in the livers of the rats of
Group IV is significant over all other groups.

If the addition of water has a chemical effect on
the diet solids, the liver fat levels of the animals fed
the freeze—dried diets should be close to those of the rats
in Group IV (50%‘wet). They are more comparable, however,
to those of the Group I controls, and are significantly

50

51

reduced from the liver fat values of Group IV. This
suggests that the effect of increased diet moisture on
liver fat concentration is not the result of chemical
interaction between nutrients in the food cup.

There is a significant difference found between
Groups VII (freeze-dried with agar) and VIII (freeze-dried
without agar) in the PER values in the initial eXperiment,
and this cannot be ascribed to the diet treatment. In this
experiment, the food intake of rats in Group VIII is lower
than those in the group to which they were pair fed. As a
consequence, Group VIII was in practice, not pair fed to
Group IV, but rather fed ad libitum. There were no differ-
ences found between Group VII and VIII in the replicate
experiment, and no differences in the liver composition
in either experiment, so the lowered PER initially must
be viewed as an isolated and unrepresentative situation.
The presence of agar in the diet cannot be presumed to have
any effect on the animal in the parameters measured in this

study.

SUMMARY

Freeze-drying a 9% casein, threonine-deficient diet
to which has been added 50%.water with and without agar
does not produce the same response in the weanling rat as
feeding the same diet wet. The elevated liver fat levels
observed previously upon feeding the wet diet do not appear
when the diet is freeze-dried and pair fed to the solids
consumption of the animals consuming the wet diet. There—
fore, the effect of adding moisture to the diet cannot be
caused by a chemical interaction between nutrients in the

food cup prior to ingestion.

52

PART III

THE EFFECT OF ADDING 50%'WATER TO LOW PROTEIN,
THREONINE-DEFICIENT DIETS ON THE LIVER XANTHINE OXIDASE
ACTIVITY OF WEANLING RATS

INTRODUCT I ON

Xanthine oxidase activity of the liver has been used
by many investigators as an indication of the adequacy of
dietary protein. Litwack et a1. (1950) fed rats 18% casein
diets supplemented with 0.25%.DLHmethionine for 7 days. At
the end of this period, half the rats were changed to a
protein-free diet with sucrose substituted for casein.
Within two days, the xanthine oxidase activity fell to only
25% of controls, and within 5 days was completely_lost.
Muramatsu and Ashida (1962) experimented with varying levels
of casein in the diet. They fed weanling rats 25% casein
for 4 to 5 days followed by diets containing various amounts
of casein from 0% to 50%” in 10%.increments. The xanthine
oxidase activity increased with each increment of casein,
from zero activity at 0% casein to a maximum at 20% casein
Of 550 microliters of oxygen consumed per hour per gram of
tissue. The curve obtained when the xanthine oxidase
activity was plotted against the percent casein in the diet
Paralleled that when the liver nitrogen was plotted against
Percent casein in the diet. The authors concluded that the

53

 

54
xanthine oxidase activity was indicative of general protein
metabolism in the liver.

Since the xanthine oxidase activity is altered with
the level of dietary protein, and if there are differences
in enzyme activity when the protein provided by two dietsis
the same, then this would indicate differences in the util-
ization of protein. If the addition of water to the basal
diet does in fact increase the efficiency of protein
utilization, then this should be reflected in an increased
xanthine oxidase activity in the livers of the rats fed the
wet diets. The colourimetric method of Litwack, Bothwell,
Williams and Elvehjem (1953) was used to measure the

activity of this enzyme system.

7~—+——r_.. . HT .~*.;-r—'.-,-.v.“"_l’.- .
_ 1‘:

1

METHODS
There were two diets used in the first of these
experiments the basal diet fed dry (Group I), and the basal W‘

diet plus 50% moisture (Group II). A total of 84 animals

 

was used. Immediately upon arrival, 12 animals were sacri-
ficed. The livers were excised and weighed, and those from f L
6 rats homogenized with 5 volumes of a sodium—potassium

phosphate buffer (pH 7.4) in a Potter-Elvehjem homogenizerl

and assayed for xanthine oxidase activity by the colouri-

mentric method of Litwack et a1. (1953). These data were

reported as "zero time" values. The livers of the remain—

ing six were homogenized with distilled water and analysed

for moisture, fat and nitrogen. The rest of the animals

were divided:mm)two groups and fed either the basal diet

dry (Group I), or the basal diet wet (Group II). Twelve

animals from each group were sacrificed at two days, seven

days, and 14 days, six used for the enzyme assay, and six

for moisture, fat and nitrogen analysis as described above.

