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TISSUE B§STREBUT18N OF ZlfiC iN
THE RAT 5123 RELATED T9 BSETARY
ZEN-C REQUEREfiiENT

Thesis far the Degree of M. S.
MECHEGAfl STATE UNIVERSE‘E'Y
BRUNA E. RUKSAN
1968

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ABSTRACT

TISSUE DISTRIBUTION OF ZINC IN THE RNT AS
RELATED TO DIETARY ZINC REQUIREMENT

By Bruna E. Ruksan

Two experiments, involving a total of 89 weanling rats, were
conducted to determine the requirement of zinc for normal growth. The
relationship between the level of zinc in the diet with appetite,
growth rate, food efficiency and zinc concentration of the tissues
was studied. A study on the interrelation between copper and zinc
for low concentration in the diet was also included.

In the first experiment anorexia and growth retardation was
observed in the group on the basal diet, and two animals of this
group died after a few days with diarrhea. The relation between
zinc content of the diet and appetite, growth rate, food efficiency
and bone zinc was highly significant (P<(0.01). No differences were
observed in the zinc content in kidneys and liver with the zinc in
the diet. However, capper concentration of liver decreased with the
increment of zinc in the diet.

In the second experiment, part A the results obtained for
appetite, growth rate and food efficiency confirmed the observation
of the first experiment and indicated that the dietary requirement
of the rat for zinc approximates 10 ppm.

From the previous observations on the significant increase
in zinc content of bones with the zinc level of diet, the group of
rats on basal diet was interchanged with the groups on 10 ppm.and
15 ppm zinc diets, in order to study the effect of previous diets on

growth rate, appetite, food efficiency, zinc content of bones, liver

Bruna E. Ruksan
and kidneys and copper concentration of kidneys and liver.
0n the repletion with zinc in the deficient group, a high incre-
ment in appetite, growth rate and zinc content of bones was observed.
While on depletion with basal diet in the 10 ppm and 15 ppm zinc diet
groups, the appetite was decreased, the weight was arrested and the
zinc content of bones decreased significantly, but neither liver nor

kidney concentration of zinc was affected.

TISSUE DISTRIBUTION OF ZINC IN THE RAT AS
RELATED TO DIETARY ZINC REQUIREMENT

By

,3, (3%“
Bruna EEDRuksan

A THESIS

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

MASTER.OF SCIENCE
Department of Biochemistry

1968

ACKNOWLEDGMENT

The author wishes to express her sincere appreciation to Dr. R.
W. Luecke for his guidance and interest throughout the course of this
investigation.

The author also wishes to thank the members of Department of
Biochemistry for their helpful suggestions and assistance.

Acknowledgment is also due to the Food and Agriculture Organi-
zation of the United Nations (FAQ) and Instituto Nacional de Tecnologia
Agropecuaria (INTA)-Argentina for the fellowhsip, which made possible
this project.

The author is especially grateful to her parents, relatives,
friends and members of FAD—INTA.Project for the encouragement, under-

standing and love throughout the course of these studies.

ii

I.
II.

III.

TABLE OF CONTENTS

INTRODUCTION . . . . .

REVIEW OF LITERATURE .

Zinc an Essential Micronutrient . . . . . . . . . . . .

BiOChemical metions Of Zinc o o o o e o o o o o o o o

Zinc-Enzyme Interrelations o e e e o e o o e o o o o

Zinc-Hormone Interrelations . . . . . . . . . . . . .

Interrelation with Other Nutrients . . . . . . . . . . .

Zinc in Tissue . . . .
EXPERIMENTAL PROCEDURE

General 0 O O O O O 0

Trace Element Assay of Tissue . . . . . . . . . . . . .

Experiment I o o o o e

The Effect of Varying Levels of Dietary Zinc . . . .

Experiment II . . . .
Part A. The Effect

Part B. The Effect
Level Diets . . . .

RESULTS AND DISCUSSION
Experiment I . . . . .
Experiment II . . . .

Part A . . . . . .

PartB O O O O O O

of Varying Levels of Dietary Zinc

of Interchanging Different Zinc

O C O O O O O O O O O O O O O O 0

iii

Page

12
15
23
3o
30
31
32
32
33
33

37

45
45
49

VI.
VII.

VIII.

S UWY O O O O O C 0

CONCLUSIONS . . . . .

LITERATURE CITATIONS

APPENDIX

iV'

Page
55

‘63

69

Table

8.

LIST OF TABLES

Composition of Basal Diets . . . . . . . . . . . .
Composition of Salt Mix . . . . . . . . . . . . .
Composition of Vitamin-Glucose Mix . . . . . . . .

Response of Rats Fed Different Levels of Zinc in
Experimentl................

Effects of Variation of Zinc Content of Diets on
Concentration of Zinc in Bones . . . . . . .

Effect of Different Levels of Zinc in the Diet on
Copper and Zinc Concentration in Livers and
KidnGYSooeooceoooooeoooeoo

Response of Rats Fed Different Levels of Zinc in
ExporimentII................

Beeponse of Rats Fed Different Levels of Zinc to
Switchover of the Diets. Experiment II-Part

Page

35
36

39

#2

43

#6

52

Figure
1.

2.

LIST OF FIGURES

Exp. I. Growth Curves of Rats Fed Different Levels

OfZiflCooooooooooooooeeeoooeoo

Relation of Weight Increase to Zinc Diet Levels

Relation Between Bone Zinc and Zinc Diet Levels

Page

41
1+7
1+8

LIST OF APPENDIX TABLES

Table Page

1. Zinc Content of Diets by Analysis of Experiment I
andEmerimentIIeooeoeooeoooooeooo69

2. Experiment I. Gain Weight, Food Intake, Bone Ash,
Zinc in Bones, Livers and Kidney, and Copper
inleerSandKidneyS................7O

3. Experiment II. Gain Weight, Food Intake, Bone Ash,
21110111801193.9000...eooooeeococo72

vii

I. INTRODUCTION

The zinc requirement in the diet by the animals for the normal
growth and development is variable and depends upon the other nutrients
present and their concentration in the diet.

The value existing for the zinc requirement for rate, for normal
growth fed a diet with egg white as a protein source is 12.6 ppm of
zinc (Forbes and Yohe, 1960). Luecke gt_gl, (1968) showed that high
levels of supplementary biotin were required to prevent symptoms of
biotin deficiency from appearing and that resulted in an improvement
of growth of rats on the zinc supplemented diet but not in the animals
on deficient diet.

There are conflicting reports in the literature on the antagonism
existing between zinc and copper at low concentrations. Davis (1958)
reported that increasing the level of zinc in the diet causes a depres-
sion of the copper values in the liver, but only when the copper levels
in the diet approach those of low normal levels of around 5 to 10 ppm.
This was not observed by Bunn and Matrone (1966) who found that the
liver copper concentration only decreased by the action of zinc and
cadmium together, but no effect was Observed by either one individually.
Reinhold.gtngl. (1967) did not find any effect of low zinc intake upon
the concentration of copper in the tissues.

Macapinlac gt_gl, (1965) observed zinc concentrations in bone
to decrease by two-thirds in rats when fed diets low in zinc. Also it

was suggested by many investigators that bone might play an important

2
role in stored zinc, releasing it for use by other critical organs.

From the studies by Forbes, (1961); Likuski and Forbes, (1965);
Fox and Harrison, (1964) and Hurley‘gt‘gl., (196A) the zinc concentra-
tion in bone ash appeared to be a sensitive reflection of zinc absorp-
tion, especially at suboptimal intake in the young growing animals.

The present series of investigations were initiated in order to
1-determine the effects of supplemental zinc fed at various levels on
appetite, growth rate and food efficiency to weanling rats fed egg-
'white protein diet supplemented with biotin. 2--Evaluate the zinc
concentration in tissue in relation to the zinc content of the diet as
a possible use as an aid in the diagnosis of zinc deficiency. 3--The
study of interrelation of zinc-copper in tissues was also included in
these experiments in order to obtain more information on the antagonism
of these elements at low concentration in the diet. In order to attain
these objectives analysis for copper and zinc in tissues were made, in

addition to collecting growth and food consumption data.

II. REV IEW OF LITERATURE

Zing g2 Essential Micronutrient

Zinc was first demonstrated to be an essential nutrient for liv-
ing organisms when Raulin (1869) showed that it is necessary for the
growth of Aspergillus‘gigg_. This finding was confirmed forty years
later by Bertrand and Javillier (1911). Previous attempts by Bertrand
and Benson (1922) and others to demonstrate the essentiality of zinc in
animals were unsuccessful because the purified diets which they
employed in their experiments were deficient in other essential nutri-
ents. Convincing evidence concerning the need for zinc for the normal
growth and development of mice was reported by Bertrand and Bhattacherjee
(1934. 1935) and Todd 3; 2;. (1934) and in rats by Stern g_t_ 5;. (1935)-

Follis‘gtngl, (1941) in their histological studies of zinc defi-
cient rats found alterations in the skin consisted of a hyperkeratiniza-
tion, thickening of the epidermis, intra and intercellular edema and
loss of hair follicles with preservation of the subaceous glands.