 

1V. R. Potter and C. A. Elvehjem, J. Biol Chem 114,

495 (1936).

55

WZS'S

 

 

56
Because of the extremely low enzyme activity
(approaching zero) found in the livers from animals fed
both diets I and II, any difference between these groups
was completely obscured. The low protein, threonine—

.a
deficient diets, wet or dry, were apparently sufficiently ET;
severe nutritionally to cause a rapid and precipitous fall
in the xanthine oxidase activity. In order to measure any
differences which might in fact exist, a second eXperiment 7
was designed in which all the rats were fed the basal diet
supplemented with 0.36%.DL-threonine at the exPense of
sucrose for a period of seven days. At the end of this time,

12 animals were sacrificed, six were used for the assay to
provide "zero time" values, and six for moisture, fat and
nitrogen analysis. Of the remaining animals, 12 were fed
the basal threonine-deficient diet fed dry (Group I), and
12 the basal threonine-deficient diet fed wet (Group II).
After four days of feeding these eXperimental diets, the
animals were sacrificed and treated as described for the
enzyme assay and the liver analysis.

Standard errors of the means were calculated for all
data, and the Student's t test used as a measure of signifi-
cance. Unless otherwise stated, P < 0.01 was accepted as a

significant difference between means.

RESULTS

Experiment 1

 

The weight gain and food and water consumption data

ufio'ofl'fl 23.477 i: I F!

for the rats used in the enzyme analysis are presented in
Table l. The same data for the animals used for the liver
analysis are seen in Table 2. No significant differences
are observed between groups fed the basal diet dry or the
basal diet plus 50%.water for the same experimental period.
Rats fed the dry diets consumed more water, as expected
since the water consumption figures do not include the
water provided by the diet.

Table 3 shows the liver composition data. No sig—
nificant differences are observed between the wet and the
dry groups in either percent moisture or nitrogen. The
only significant difference in percent fat is found in those
animals fed the wet diet for the 2 day period: liver fat is
increased significantly (P < 0.01) over that of the group
fed the dry diet.

The enzyme analyses are reported in Table 4 as mic-
romoles of xanthine disappearing per hour per gram of liver

57

58
tissue. The zero time value is 5.67 micromoles per hour per
gram tissue, and there is a rapid fall in enzyme activity in
animals fed both the wet and the dry diets. At 2 days, the
decrease in enzyme activity in both groups is significant at
5%, and at 7 days, significant at 1%. By 14 days, there is
virtually no activity left, and it is not possible to calcu-
late standard errors for these data. Though the enzyme
activities are higher numerically in livers from rats fed
the wet diet as compared with the dry, the differences were.
not significant. The failure to establish significance
might be due to the extremely low activity of this system
in both groups. Litwack et a1. (1953) using the same 1 ml
homogenate system, observed a range in enzyme activity for
normal rats from 7.8 to 22.0 micromoles per hour per gram
of tissue.1 The threonine—deficient diet consumed by the
animals in this study apparently causes such a severe re-
duction in the xanthine oxidase activity that any differences
due to the feeding of the diet wet or dry are obscured.
Consequently the experiment was repeated with modifications

to obtain more readable results. In the second exPeriment,

 

1These values are probably from adult rats, although
the authors do not specify. In this laboratory a three week
old stock rat had a xanthine oxidase activity of 8.8 micro-
moles per hour per gram tissue.

 

THESIS

 

59
an attempt was made to place the animals in better nutri—
tional condition prior to imposing the amino acid imbalance.
To do this, the amino acid imbalance was corrected though

the diets were still low in protein (9%.casein). The animals

.'v.'\‘ ‘P’

were fed the basal diet supplemented with threonine for 7
days, then changed to the basal diet deficient in threonine,
fed either wet or dry as before, for a period of four days.

The following results were obtained.