The early work with the rat and mouse indicated that the zinc
requirement is less than 5 ppm.of diet. Therefore, it was generally
assumed that a deficiency of this element would not occur in other
species such as swine, cattle and poultry when fed diets made up of
natural feed ingredients such as corn, soybean oil meal (Luecke, 1965).

In 1953 Kernkamp and Ferrin, reported the incidence in pigs of

a dermatitis termed parakeratosis which had been experienced for

L,

eleven years. The indidence of the disease was as high as 60 percent.
Thomas and Eden (195A) also reported the occurrence of a skin disease
in pigs in Great Britain which they termed nutritional dermatitis. A
relationship of zinc to parakeratosis was suggested by data reported by
Curtin (195”). There were no cases of dermatitis in pigs receiving a
diet containing cottonseed meal when supplemented with 2 mg of zinc
per lb of diet (Brinegar and Hunter, 1955).

Tucker and Salmon (1955) elucidated the essential role of zinc
in the prevention and cure of this disease. They suggested that the
levels of calcium or phosphorous or both are contributing factors in
the incidence of the disease. The influence of Ca and P as well as
other nutrients on zinc metabolism were studied later by'Luecke‘gtwgl.
(1956-1957), Lewis 233;. (1957), 13.1113 and Philp (1957), and others.

Fbllowing the discovery of the essentiality of zinc in the
nutrition of pigs investigations were extended to other animals.
O'Dell and Savage (1957) have briefly reported that added zinc stimu-
lated the growth and the bone length of chicks fed a semipurified soy-
bean protein diet containing approximately 50 ppm of zinc. They con-
cluded that the zinc in soybean protein was not available in compari-
son with the zinc of a similar diet containing casein. This study was
confirmed by Morrison and Sarett (1958), O'Dell gt 3;, (1958) and Young
21. al- (1958).

Supplee gtugl, (1958) observed that zinc and.potassium were
needed for growth, prevention of perosis and normal feathering in
poults fed a soybean protein ration. Kratzer‘gt.gl, (1958) confirmed
the importance of zinc in rations for poults.

The abnormal bone condition appears to arise from a failure of

5
cartilage development in the epiphyseal plate region of the long bones

and decreased osteoblastic activity in the thin bony collar (Keinholz
gt ;l_. 1961).

Few studies have been made on the nature and occurrence of this
deficiency in ruminants. The first indication that zinc deficiency
could arise in grazing cattle came from Guiana where Legg and Sears
(1960) found that it was responsible for outbreaks of parakeratosis in
cows and yearlings and poor growth in young stock, since these manifes-
tations reSponded to oral or parenteral administration of zinc.

Haaranen and Hyppola (1961) described work done in Finland where
a syndrome characterized by skin lesions, poor reproduction and low
milk production in housed dairy cattle responded to zinc therapy.

In the Netherlands, Grashius (1963-196A) noted a syndrome, where
the affected animals had a poor reproductive performance, low milk
yield, eczematous scabs and loss of hair around the muzzle, neck and
tail. The zinc contents of herbage grazed by affected stock was 19
to 83 ppm. Underwood (1962) has suggested that the minimum zinc
requirement for grazing cattle is about 30 ppm while Haaranen (1963)
indicated a requirement of about 45 ppm. Hartmaus (1965) who reinves-
tigated the problem on these Finish farms, vigorously contested Grashius's
findings claiming that the condition was probably attributable to "poor
management" and not to a deficiency of zinc (Mills gt_2l., 1967).

In 1960 Halsted and Prasad described a sindrome occurring in
human males characterized by severe iron deficiency anemia, hypogonad-
ism, dwarfism, hepatosplenomegaly and geophagia, which they observed in
villagers in Iran suffering from.malnutrition. Despite hepatosplenomegaly

the liver function tests were normal except for serum alkaline phOSphatase

6
which was consistently elevated. They pointed out the possibility of
zinc deficiency as an explanation of hypogonadism, dwarfism and changes
in alkaline phOSphatase.

One year later "hypogonadal dwarfism" was described in male sub-
jects residing in the Egyptian Nile Valley with severe iron deficiency
anemia, schistosomiasis and ancyclostomiasis (Prasad, 1961). The zinc
level of plasma, red cells and hair was reduced in these subjects and
65zinc turnover studies appeared to confirm the presence of zinc defi-
ciency. Minor differences between the patients of Egypt and Iran
include the following:

A. Geophagia was common in Iran but was not found in Egyptian

cases.

B. Bookworm and schistosomal infection were not present in

Iranian cases but were in Egypt (Prasad‘gtngl., 1963).

Coble gt a_l_. (1966 b) concluded that multiple factors are present
that may affect the growth and development of rural male Egyptians.
Inadequate information on development potential and effect of environ-
ment on these subjects and the observation that they ultimately obtain
normal maturation and stature without therapy or change in zinc levels
further complicate definition of the role of zinc in their delayed
maturation.

The possibility remains, however, that the slower growth curves
and smaller ultimate statures of rural male Egyptians in comparison to
their upper socioeconomic Cairo counterparts are in part a result of
inadequate zinc nutrition. A recent finding of inadequate growth horu
mone rise following insulin induced hypoglycemia in both retarded and

control subjects with low plasma zinc levels indicates that a defect in

7

pituitary reserve is present (Coble at g” 1966 a).

Biochemical Functions of Zinc

Zinc-Enzyme Interrelations:

Keilin and Mann (1939-19A0) showed zinc as an integral part of
the enzyme carbonic anhydrase which catalyzes the reversible reaction
between carbon dioxide and water. It was demonstrated that erythrocyte
carbonic anhydrase also catalyzes the hydration of acetaldebyde and
pyridine aldehydes. The complete inhibition by acetozolamide also
suggest the necessity of the zinc ion for these processes (Pocker and
Meany, 1967).

Carbonic anhydrase has been isolated from various sources,
mainly mammalian red blood cells. Bovine erythrocyte carbonic anhydrase,
homogeneous by ultracentrifugal and electrophoretic analysis was pre-
pared by Lindskog (1960). In 1962 Lindskog and Helmstrom, indicated
that only one zinc ion in each molecule of erythrocyte bonine carbonic
anhydrase, is an obligatory component for the enzymatic hydration of C02.

Since the first evidence in 1939 of the fundamental role for
zinc in metabolism, about fifteen to twenty zinc-containing metallo-
enzymes have been isolated and purified from a variety of organisms and
tissues from.diverse species, in the last fifteen years, thereby indi»
cating the general metabolic importance of the element.

Vallee and Neurath (195h-1955) have demonstrated that zinc is a
part of carboxypeptidase of bovine pancreatic juice. This enzyme
degrades polypeptides in a sequential fashion, beginning at the C tern

minus. Two carboxypeptidases A and B, are involved in protein digestion

8

in the duodenum. Carboxypeptidase A, exhibits maximal catalytic active
ity with C terminal aromatic residues, while carboxypeptidase B is
specific for C terminal basic residues. Carboxypeptidase A is a metal-
loenzyme of molecular weight 3h,300 containing one atom.of zinc per
molecule of protein (Mahler, 1966). It occurs in the pancreatic juice
in the form of a zymogen (Coleman‘gtmal., 1960). This finding explains
previous observations in which it was demonstrated that about 6.5 per-
cent of the dose of 65zinc administered to dogs was eliminated in their
pancreatic juice within five days (Vallee, 1937). A variety of chelat-
ing agents such as phenanthroline (Quiocho and Richard, 1966);
acetylation and iodination of tyrosine residues (Coleman gt_§l,, 1966);
substitution of other metal ions for the native zinc atom.(Coleman and
Vallee, 1960) and irradiation with ultraviolet light (Piras and Vallee,
1966, 1967 a, b) alter the catalytic Specificity of carboxypeptidase A.

Because of the similarities in composition and mechanism of
action.of’carboxypeptidase A and.B, it was of interest to compare the
structure of the active center peptides of both carboxypeptidases.
The lack of homo10gy between the cysteinyl peptides of carboxypeptidases
A isolated by Sampath‘gt‘gl. (1963) and carboxypeptidase B studied by
‘Wintersberger (1965) may suggest that these two enzymes have evolved
independently. Alternatively, it might be suggested that the differ-
ence in amino acid sequence in the region of the zinc-binding thiol,
merely serves as a support for the crucial metal atom but, itself, is
not a part of the active site.

Alcohol dehydrogenase of yeast (Vallee and Hoch, 1955 a, b) and
of the equine liver (Theorell gt_gl,, 1955; Vallee and Hoch, 1956) was

found to contain four and two moles of zinc per mole of protein reSpec-

9
tively and cannot be removed without causing irreversible changes in
protein structure. Removal of the zinc of yeast alcohol dehydrogenase
leads to dissociation of the molecule into subunits (Kagi and'Vallee,
1960), while in horse liver alcohol dehydrogenase (LADH) protein dena-
turation and aggregation results (Druyan and Vallee, 1962). Apparently
the metal-protein interaction is responsible in part of the tertiary
and quaternary structures of these enzymes.

The LADH enzyme has a molecular weight of 84,000, binds two
moles of coenzyme per mole of protein and catalyzes the reversible
oxidation of alcohols to aldehydes. The SH groups of two cysteinyl
residues per mole of protein have been found essential for the activ-
ity of this enzyme, one per active enzymatic site. Carboxymethylation
of these groups with iodoacetate inactivates the enzymes, and the prior
binding prevents the reaction (Li and Vallee, 1965).