Experiment 2

 

Tables 5 and 6 show the weight gain, food and water
consumption data for the animals used for the enzyme and
liver analyses reSpectively. No signifiCant differences are
observed between the rats fed the deficient diet, wet or dry.
These data corroborate those from Experiment 1.

Liver composition data are presented in Table 7.
There are no significant differences between any of the groups
in percent moisture or nitrogen. However, there is a signifi-
cant increase (P < 0.01) in liver fat concentration in the
group fed the wet deficient diet as compared with the dry
deficient diet.

Table 8 shows the enzyme activity data. Feeding the
basal diet supplemented with threonine causes no significant

decrease in the enzyme activity from the "zero time" values.

“£2.77sfi-.;{m.~..éwxfix :27” .- _

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60
When animals are then fed the deficient diet dry for 4 days,
there is a decrease in enzyme activity significant at the 5%
level. However, when rats are fed the wet threonine—

deficient diet, this depression in xanthine oxidase activity

 

is not seen. In these animals xanthine oxidase activity is

maintained at the ”zero time" level, even though the diet

 

protein is less adequate. f .

61

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64

Table 4. Xanthine oxidase activity in rats fed low protein,
threonine-deficient dietswith and without the
addition of 50%.water.

 

Xanthine Oxidase Activityl

 

 

Days on
Diet Dry Diet wet Diet
0 5.67 i 0.69
2* 2*
2 2.45 i 1.0 3.09 i 0.78
** **
7 2.24 :_0.9 2.27 :_0.37
14 0.72 0.0

 

1Xanthine oxidase activity calculated as micromoles of
xanthine disappearing per hour per gram.of tissue.

25E

*Difference significant from zero time value at P<0.05.

**Difference significant from zero time value at P<0.01.

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67

Table 7. Liver composition of rats fed low protein,
threonine-supplemented diets, and low protein,
threonine-deficient diets with and without the
addition of 50%‘water.

W

Liver Composition

 

w aw; unsung—no um £2.41
..
I

 

Moisture Fat Nitrogen
Group %. %.dry weight %.fresh weight
threonine- . 1 l l
supplemented 67.7 :_3.0 11.6 i 2.1 2.84 i 0.3
threonine-
deficient, dry 71.8 :_0.9 9.65: 1.0 2.30 i 0.1
threonine-

deficient, wet 69.6

|+
N
R)

14.0 i.0~7** 2.37 + 0.1

 

lSE

**Significant difference from threonine-deficient dry group
at P < 0.01.

.'

68

Table 8. Xanthine oxidase activity in rats fed low
protein, threonine-supplemented diets, and
low protein, threonine-deficient diets
with and without the addition of 50% water.

W

 

Group Xanthine Oxidase Activity1
. . . 2
lnltlal 5.67 i 0.69
threonine-
supplemented 5.46 i_0.49
threonine-
deficient, dry 4.07 i 0.28*
threonine-
deficient, wet 5.28 i_0.60

 

l . . . . .
Xanthine ox1dase activ1ty calculated as micromoles of
xanthine disappearing per hour per gram of tissue.

28E

*Difference significant from zero time value at P < 0.05.

mzsm

 

 

 

 

DISCUSSION

The xanthine oxidase activity of the liver is very
sensitive to the quality and quantity of the protein present
in the diet (Litwack et al., 1952). In this study the
xanthine oxidase activity is extremely labile in threonine
deficiency. After two days of feeding the deficient diet,
the enzyme activity is so low that differences between the
wet and dry treatments could not be measured.

When the animals are fed the threonine-supplemented
diet for a week, the enzyme activity does not decrease from
"zero time" values, although it does not increase as it
would in a normal animal because the diet is still low in
protein. However, the animal is in a sufficiently improved
nutritional state to better withstand the stress of the
deficient diets. When 50% water is added to the diet, no
significant decrease in enzyme activity is noted. The de-
crease obtained by feeding the same diet dry is significant
at 5%, and there is no difference between the enzyme activi-
ties of the animals fed the threonine supplemented diet and
the deficient diet plus water. Thus the addition of moisture

69

Ell

 

70
to a low protein, threonine-deficient diet improves the
"protein quality?.