Alcohol dehydrogenase has been purified from human liver also
and identified as a metalloenzyme by Von wartburg gtugl., (1964). It
is quite similar to the horse liver enzyme enzymatically and structurally
and has broad substrate specificity, including methanol and ethylenegly-
col.

Glutamic dehydrOgenase of beef liver, which catalyzes the rever-
sible oxidative deamination of glutamate, was reported by Adelstein
(1957) to be a zinc metalloenzyme. The average zinc content was 3.42
1.0 atoms per molecule. Like alcohol dehydrogenase it appears to bind
one NAD molecule per atom of zinc. Glutamic dehydrogeanse, having a
molecular weight of 1,000,000 is a much larger molecule than other
pyridine nucleotide dependent enzymes that have been studied.

Lactic dehydrogenase of mammalian tissues has also been found

10
to be a zinc metalloenzyme (Vallee and wacker, 1956).

The detection of zinc in these enzymes may explain the high con-
centration of this element in the liver and the retina, since it has
been shown that LADH oxidizes vitamin A and reduces retinene probably
being identical with retinene reductase. Thus far, no other metals
have been found to occur in similar dehydrogenases (Vallee, 1959).

In addition to the dehydrogenases presented, more recent inves-
tigations have shown that D-glyceraldehyde-3-phosphate dehydrogenase,
isolated from'bovine, crayfish and bakers yeast is a zinc metalloenzyme,
with two moles of zinc per mole of enzyme, apparently bound by means of
cysteine and histidine (Keleti, 1964).

17-76Lhydroxysteriod dehydrogenase activity was stimulated by
zinc ions for the placental (Langer and Engel, 1958), and adrenal
(Dahm and Brenner, 1964) enzymes. No significant stimulation was
observed in the case of testicular 20-cr-hydroxysteroid dehydrogenase
(Shikita _e_t _a_J_.., 1967).

The isolation of a zinc-dependent hexokinase from NeurosEora
‘grgggg,led to the suggestion that this is a zinc metalloenzyme. Three
to fourfold increases were found in zinc-sufficient, as compared to
zinc-deficient organism. The hexokinase was not isolated in homogen-
eous form and, indeed, showed significant phOSphoglucose isomerase
activity. The partially purified enzyme was inhibited by EDTA (Medina
and Nicholas, 1957).

The role of zinc in regulating the activity of alkaline phospha-
tase has been of continued interest since some preparations of this
enzyme have been reported to constitute zinc metalloenzymes and also

to exhibit properties of metal-enzyme complexes.

11

Mathias (1958) has established that alkaline phOSphatase from
swine kidneys is a zinc enzyme. The enzyme contains significnat
amounts of zinc, magnesium and copper. He demonstrated a direct pro-
portionality between increases in specific activity and zinc content
of the enzyme. The best preparation contained 0.15 percent of firmly
bound zinc and removal of any part of the remaining zinc by dialysis
resulted in directly proportional losses in enzymic activity.

Reynolds and Schlesinger (1967) in their study of the refolding
and reassociation of subunits of alkaline phosphatase from.Escherighia
ggli,found that the active dimer is a zinc metalloprotein and contains
three zinc atoms per cimer. The metal is not required to form the

refolded monomer, but is essential for dimerization process.

unfolded monomer (pH 2) active dimer
(extended coil)
-H+ 1 Int-H+

unfolded monomer (pH 4) $4 folded monomer (pH 6-8) '7? folded

- +Zn monomer Zn2+

(globular)

Snaith and Levvy (1968) have found.that.a-mennosidase is an active
zinc-protein complex readily dissociable at pH 5, the pH optimum enzyme
activity and the zinc ion can be displaced by cadmium, cobalt and other
bivalent ions with almost complete loss of enzyme activity. These
observations apply equally to the enzyme from rat epididymis, jack
bean meal and the limpet, Pgtilla vulgata. <X-Mennosidase seems to be
present in all mammalian tissues. Its activity is high in the male
genital tract where it responds directly to androgens.

In addition to the metalloenzymes, there have been numerous

12
reports on a variety of enzymes whose activity is increased by addition
of zinc ions (Vallee, 1959). Most of them lack Specificity as avi-
denced by the fact that they are activated by other ions also. Some
of these zinc enzyme complexes are: arginase, carnosinase, histidine
deaminase, lecithinase, aminopeptidase, enolase, yeast aldolase,
oxalacetic decarboxylase, several peptidases and serum intestinal
phosphatase.

Harvey‘gt.gl. (1967) purified a phOSphodiesterase from carrot
that possesses exonucleolytic activity which degrades both oligodeoxy-
ribonucleotides and oligoribonucleotides starting from a free hydroxyl
on the 3‘ end and producing 5' mononucleotides. Divalent ions Mg2+,

2+, Ca2+ and Zn2+ increase activity while EDTA inhibits.

Mn
Requirement for zinc for DNA synthesis has been demonstrated by
Fujioka and.Lieberman (1964). ‘Wacker (1962) found that RNA of Epglena
gracilis is decreased; that of amino acids is increased and the DNA
content is doubled.
The formation of either polysomes or non-specific aggregation

of ribosomes under the influence of zinc ions was obtained from the

seed of Pesim arvense by Barker and Rieber (1967).
Zinc-Hormone Interrelations:

The significant amount of zinc in the pancreas and the role of
zinc in the crystallization of insulin led to Speculation that there
might be some functional relationship between zinc and the secretion
of insulin by the pancreas. In 1938 Scott and Fisher reported that
the pancreas of the diabetic contained some 50 percent less zinc than

did that of nondiabetics. Later it was showed that when the results

13
were expressed in a fat-free basis, there was no significant difference
between zinc content of diabetic and normal pancreas.

The relation of zinc to the pancreas and insulin has been
studied histochemically, using dephenylthiocarbazone. ‘Variable amounts
of ainz were found in both the alpha and beta cells of the islets of
Langerhans. The amount of zinc in the beta cells varied directly with
insulin secretion in reSponse to physiological stimuli. However the
mechanisms for insulin production were not inactivated by irradiation
of the islets with 65zinc, or by injecting 2 to 3 mo of 65zinc to the
rat. Nor does 65zinc administration produce any changes in the blood
sugar level in rats. Consequently the possible biochemical interrela-
tionship between insulin and zinc is still an unsolved problem.

Some findings suggest a relationship between zinc content of the
alpha cells and glucagon secretion. However, further research will be
required to establish a zinc-glucagon relationship.

Reports have appeared in the literature suggesting a relation-
ship between zinc and other hormones, eSpecially the adrenocorticotro-
phic hormone (ACTH). Treatment of ACTH preparation with zinc (Zn(0H)2)
increases and prolongs its physiological action as in insulin.

Although pure preparations of growth hormone from pituitaries contain
practically no trace of zinc. The problem remains unsolved according
to Orten (1965).

Since the first suggestion by Bertrand and Vladesco (1920-1921)
that high concentration of zinc of the genital tissue might be involved
in male reproductive function. The high concentrations of zinc in male
sex organs and fluids of various species was confirmed by many investi-

gators. But the biochemical forms and functions of zinc in these glands

14
and their secretions have been only partially elucidated and only a few
functions are known. The carbonic anhydrase content of these tissues
is high, but only 3.5 percent of the total zinc of the dorsolateral
rat prostate can be accounted for by this enzyme. Recently, a highly
active cX-mannosidase was isolated from the male genital tract and
it responds directly to androgens (Snaith and.Levvy, 1968).

Rullar‘gt_gl. (1957) found that zinc deficiency in rats results
in degeneration of the testes, hypoplasia of the coagulating glands,
the seminal vesicles and prostate, and relative or complete decrease
in the numbers of Sperm in the epididymes. All changes produced by
zinc deficiency, except the testicular atrophy, were reversed when
supplemental zinc was added to the diet. If testicular atrophy had
occurred, neither testis nor epidymis regained normal size, function
or zinc concentration. It appears that the impaired development of
the male accessory sex organs in rats on zinc deficient diet may be a
result of severe inanition which in turn causes reduced gonatropin
secretion from the pituitary and a consequent fall in androgen produc-
tion.

Savage (1968) observed that the zinc requirement of hens for
egg production is appreciably lower than the amount required for hatch-
ability or for optimum growth and feathering in chicks and poults.
Since as the hens become depleted of body stores of zinc, embryos show
skeletal anomalies which are characterized by faulty limb and trunk

development.

15
Interrelation With Other Nutrients

There are several factors which affect the ability of the animal
to utilize zinc as it is present in foods (Luecke, 1965). Since the
suggestion by Tucker and Salmon (1955) that the level of calcium or
phosphorous or both are contributing factors in the incidence of para-
keratosis. A number of investigators became interested in the relation
between calcium, zinc, iron, phosphorus, phytate, vitamin D, copper,
cadmium and other nutrients.

The ability of animals to absorb zinc varies with the chemical
form in which it is ingested. Zinc in the form of the oxide, carbon-
ate, sulfate or metal appears to be equally available to the chick,
whereas the zinc in the ores sphalerite composed mostly of zinc sulfide
and franklinite of oxides of zinc, iron and manganese are largely
unabsorbed. Since zinc as zinc oxide is quite available, therefore, it
would appear that the crystal structure that results from the mixture
of these metallic oxides prevent the zinc in franklinite from.being
available for chicken (Edwards, 1959).

Different protein sources also vary greatly in their capacity
to supply zinc for the needs of rats, chicks and pigs. Soybean meal
was the principal source of protein in the rations associated with a
zinc deficiency. In 1960 Lease gt_gl, found that the feeding of
sesame meal rations produced gross sings of zinc deficiency although
the rations contained about 52 ppm of zinc.

Some peculiarity of plant proteins was suspected as a factor
in the development of a zinc deficiency (Anonymous 1957; 1961; 1967).

This was shown by the observation that purified rations containing

16
casein produced good gorwth and normal skin in swine even when the level
of zinc was no greater than 10 ppm. The chicks fed a similar diet,
with the exception that the protein source was casein and gelatin,
instead of soybean, showed no improvement in growth when zinc was
added.

Phytate was early suspected as the factor in soybean and sesame
meals that might be affecting the zinc availability. The confirmation
for this was obtained by the addition of phytic acid to casein rations
fed chicks that increased their requirement for zinc (Anonymous, 1964).

In another study to evaluate the interrelation between zinc,
calcium and phytate, young pigs were fed a casein diet to which 1.4
percent phytic acid was added. The ration contained 14 ppm of zinc
and 1.5 percent of calcium. The weekly weight gain decreased from
1.68 to 0.25 pounds when phytic acid was incorporated in the diet.

The growth depression could be alleviated by the supplementation of the
diet with 100 ppm of zinc (Oberleas‘gtigl., 1962).

Byrd and Matrone (1965) studied the influence of calcium on the
incorporation of zinc into the phytate complex. They found that when
the molar ratios of calcium and zinc were 1 to 1 or 2 to 1, the calcium
decreased the absorption of zinc, by the phytate, but when the ratio of
calcium to zinc was 100 to 1, the presence of calcium in the solution
increased the incorporation of zinc into the complex to the point where
99 percent of it was present as the phytate. In the absence of calcium,
none of the zinc was incorporated into phytate. Since rations that are
associated with the development of parakeratosis generally contained
30 to 100 ppm of zinc and 1 to 2 percent of calcium, it was postulated

that calcium initiated a coprecipitation with zinc to form insoluble

17
phytates. The adverse effect of the phytic acid can only be overcome
by adding enough zinc to the ration.

When the calcium and zinc were both present in the same solution
during precipitation a greater amount of phytate precipitate was formed
(Oberleas 23,3l,, 1966). This suggestion stems from the work of Byrd
and Matrone (1965) who found a greater zinc loss from solution when
the molar ration for these two elements was 100 to 1. All of the pre-
vious evidence has emphasized the inhibitory effect of calcium on zinc
absorption in the presence of phytates. It would appear that the
deleterious influence of plant proteins in zinc absorption was related
to their phytate content. Plumlee gt pl. (1960) and Smith e_t. 9;. (1962)
have found that the utilization of zinc in various proteins is corre-
lated with the level of phytic acid in the protein. They also
reported an improvement in zinc utilization when EDTA (ethylenediamin-
etetracetic acid) was added to a soybean protein ration.

Kratzer and Starcher (1963) suggested that EDTA improves the
effectiveness of added zinc as well as increases the utilization of
zinc already present in the basal diet. Other similar chelating
agents were studied and their effectiveness were determined by Vohra
and Kratzer (1964).

It is interesting to speculate concerning the presence in common
foods of naturally occurring chelating agents capable of reducing or
enhancing the biological availability of zinc and other trace elements
(Pond, 1965). Scott and Zeigler (1963) presented results indicating
that certain natural feedstuffs, such as casein, liver extract, contain
a factor or factors capable of improving both absorption and metabolic

utilization of zinc in similar way to ethylendiaminetetracetic acid.

18
It appears that the active principles in the natural foodstuffs may be
natural chelates which normally function to improve tranSport and
utilization of required mineral nutrient.

Smith gt_gl, (1960) found that pigs fed the autoclaved ration
exhibited improved performance, a lower incidence of parakeratosis, a
greater zinc retention and a lowered zinc excretion. Autoclaving
reduced the chick's or poult's need for added zinc on isolated soybean
protein or sesame diets (Kratzer‘gtugl., 1959; Lease gt_£l,, 1960) and
it has been suggested that this reduction is due to destruction of
phytic acid (O'Dell gt_gl., 1964). They found that autoclaving for
four hours reduced the phytic acid of isolated soybean protein to 13
percent of the original amount. Lease in 1966 studied the effect of
autoclaving sesame meal on its phytic acid content and on the avail-
ability of its zinc to the chick. The author feund that after auto-
claving for two hours phytic acid was not reduced. Autoclaving for
four hours reduced the phytate from 1.00 to 0.78 percent which was not
statistically significantly different from the unautoclaved meal.

The difference may be due to a difference in zinc-binding factors
between the two materials or to destruction of phytic acid in the iso-
lated soybean protein concomitant with, but not related to the release
of zinc.

Oberleas gtugl. (1962) concluded that the action of phytate on
zinc availability takes place in the gastrointestinal tract rather than
at the cell level. Phytic acid also decreased the availability of
zinc in the absence of protein, with amino acids serving as the nitro-
gen source to as great an extent as when casein was present in the

diet (Likuski and Forbes, 1964). That the problem of zinc availability

19
may not be simple matter of binding to phytate is suggested by a recent
work from the university of Wisconsin (Anonymous, 1967). Dahmer _e_t .a_l_.
(1966) found that the addition of histidine to low zinc, soybean meal
ration alleviated the leg abnormality but did not affect other zinc
deficiency symptoms such as poor growth or low zinc content of bone.

Nielsen.gt_gl, (1967) studied the effect of histidine and its
various metabolites on chicks fed lowbzinc diets. They reported that
histidine at 1.0 percent and 2 percent of the diet or histamine at
0.2 percent of the diet prevented the "arthritic-like" or "perosis-
1ike" syndrome in zinc deficient chicks fed soy protein diets, while
having little or no effect on other Symptoms of zinc deficiency.
Nielsen gt_gl, (1968 a, b) continued their studies with histamine, as
'well with other anti-arthritic agents and found that they prevented
leg abnormalities, but had little or no effect on other symptoms of
zinc deficiency. The authors concluded that the leg defect was
"arthritis like" in certain reapects as it responded to various anti-
arthritic agents.

Lewis gtflgl. (1957 a) demonstrated that zinc was readily removed
from an ipflzitrg_solution during the precipitation of calcium.phos-
phates brought about by increasing from 3 to 6 the pH of solutions of
calcium, phOSphate and zinc. These conditions were said to simulate
somewhat the change in pH of the ingesta during its passage from the
pig's stomach to small intestine. Increasing the CaxP ratio of these
solutions markedly increased the amount of zinc removed from solution.
This type of phenomenon may explain the detrimental effect of a high_
calcium.diet on parakeratosis, but do not explain it completely as was

suggested by the authors.

20

Phytate occurs in nature as the calcium magnesium salt of phytic
acid, and thus as a component of such a complex and as a companion
Group II element magnesium might be expected to act as does calcium in
increasing zinc uptake in the presence of phytate. But the effect of
added magnesium on the chicks dietary zinc requirements apparently was
not influenced by variation in the magnesium, zinc, calcium and phytate
content of the protein sources. Safflower meal was the highest in
these factors and casein ration the lowest. in neither case did added
magnesium increase the chick's dietary requirement (Lease and Williams,
1966) .

Another interesting possibility concerning the absorption of
zinc is that suggested by worker and Magicowsky (1961) who have proposed
that the absorption of many divalent ions may have their absorption
influenced by the level of vitamin D in the diet. The pronounced
effect of calcium on zinc nutrition and the role of vitamin D in cal-
cium absorption and metabolism suggest a possible effect of dietary
vitam D on zinc utilization. Becker and Hoekstra (1966) found that
the effect of dietary vitamin D on uptake of oral 6Sabine was most
pronounced when the vitamin was suddenly added to the diet of previously
vitamin D-deficient rats. At 60 hours the skeletal, but not soft
tissue, uptake of oral or injected 6Ssinc was significantly increased
by the supplementation of vitamin D to vitamin D deficient rats.
However, skeletal Specific activity (% 6saainc per g) was increased by
increased by vitamin D only with orally administered 6spine. The
authors concluded that the increased absorption of dietary zinc attri-
buted to vitamin D probably results not from a direct effect of the

vitamin, but from a homeostatic reSponse to the increased need for

21
zinc which accompanies inhanced growth and calcification.

A number of observations concerning copperbzinc interactions
have been made in connection with studies that have dealt with the
effects of excessive dietary zinc. Zinc toxicity in the rat is mani-
fested by a hypochromic, microcytic anemia and poor growth (Sutton and
Nelson, 1937; Smith and Larson, 19h6) indicating that the interaction
is not a simple one and that the zinc toxicity syndrome has an effect
not only on copper metabolism but also on iron metabdlism as well.