This increase in the xanthine oxidase activity may
be indicative of an increased utilization of the restricted
diet protein. Yoshida et a1. (1966) suggests that there is
an increased incorporation of the limiting amino acid into
protein in the livers of animals fed a threonine-deficient
diet as compared with control diets. This effect is seen
within a very short time, 3.5 to 8 hours. They prOpose a
mechanism responsive to the altered plasma ratio of amino
acids induced by an amino acid imbalance which in turn
causes a precipitous decrease in food consumption. Since
the addition of water to the diet increases the efficiency
of protein utilization, indicated by the increased xanthine
oxidase activity in rats fed the wet diet, this may more
rapidly produce the altered plasma amino acid pattern and
the "critical ratio" (see pages 12-15) in the animals fed
the wet diet than the same changes occur in those fed the
diet dry. Increased protein metabolism in the liver could
also explain the elevated liver lipids in the animals in
the wet group if more of the most limiting amino acid is
used for protein synthesis, leaving less available for the

control of fat deposition.

 

SUMMARY

Within two days after feeding weanling rats a low
protein, threonine-deficient diet dry or wet, the liver
xanthine oxidase activity is so drastically decreased that
any differences caused by the diet treatments are not mea-
sureable. When the nutritional status of the animals is
improved by feeding a 9% casein diet supplemented with
threonine for seven days prior to feeding the deficient
diet, differences in enzyme activity due to feeding the
imbalanced diet wet are observed. The animals fed the
deficient diet dry have a xanthine oxidase activity de-
creased significantly (P < 0.01) from "zero time" values,
While no such decrease is caused by feeding the deficient
diet wet. Thus the addition of 50%.water to a low protein,
threonine-deficient diet produces an increase in the
efficiency of protein utilization, and hence an improve-

ment in protein quality.

71

 

‘1.- . ‘
—JJ_~ _

GENERAL DISCUSSION AND SUMMARY

Additions of 20, 50 or 80%.moisture to a 9% casein,
threonine-deficient diet cause a decrease in weight gain,
food and water consumption and Protein Efficiency Ratio in
weanling rats. Moreover, liver lipids are elevated in
animals fed the deficient diet plus 50%.water.

These effects are apparently not caused by chemical
interaction between water and the other diet components in
the food cup prior to ingestion. When the diet containing
50%.moisture is allowed to remain at room temperature for
24 hours and then freeze-dried before feeding, liver lipids
are not elevated as observed when the diets are fed wet.

The addition of water to the threonine-deficient

diet increases the efficiency of utilization of the dietary

 

amino acids as measured by the xanthine oxidase activity of
the liver after four days of feeding the diet. This may
appear to contradict the data obtained in Part I, in which
the PER was decreased with increasing water in the diet.
However, in the imbalanced animals receiving the wet diet,
two different mechanisms are apparently in Operation to

72

73
maintain homeostasis. Initially the efficiency of util—
ization of the amino acids is increased in the animals fed
the deficient diet wet as compared with those fed the same

diet dry, using xanthine oxidase activity of the liver as

b

the criterion. However, this initial increase in the

efficiency of protein utilization is followed by a decrease

 

in the food intake in response to the altered plasma amino ii
acid pattern (Yoshida et al., 1966). This pattern is more

rapidly distorted towards the "critical ratio" (see pages

12-15) in the rats fed the wet diet than is the case with

those fed the dry diet. Thus the increased availability of

the amino acids in the imbalanced diet induced by the

addition of water serves only to aggravate the situation

caused by the imbalance, and over the two week experimental

period results in a significantly decreased food consumption,

weight gain and PER.

BIBLIOGRAPHY

Deshpande, P. D., A. E. Harper and C. A. Elvehjem 1958a
Amino acid imbalance on low fibrin diets. J. Biol.
Chem., 230: 327.

Deshpande, P. D., A. E. Harper and C. A. Elvehjem 1958b
Amino acid imbalance and nitrogen retention. J.
Biol. Chem., 230: 355.

Hankes, L. V., L. M. Henderson and C. A. Elvehjem 1949
Effect of cystine and threonine on growth of rats
receiving tryptophan-deficient rations. J. Biol.
Chem., 180: 1027.

Harper, A. E., D. A. Benton, M. E. Winje and C. A. Elvehjem
1954c "Antilipotropic" effect of methionine in
rats fed threonine-deficient diets containing
choline. J. Biol. Chem., 209: 159.