Davis (1958) reported that capper has a marked and inverse rela-
tionship to zinc, at least within the liver of animals. It was noted
that when copper levels in the liver rose the values above 3,000 ppm,
there was a reduction of zinc from a normal value of 300 ppm to
levels approaching 1 ppm. Conversely, increasing the level of zinc in
the diet caused a depression in the copper concentration of the liver,
but only when copper levels in the diet approached those of borderline
or normal of around 5 to 10 ppm.

Cox and Harris (1960) demonstrated that excess of dietary zinc
results in an accumulation of zinc in the liver with an early and
marked loss of liver iron. The data suggest that the reduction of iron
is responsible for the production of the anemic condition and presumably
the depression of the activity of some iron containing enzymes. A
lowered.1iver copper may also occur and the data indicate that it may
be the result of the reduced liver iron rather than an effect of the
zinc.

Van Campen and.Scaife (1967) studied the site or sites at which
zinc interferes with copper. They found that high levels of zinc

depressed the absorption of 6L‘copperwhen both zinc and the 6L‘cOpper

22
were placed directly into the isolated duodenum segment. If the 6Ll'cOpper
'was put into intestinal segment and the zinc was given intraperitoneally,

no depression in 6Q

copper absorption resulted. The results indicate
that zinc does not depress copper absorption by first building up to
critical levels in some non-intestinal tissue, and subsequently, inter-
fering with copper absorption; since the tissue zinc concentrations
were comparable in both groups of rats given either intraperitoneally
and intraduodenally zinc. The evidence, rather indicates that the
depression of copper absorption by high.levels of zinc is mediated
either in or on the intestine.

Hoefer gt E;- (1960) and Ritchie gt 5;. (1963) have published
that rather high levels of copper (125—250 ppm) will alleviate the
parakeratosis syndrome in swine. In some cases a copper supplement
was nearly as effective as a zinc supplement. Wallace _e_t_ 3;. (1960)
presented fragmentary evidence of such and effect of copper. ‘While
O'Hara‘gt‘gl. (1960) on the contrary fbund that zinc deficiency
appeared in pigs fed a high level of copper but not hathose receiv—
ing a similar diet without copper. 'Work at Wisconsin has repeatedly
shown no beneficial effect of copper in curing or preventing zinc
deficiency in swine. This confusing picture is difficult to ration-
alise. The mechanism is not presently understood, but the importance
of dietary agents of various proteins may also play an important role
(Hoekstra, 1960).

The cadmiumpzinc antagonism has been most widely studied, but
Hill gtugl. (1963) have demonstrated in the chick that copper and iron
are also antagonized by cadmium. The cadmium toxicity resulted in a

reduced growth rate, mortality, microcytic hypochromic anemia and

23
atony and elongation of the gizzard. The growth depression and gizzard
abnormality were corrected by increased dietary zinc. The mortality
was reversed by added copper and increased dietary iron partially cor-
rected both the mortality and the growth depression, indicating a
previously unsu3pected iron component of cadmium toxicity.

Cadmium appears to be very firmly bound in the body, as evidenced
by studies showing a virtual absence of cadmium turnover in mice
(Cotzias ELHEL- 1961) and.by the gradual accumulation of cadmium in
the tissues of animals and man throughout life (Schroeder>g§_gl,, 1963).

The suggestion that cadmium competes with zinc in important
cellular sites is supported by the permanent sterility produced in
male animals by injecting a single dose of a cadmium salt and its pre-
vention or at least its delay, by simultaneous administration of large

amounts of zinc (Hoekstra, 1964).
Zinc in Tissue

Lechartier and Bellamy (1877) noted the presence of zinc in the
biological matter. In the same year Raoult and Breton confirmed the
occurrence of zinc in human liver. For fifty years the interest in
the biological role of this element was only sporadic. Difficulties
in methodology limited most investigations to qualitative determina-
tions, but it was realized that the element was a common constituent of
plants and animals.

Lutz (1926) critically examined the available literature on the
distribution of zinc in biological matter and concluded, "in few if
any, natural biologic materials which have been analyzed has there

been reported failure to detect zinc. we may, therefore, consider as

24

established the fact that zinc is a universal and normal mineral con-
stituent of all biological material." He found it present in all
organs of the rat, cat and man, and that bone, skin and hair contain
high zinc levels compared to most soft tissues of the body. The
methods employed by subsequent workers were also diverse; the precision,
accuracy and the bass lines comparison were quite variable.

The quantitative and qualitative aSpects of the zinc content
of animal tissues have been treated in detail by'Vallee (1959) and
by Underwood (1962). The concentration in most of the soft tissues
of the body approximates 20.30 ppm, which is some 10-15 times that of
copper and less than half that of total iron. In contrast to the
copper, the mammalian newborn does not consistently have higher con-
centrations of total body zinc than do mature animals of the same
species. There is no evidence of appreciable fetal storage of zinc
as normally occurs with iron and copper. During suckling whole body
zinc concentration rises substantially from newborn levels in the rat
and the pig but not in the cat or the guinea pig. The liver and
spleen of the rat, rabbit and pig contain higher levels of zinc at
the end of the suckling period than at the beginning, whereas in the
kitten which is born with higher levels in these organs than the other
species, there is a fall during suckling (Widdowson, 1950; Spray and
'Widdowson, 1951). The importance of the colostrum milk which is 4 to
5 times richer in zinc than later milk, was demonstrated by fostering
newborn mice on the mothers in later lactation. A pronounced reduc-
tion in whole body zinc was achieved (Nishimura, 1953). This indicates
that the young mammal can readily obtain its zinc requirement from

maternal milk.

25

Zinc occurs in all living cells in varying concentrations. The
zinc concentration in most of the organs approximates 25 ppm on the
freshaweight basis and considerably higher concentration of zinc is
found in bone, hair and wool and portions of the prostate and the eye.
Except in the liver and kidneys zinc concentration is not appreciabLy
changed by alteration of zinc intake, but the capacity of the body to
store zinc in any of its organs other than in bone is limited (Keinholz
gig” 1964; Moses and Parker, 1961+; Hoekstra 939;” 1956; and Turk,
1965).

In the young growing animal the zinc concentration in the bone
ash is a sensitive reflection of zinc absorption, especially at sub-
optimal intake levels (Forbes, 1961; Likuski and Forbes, 1965; Fox and
Harrison, 1960 and Hurley gt_gl,, 1960).

The major portion of zinc in whole blood is in the erythrocytes
(75-85 percent) where it occurs mainly as a constituent of carbonic
anhydrase. Determination of zinc content and carbonic anhydrase activ-
ity have implied that they are mutally dependent variables (Vallee‘gt
.g;., 1949). Erythrocytes are rich sources of pyridine nucleotide
dependent dehydrogenases, it is possible that the fraction of zinc may
be associated with some of these enzymes (Vallee gt_§;,, 1956).

Leucocytes contain 3 percent of whole blood zinc, but each
leucocyte contains 25 times as much zinc as each erythrocyte. Human
leucocytes contain a metalloprotein, with a zinc to protein ratio of
3 mg of zinc per g of protein, however, no enzymatic activity in this
complex nor any correlation between the zinc content of leucocytes
and the activity of several zinc containing enzymes has been demon-

strated (Underwood, 1962).

26

Halsted‘gt.al. (1968) found that the blood platelets contain a
significant amount of zinc. This, in part, accounts for the fact that
serum zinc is 16 percent higher than is plasma zinc.

In blood serum, zinc exist in at least two fractions, a firmly
bound form which is said to amount to 34 percent, and a loosely bound
zinc amounting to 66 percent of the total zinc content. The firmly
bound zinc protein is a globulin that satisfies the criteria of metal-
protein complex. The looselyabound complex appears to be concerned
primarily with zinc tran5port. Neither substance has been shown to
exhibit enzymatic properties (Vikbladh, 1951).

Several studies have shown that large oral doses of zinc sig-
nificantly increase whole blood and plasma zinc in rats and rabbits
(Underwood, 1962). Dreosti‘gt.al. (1968) recently reported that in
rats receiving a zinc deficient diet for only one day, showed a plasma
zinc decrease of 38 percent (from 95.9 to 60.1 pg percent) in pregnant
animals and 55 percent (from 110.3 to 50.0 pg percent) in weanling
males. Food restriction elicited a similar but less marked re5ponse
in the pregnant females, but not in the young males. Miller and Miller
(1962) reported that zinc-deficient calves exhibit a reduced blood zinc
content.

The tissue of the eye, eSpecially the choroid of carnivora and
particularly the tapetum lucidum, contains higher concentrations of
zinc than any other animal organ; this amounts in some species to as
much as 13 percent of the dry tissue. The function of zinc in eye
tissues remains unexplained. Some zinc in the retina may be incorpor-
ated in part in the retinene reductase which catalyzes the interconver-

sion of vitamin A alcohol and aldehyde: and may point to a vitamin A

27
zinc interrelationship, since no other metals have been found to occur
in similar dehydrogenases (Forbes, 1967; Vallee, 1959).

Since the reports by Bertrand and Vladesco (1921), several
groups of workers have confirmed the finding of high concentration of
zinc in the male sex organs and fluids of various species. The pros-
tate gland in particular concentrates zinc, although there are wide
variations between Species in the distribution of this element in the
gland. Zinc deficiency in rats results in degeneration of testes,
hypoplasia of the coagulating glands, the seminal vesicles and pros-
pate, and.relative or complete decrease in the numbers of Sperm in the
epididymis. All changes produced by zinc deficiency except the testi-
cular atrOphy, were reversed when supplemental zinc was added to the
diet (Millar‘gtngl., 1958, 1960). There is a correlation between zinc
content and carbonic anhydrase activity of prostatic tissue; the rdle
of zinc in normal Sperm function remains obscure since zinc deficiency
produces testicular degeneration and aspermia, as well as markedLy
reducing the zinc concentration in the total male reproductive tract.
The carbonic anhydrase accounts for only 3 percent of the total zinc
present. There is no correlation between zinc content and the activ-
ity of either acid or alkaline phOSphatase. It is interesting that
the necrotizing effect of dithizone on the prostate of the rat and the
dog is correlated with formation of a fine intracellular deposit of
zinc dithizone. This indicates that a large portion of the zinc
present is not firmly bound to protein that it is unavailable for
chelation (Forbes, 196?).

In normal liver and mammary tissue cells zinc is present in the

nuclear mitochondrial and "supernatant" fraction, with the highest

28
concentrations occurring in the supernatant fluid and microsomes (Thiers
and Vallee, 1957; Bartholomelew.gt‘gl., 1959). The data reported on
the intracellular distribution of zinc in rat liver in terms of micro-
grams of zinc per milligram of nitrogen was found: nuclei, 0.77;
mitrochondria, 0A2; microsomes, 0.65; supernatant, 2.0; and recon-
stituted whale liver, 1.05 (Edwards gt_al,, 1961; Cotzias and Papava-
siliou, 1964). These findings are consistent with the view that the
majority of the known zinc-containing enzymes are in the supernatant
fraction of the liver cell (Thiers and Vallee, 1957).

It is apparent that zinc accumulates in cartilage of the bones
at sites of calcification and, once deposited in the calcified tissue,
is firmly bound (Haumont, 1961; Vincent, 1963; 1965; Gilbert and
Taylor, 1956). Haumont and McLean (1965) in a later histochemical
and autoradiographic study of zinc and the physiology of bone estab-
lished that the stain produced by dithizone in their sections, shown
to be associated with the locus critical for calcification, results
from a reaction with zinc. From this they concluded that zinc is in
some way bound to the initiation of mineralization of preosseous
tissue, whether in the formation of osteous, or in endochondral or
subperiosteal ossification. 'While their autoradiographic studies
showed that zinc is present in calcified tissue; and it has proved
that zinc is progressively incorporated within the preosseous tissue
as mineralization occurs and that it is still present in the fully
calcified tissue.

Alexander and Nasbaum (1962) found that the shaft of the rat
femur had a lower zinc concentration 330 ppm of ash than the head of

the femur, #30 ppm, but they did not find an elevation of zinc in the

29
bones with age, in order to confirm the previous observation reported
by Taylor (1961).

These observations do not tell whether zinc in bone is associated
with the bone mineral or with the organic matrix. 'While the evidence
is not conclusive, all indications point to a Specific effect, on an
organic constituent of bone, rather than to any association with the
mineral itself. Since it appears to play a part in the sequence of
events leading to calcification, and in view of its known association
with hormones and enzyme systems, it may be fair to assume that it
helps to catalyze the calcification process, but such a mechanism, if

it exists, has not been demonstrated (Haumont and McLean, 1965).

III. EXPERIMENTAL PROCEDURE
General

Weanling male rats of the Sprague-Dawley strain were obtained
from a local breeding farm around 21 days of age and immediately
placed on experiment. The general procedure for lotting, management
and record keeping was essentially the same in the two experiments.
All lots were distributed as evenly as possible for weight and the
animals were kept in individual stainless steel cages with free access
to deionized distilled water. Food was provided‘ag,libitum.in alumi-
num feeders with stainless steel covers. Food consumption and growth
data were collected every 98-72 hours.

Spray dried egg white solid was used as the source of protein
since it has a very low'zinc content. The vitamin-glucose mixture of
the basal diet was modified by increasing the level of biotin so that
the complete diet contained 4 mg/kg of this vitamin according to the
previous observations of'Luecke gtmal. (1968). The authors observed
that the increase was necessary since preliminary studies with the un-
modified diet containing 0.2 mg of biotin per kg resulted not only in
growth failure but the appearance of scaly seborrheic type of dermatosis,
often during the second week of the experiment. They also observed
other symptoms included progressive alopecia, particularly in the
areas around the mouth and eyes in some instances a Spastic gait and
kangaroo-like posture.

In general, all the overt symptoms suggested a clinical manifes-
30

31
tation of biotin deficiency, but it was never noted in the zinc-supple-
mented group. They found that the use of the modified vitamin-glucose
mixture containing high levels of biotin did not alter the poor growth
shown by the zinc deficient group, but resulted in marked improvement
in growth of the supplemented group.

All diets were adequately fortified with minerals to provide
the required levels except for zinc. The zinc was supplemented at
different level to the diets as zinc sulfate. Analysis for zinc and
copper were performed on all rations throughout the study. The methods
for determining zinc and copper of the diets were essentially the same
as those used for liver and kidney assays.

The statistical results have been computed by the CDC 3600
available for research work at the Computer'Center of Michigan State

University.

Trace Element Assgy of Tissue

The rats were placed under light anesthesia with ether and
killed by removing as much blood as possible by heart puncture. Liver,
kidneys and bones were removed within a few minutes after exsanguina-
tion and washed several times with deionized water, placed in metal-
free Saran and frozen with dry ice. The tissues were stored in the
frozen state until analysis were performed. Adhering tissue was
removed from bones after boiling in water for one minute. They were
then fat extracted first with alcohol and.later with ether and RBhOd
at 600°C. The ash was taken up in concentrated hydrochloric acid,
suitable dilutions made and the analysis carried out with a Perkin-

Elmer Model 303 atomic absorption Spectrophotometer using a single

32
zinc cathode tube with absorption measured at 213.7 A.

Dry matter was determined on the liver and kidney tissue in an
oven at 90°C. The nitric-perchloric acid method was utilized for wet
washing. The procedure followed was similar to that outlined by Johnson
gt al. (1959), except that a smaller amount of tissue was used (0.3-1.2
g for kidneys and 0.8-9.1 g for liver on the fresh basis). All analysis
were made in duplicate.

After digestion was complete and the residue had been evaporated
to dryness, it was dissolved in deionized water with addition of hydro-
chloric acid and transferred to a volumetric flask. For zinc additional
dilutions were made. The determination of copper was done on the original
dilution by atomic absorption Spectrophotometry using a single copper
cathode tube with absorption measured at 324.1 K. Control blanks were
determined simultaneously on the purity of cleaning and digesting

reagents.

mm;

The Effect of Varying Levels of Dietary Zinc:

The purpose of this experiment was to determine the zinc require-
ment of growing rats fed the egg white protein diet, and its effect on
growth, appetite and tissue concentrations of zinc and copper.

The analysis of tissues for copper and zinc were made to study
the relationship of zinc content of the diet with tissue concentration
and the copper-zinc interrelationship. The interest in the determina-
tion of relationship of zinc content of the diet to the zinc content of

tissues arose in part, from the need of a sensitive method for the

33
diagnosis of zinc deficiency which might prove superior to blood serum

levels, since the low serum yield in small animals and frequent hemoly-
Sis of the cellular material proved troublesome.

The determination of relationship between copper and zinc in the
tissues at low levels of dietary zinc was the other point of this study
since there is little existing information on this point.

The composition of the basal zinc-low diet is shown in Tables 1,
2 and 3. The diet was found by analysis to contain 0.78 ppm of zinc.
All animals were fed to appetite, and deionized water was available at
all times.

Forty weanling male rats were equally divided into 5 groups of
8 animals each, and fed basal diet supplemented with zinc sulfate, at

levels of 0, 5, 10, 15 and 20 ppm.

Experiment II

Part A
The Effect of Varying Levels of Dietary Zinc:

This experiment was designed by similarly to Experiment I in
order to confirm the values obtained and to provide additional data
on weight gain, appetite, food efficiency and zinc content of bones.
Smaller weanling rats which are presumably more sensitive to zinc
deficiency were used.

A total of 49 rats was used in this experiment in which zinc
sulfate was added to basal diet to provide 0, 5, 7.5, 10, 12.5 and 15
ppm of zinc. Eight animals were placed on each treatment, except for

the zero group which had nine. Details of feeding and caging the

TABLE 1

Composition of Basal Diets

 

 

 

 

u
in

 

 

 

 

Ingredient Percentage of Diet
Glucose monohydratea 57.7
Egg white solids (spray-dried)b 20.0
Corn oil ' 10.0
Cellulosec 3.0
Salt mixd (Table 2) 3.7
Vitamin-glucose m" (Table 3) 5.0
Vitamin A, D, E and K oilf 0.5
Ethoxyquing 0.1

 

aCerelose, Corn Products Company, Argo, Illinois.
bGeneral Biochemicals, Inc., Chagrin Falls, Ohio.
cSolka Floc. Brown Company, Berlin, New Hampshire.

dSimilar to that used by Phillips, P. H. and E. B. Hart. J.