Harper, A. E. 1959 Amino acid balance and imbalance. I.
Dietary level of protein and amino acid imbalance.
J. Nutr.,, 68: 405.

Harper, A. E., P. Leung, A. Yoshida and Q. R. Rogers- 1964
Some new thoughts on amino acid imbalance. Fed.
Proc., 23: 1087.

Henderson, L. M., O. J. Koeppe and H. H. Zimmerman. 1953
Niacin-tryptophan deficiency resulting from amino
acid imbalance in non-casein diets. J. Biol. Chem.,
201: 697.

Hunt, J. N. 1959 Gastric emptying and secretion. Physiol.
Rev., 39: 491.

Keane, K. W., C. J. Smutko, C. H. Krieger and A. E. Denton
1962 The addition of water to purified diets and
its effect on growth and protein efficiency ratio
in the rat. J. Nutr., 77: 18.

74

75

Keane, K. W., C. J. Smutko, C. H. Krieger and A. E. Denton
1963 Water addition to purified diets. II. Effects
of water and carbohydrate on protein efficiency
ratio in rats. J. Nutr.., 81: 87.

Krehl, W. A., P. S. Sarma and C. A. Elvehjem 1946 Effect
of protein on the growth of rats on niacin-trypto-
phan deficient rations. J. Biol. Chem., 162: 403.

Kumpta, U. S., A. E. Harper and C. A. Elvehjem 1958 Amino
acid imbalance and nitrogen retention in adult
rats. J. Biol. Chem., 233: 1505.

Kumpta, U. S. and A. E. Harper 1961 Amino acid imbalance.
VII. Effects of dietary additions of amino acids
on food intake and blood urea concentration of rats
fed low protein diets containing fibrin. J. Nutr.,
74: 139.

Kumpta, U. S. and A. E. Harper 1962 Amino acid imbalance
IX. Effect of amino acid imbalance on blood amino
acid pattern. Proc. Soc. Exp. Biol. Med., 110: 512.

Litwack, G., J. N. Williams, Jr., P. Feigelson and C. A.
Elvehjem 1950 Xanthine oxidase and liver nitrogen
variation with dietary protein. J. Biol. Chem.,
187: 605.

Litwack, G., J. N. Williams, Jr., L. Chen and C. A. Elvehjem
1952 A study of the relationship of liver xanthine
oxidase to quality of dietary protein. J. Nutr.,
47: 299.

Litwack, G., J. N. Williams, Jr., J. W. Bothwell and C. A.
Elvehjem 1953 A colourimetric assay for xanthine
oxidase in rat liver homogenates. J. Biol. Chem.,
200: 303.

Morrison, M. A. and A. E. Harper 1960 Amino acid balance
and imbalance. IV. Specificity of threonine in
producing an imbalance in diets deficient in niacin
and tryptophan. J. Nutr., 71: 296.

Muramatsu, K. and K. Ashida 1962 Protein level and liver
enzyme activities. J. Nutr., 76: 143.

 

 

76

Salmon, W. D. 1954 Tryptophan requirement of the rat as
affected by niacin and the level of dietary protein.
Arch. Bioch. Biophys., 51: 30.

Sanahuja, J. C. and A. E. Harper 1962 Effect of amino acid
imbalance on food intake and preference. Am. J.
Physiol., 202: 165.

Sauberlich, H. E. 1956 Amino acid imbalance as related to
the methionine, isoleucine, threonine and tryptophan
requirement of the rat or mouse. J. Nutr., 59: 353.

Sauberlich, H. E. and W. D. Salmon 1955 Amino acid imbalance
related to the tryptophan requirement of the rat.
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Singal, S. A., S. J. Hazan, V. P. Sydenstricker and J. M.
Littlejohn 1953a The production of fatty livers
in rats on threonine- and lysine-deficient diets.
J. Biol. Chem., 200: 867.

Thomas, J. E., J. O. Crider and C. J. Mogan 1934 Reflexes
involving the pyloric sphincter and antrum and their

role in gastric secretion. Am. J. Physiol., 108:
683.

Yoshida, A., P. Leung, Q. R. Rogers and A. E. Harper 1966
Effect of amino acid imbalance on the fate of the
most limiting amino acid. J. Nutr., 89: 80.

 

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