Biol. Chem., 109:657. 1935. Reagent grade salts were used and ZnCl2
was omitted.

eComposition similar to that used by Forbes, R. M. and M. Yohe.
J. Nutr., 70:53. 1960. Except that the level of biotin was increased
from 0.004 to 0.080 g/kg of mixture to prevent biotin deficiency.

fVitamin A and D concentrate: 2000 IU vitamin A and 250 IU
vitamin D with the addition of 10 mg menadione and 600 mg <x-tocopherol
per 100 g of A and D oil.

gSantoquin (1-2 Dihydro-6-ethoxy-2,2,’+ trimethquuinoline)
Monsanto Chemical Co.

35

TABLE 2

Composition of Salt Mix

 

 

 

Ingredient Percentage of’Salt Mixture
Calcium carbonate 30.000
Calcium phOSphate, monobasic 7.500
Cobalt chloride 0.005
Copper sulfate 0.030
Ferric Citrate 2.750
Magnesium sulfate 0.510
Potassium iodide V 0.080
Potassium.ph08phate, dibasic 32.200
Sodium chloride , 16.700
Zinc chloride 0.025

 

The mineral mix was provided by G.B.I. under the name of Phillips
and Hart No. Q Salt Mix. It was made with reagent grade salts from
which zinc chloride was omitted. The original Phillips and Hart diet

was modified by addition of cobalt chloride and manganese sulfate was
omitted.

36

TABLE 3

Composition of'Vitamin-Glucose Mix

 

 

 

 

 

 

 

Ingredient Amount
Vitamin B12 in mannitol 0.00 mg.
Biotin 8.00 mg.
Folio acid 1.00 mg.
Pyridoxine HCl 8.00 mg.
Riboflavin 12.00 mg.
Thiamine mononitrate 20.00 mg.
Calcium pantothenate 32.00 mg.
Nicotinic acid 50-00 m8-
Choline chloride 3.000.00 mg.
Cerelose to complete 100 g.

 

Composition similar to that used by Forbes, R. M. and M. Yohe.
J. Nutr. 70:53, 1960, except that the level of biotin was increased
from 0.000 to 0.080 g/kg of mixture to prevent biotin deficiency. The
chlorotetracycline was omitted.

37
animals were Similar to that utilized in the preceding experiment and

the basal diet had the same composition.

Pgrt B
The Effect of Interchanging Different Zinc Level Diets:

It was decided that the results obtained merited further inves-
tigation. The animals of groups fed 0, 10 and 15 ppm of zinc in the
diet were utilized for further study.

The rats of zero ppm of supplemental zinc were fed 10 and 15
ppm.of zinc while the rats of these latter two groups were placed on 0
ppm zinc diet. Two animals of each group were killed as controls,
four rats of contrdl group were placed on 10 ppm zinc diet and the
other three on 15 ppm zinc diet. Three animals of 10 ppm.of zinc
diet and four of 15 ppm zinc diet were placed on the basal diet, while
the other two of each group were kept on the original diet as positive
controls. No control was carried with zero ppm zinc group, because of
insufficient number. On the other hand the animals were very weak, and
might not have survived the additional three weeks of the experiment.
The animals were killed at first and third week of the experiment for a
study of the concentration of the zinc in the tissues.

In this part consequently the effect of the interchange of diets
on appetite and growth of rats was studied. Second it was important to
determine to what degree the animal's previous food intake history
played on growth and appetite. Other investigations have suggested
that the bone may serve to store excess zinc, which to some degree, can
be released for the use by other critical tissues (Savage,‘gtual., 1960).
Accordingly the zinc concentration in the bones and liver was studied

after one and three weeks of switchover.

IV. RESULTS AND DISCUSSION

W

Differences resulting from the supplementation of different
levels of zinc to the basal diet were highly significant (P (0.01) for
fecd intake, growth and food efficiency and are Shown in Table h.

The poor growth of the rats on the basal diet was accompanied
by a unkempt aspect, the hair was greasy, Sparce and coarse, and a
red brownish color appeared on the head of some of the animals of
this group. Also two rats developed diarrhea at twenty-first day and
one died on the twenty-fourth and the other on the twenty-seventh day.
But neither the diarrhea or other symptoms were observed in any of the
rats of zinc supplemented groups, even with the group on 5 ppm zinc
that did not grow at a normal rate.

Anorexia was observed early in the rats on basal diet. After
only 96 hours the animals consumed 50 percent of the amount consuming
during the first 48 hours, and approximately 40 percent of the amount
consumed by the animals fed 10 ppm or more of zinc in the diet. The
same low food intake was maintained for the overall four weeks of the
experiment by this group. The relative intake declined to one-fourth
of the 10 ppm of zinc diet by the end of third week. Similar observa-
tions were reported by Macapinlac gt_gl, (1965).

Prasad 23,5l, (1967) suggested that the difference in weight

gain can be accounted for partly on the basis of anorexia; however

38

39

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approximately three times as much food was required by 0 ppm zinc diet
group for each gram of body weight gained as was required by 10 ppm or
higher zinc level diet, suggesting a metabolic derangement of some
type.

Analysis of the residual food of the basal diet group was made
to determine how much contamination from the environment was present
by the method of feeding utilized. The zinc content in the residues
were 1.52 ppm and 1.04 ppm of zinc in the diet after first and second
week respectively, while the original determination gave 0.78 ppm.of
zinc for the same diet. Even though the analysis for the other diet
residues were not performed it is possible to assume that after the
first week the concentration of zinc in the diets should be very
similar to its original concentration at all times.

The growth curves of the experimental rats on different diets
are shown in Figure 1. From these results and those presented in
Table h it would appear that under the conditions of the experiment
the requirement of the rat for zinc approximates 10 ppm of diet.

Vallee (1959) reported that the zinc content of soft tissues is
normal, but that of bone is lowered in zinc deficiency. Similar obserb
vations were made by other investigators. Savage‘gtmal. (196h) found
that the bones of zinc depleted chicks contained about one-third as
much zinc as controls. Macapinlac (1965) found that the zinc concen-
tration of the deficient animals were markedly reduced in the testes
and the femur, but not in the other tissues examined. Taylor (1961)
indicated that the zinc content of the femur, pelvis and humerus
expressed as pg of zinc/g wet bone do not differ significantly from

each other at any age. They also observed that the zinc concentration

41

Group 1 Basal Diet

 

_-_- Group2 5pmen

k—uGroup 3 10 ppm Zn

__ _ _Group 4 15 ppm Zn

200- -— -Group 5 20 ppm Zn

 

 

 

 

 

E 1‘ ,/
$4 ,/
8 /
’/'
1‘.) "/’/
no
«4
:33
>. 100
"U
o
m
1 u n in
o 1 2 3 4

Weeks on Study

Figure 1. Experiment I--Growth Curves of Rats Fed Different Levels of
Zinc.

42

in bones increased with the age, but this was not confirmed by Alexander
and Nusbaum (1962) who found 30 percent more zinc in the ends than in
the shaft and no variation with age in their experiment conducted over
414 days.

Femur was utilized for the zinc and percentage of ash determina-
tions. The results are shown in Table 5. The zinc concentration of
the bones of the different groups were highly correlated with the zinc
concentrations in the diet (P < 0.01). Also a significant decrease
(P < 0.01) was observed in the percentage of ash with the zero ppm zinc
diet group. Hurley’gtual. (1964) also observed a decrease of ash content

of femurs in deficient females.

TABLE 5

Effects of'Variation on Zinc Content of Diets
on Concentration of Zinc in Bones

 

Item 0 ppm 5 ppm 10 ppm 15 ppm 20 ppm
No. of rats 8 8 8 8 8
Days fed diet 28a 28 28 28 28

Percentage of ashb 57.1io.5 60.3io.6 60.1to.5 60.3to.3 60.7io.2
Zn concentrationc 70.833.5 87.512.8 186.534.1 238.214.4 256.“:3-8

aExcept for the two rats that died on days 24 and 27.

 

bPercent on the fat-free, dry basis.

cExpressed in ppm of zinc on the dry, fat-free basis.

The zinc content of the liver and kidneys showed no significant
differences (Table 6), exoept for the content of zinc in liver of the

two animals that died before the experiment was finished (Appendix

43

Table 2). The lack of significant differences in the zinc content of
the liver and kidneys was observed previously by Savage 2£.£ln (1964)
with chicks, who suggested that these critical organs might hold zinc
tenaciously. Keinholz gt,al, (1964) found that 10 ppm dietary zinc
maintained zinc concentration of many body components of hens equal to
those produced by feeding 70 ppm zinc diet and, if the animal grows
and develops at all, its tissues maintain the normal zinc concentra-
tion. There was no tendency for the rats fed excess zinc to store the

element in liver or kidney as was the case with some other nutrients.

TABLE 6

Effect of Different Levels of Zinc in the Diet
on Copper and Zinc Concentration in Livers and.Kidneys

Item 0 ppm 5 ppm 10 ppm 15 ppm 20 ppm

 

Liver coppera 15.2ti.7 9.7to.5 5.6to.9 6.8to.6 5.uto.5

Liver zincb 93.638.4 77.912.6 77.612.7 83.512.0 84.932.5
Kidney oopper° 23.012.3 19.111.6 17.oto.2 16.1to.5 17.111.3
Kidney zincd 87.910.6 79.6:2.5 87.211.4 92,511.? 86.112.7

 

3The value is the average of the analysis for 8 livers and is
expressed in ppm of cOpper on dry weight basis.

‘bIdem as for c0pper.

°The value is the average of the three pairs of kidneys and is
expressed in ppm of copper on dry weight basis.

dIdem as for copper in kidneys.

A number of observations concerning copper-zinc antagonism have
been reported in connection with the effects of excessive dietary con-

centration of one or the other of these elements in the diet, but only

44
a few studies have been made of their interactions with low zinc diets.
Davis (1958) observed that increasing the level of zinc in the diet
causes a decrease of copper values in the liver, but only when the
copper levels in the diet approach those of a low normal value of about
5 to 10 ppm.oopper.

Reinhold.gt_al, (1967) found no evidence of low zinc intake on
the copper concentration in liver and kidneys, but their diets con-
tained higher concentration of copper based on the mineral mix composi-
tion bases, since there was no copper analysis reported for the diets
fed.

The depression of the copper values in the liver observed was
highly significant (P < 0.01). The copper content in liver for the
groups fed 10 ppm, 15 ppm.and 20 ppm.of zinc in diets were 5.6 ppm,

6.8 ppm.and 5.4 ppm of zinc which are below the 10-50 ppm of copper
concentration in liver reported fer normal adults rats or humans
(Underwood, 1962). A decrease in the copper content of the liver of
this group may have been due to dilutional phenomena, but Van Campen
and.Scaife (1967) suggested from.their studies, that a depression of
copper absorption by high levels of zinc is mediated either in or on
the intestine; the same antagonistic effect might be present for the
low concentrations. A marked reduction in liver catalase and cyto-
chrome oxidase was observed by Van Reen (1953) for high diets, but
there is no evidence on this point, and it might be related to the

capper reduction observed in this experiment.

45
Experiment II

E

 

The results of Experiment II, part A for food intake, growth,
food efficiency and zinc content in bones are reported in Table 7.
Bones were analyzed from only three of the six groups at the end of
third week, since the other groups were retained for further study.
One rat of the group fed 10 ppm.zinc diet died on fifteenth day of
experiment but the reasons were not determined.

The values obtained for appetite, growth, food efficiency and
zinc content in bones, in general confirm the values in the Experi-
ment I. Consequently the values of both experiments were submitted
to a statistic study to obtain a more complete picture of the relation-
ship between the zinc concentration of the diets and the other variables.
The analysis of bones of the two experiments were determined in the
first experiment after feur weeks and in the second after three weeks
of experiment. However on the basis of the studies of Alexander and
Nusbaum (1962), who did not find any variation of zinc concentration
with time, the values were grouped together.

In the Figure 2 is shown the relationship of weight gain to the
concentration of zinc in the diet for all diets studied after three
weeks on the experiments.

Similar correlations were obtained for food consumption and for
food efficiency, but they are not shown. Thus the results of both
experiments indicate that the zinc requirement of the rat approximates
10 ppm of the diet.

In Figure 3 it is important to note that the linear increase of

46

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47

 

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300..

200

Zinc Content of Bones

100

 

 

l 1 L J
0 5 10 15 20 ppm.

Zinc Level of Diets

Figure 3. Relation Between Bone Zinc and Zinc Diet Levels

49
zinc concentration of the bones with reSpect to zinc content of the
diets is between 5 and 12.5 ppm. It is also observed that only a small
difference in the concentration of bone zinc occurs between basal and
5 ppm zinc diet. This indicates that the animal utilizes at maximum
the zinc available for growth and that the amount of zinc is not suf-
ficient for the requirements of normal development. The growth rate
was 50 percent of the 10 ppm zinc diet group, and consequently there is
not an excess of zinc for storage in the bones.

For the higher concentrations of zinc in the diets the incre-
ment in bone zinc content is smaller. This may be due to some regulat-
ing mechanism or to a decrease in efficiency of the absorption mechan-
isms, since the immediate requirement of the animals for normal growth
are satisfied.

Although the essentiality of zinc for growth and metabolism of
the bone has not been established its presence in the deficient animals
at similar concentration (70-80 ppm) in the kidneys and liver on a dry

basis suggests that its presence is more than accidental.
Part B

The results of Experiment II, part B, are reported in the Table
7. The initial values of food intake are the average of the last 48
hours on the original diet. The final food consumption is also the
average of 48 hours, even if it was checked every 24 hours.

The appetite of the repleted rats increased 50 percent in the
first 24 hours, about 100 percent at 48 hours and up to 300 percent at
96 hours. The consumption later increased slowly up to 450 percent

during the subsequent two weeks. Quarterman (cited by Mills, 1967) noted

50
an increase in appetite after four hours of feeding a repleted diet.
In this case the increment of food consumption was 50 percent after
24 hours; nevertheless, it was noted that after the animal tried the
new diet, the animal immediately became more interested in it. The
response of the first hours was not determined and might not be
detectable, but a change in the interest of the animals was evident in
a few minutes. The mechanism related to this quick response is being
studied by Quarterman. The current evidence suggests that enzymes
other than the presently known zinc enzymes are more sensitive to zinc
deficiency, it is possible that the same enzymes also play an important
role in repletion. The activities of amylase and of liver and kidney
catalase, which are not zinc dependent enzymes have been shown to
decrease in zinc deficiency (Robertson and Burns, 1963). It is of
interest that if amylase is involved in the depletion it may be that
the same enzyme plays some role or other similar in the fast response
due to repletion, since cx-amylase (1-4 glucan 4 glucanohydrolase) is
present in saliva and pancreatic juice.

The homeostatic mechanisms which acts at the sites of absorption
and on intestinal excretion are a reflection of mechanisms resulting in
homostatic conservation of the zinc. It was observed that a very high
absorption exceeding 80 percent occurred for the low zinc content
diets, but its importance in the fast response due to repletion is
unknown.

Similar reSponses to appetite, growth, food efficiency, was
observed for both groups of animals on 10 and 15 ppm zinc diet, even
appearing a little higher for the 15 ppm zinc diet. One rat on the

basal diet had diarrhea in the latter part of Experiment II, part A and

51
placed on 10 ppm zinc diet to determine whether it could be corrected
by administration of only a zinc supplemented diet. In the first
experiment two animals from the group on the basal diet died in a few
days from diarrhea. The rat responded to the supplemented diet
similarly to those of the other animals and the diarrhea was stopped.

The inverse picture was observed with the rats of groups from
10 and 15 ppm zinc diets when placed on the depleted diet (basal diet).
Again both groups responded quite similarly but the effect was quicker
on rats previously fed 10 ppm zinc diet. The food intake decreased to
66 percent at 72 hours for rats previously fed 10 ppm zinc diet, while
80 percent consumption was observed for the rats on the 15 ppm zinc
diet at 96 hours. More depression of food consumption was observed a
little later and subsequently a marked increase in food intake was
maintained for 24 hours in rats on the 10 ppm zinc diet and 72 hours in
rats on the 15 ppm zinc diet. After that a second depression of food
consumption occurred and the intake was maintained around 50 percent
of the controls for the last two weeks of the experiment.

The difference observed in the animals to repletion and to
depletion of zinc may be influenced by the presence of a zinc supple-
mented diet in the digestive tract, which delays the response of the
animal to the new diet.

The zinc content of the bones of deficient rats on repletion
increased from 65.3 ppm.to 78.2 ppm of zinc for the 10 ppm zinc diet
and to 135.3 for the 15 ppm zinc diet, reSpectively, after one week.
The values were 100.4 ppm and 172.5 ppm zinc for the 10 ppm and 15 ppm
zinc diet, reSpectively, after three weeks. All of the values are

expressed on dry, fat-free bone basis. Here the difference between

 

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54
the two groups of animals placed on 10 ppm and 15 ppm zinc diet was even

higher than that observed for food consumption and growth rate.

The zinc content of the bones of rats on a basal diet that were
previously on 10 ppm and 15 ppm zinc diet, decreased from 131.2 ppm to
104 ppm and from 216.5 to 143.5 ppm in one week and to 93.1 ppm.and
121.5 ppm of zinc at the end of the third week, respectively.

The zinc content of the bones decreased 30 percent during the
first week. Alexander and Nusbaum (1962) found that the ends of the
bones contained 30 percent more zinc than did the shaft. The zinc
from the ends may be released for maintaining the homostasis since at
the time of osteogenesis zinc is still free; while the zinc of calci-
fied bone is literally sequestered, neither Ca-EDTA nor dithizone can
react with it and the zinc apparently is not available for ionic

exchange (Haumont and McLean, 1965).

V. SUMMARY

W

Forty weanling rats were fed a diet containing egg white solids
as a source of protein, supplemented with zinc as zinc sulfate at
levels of 0 ppm, 5 ppm, 10 ppm, 15 ppm and 20 ppm for four weeks.

The manifestations of zinc deficiency syndrome in the rat, which
include anorexia and growth retardation, was presented by the group
on the basal diet. The appetite, growth rate and food efficiency were
significantly related to the zinc content in the diet. Diarrhea
occurred in two animals on basal diet, which died, a few days later.
But it was not observed in any of the animals of other groups.

The zinc content of the bones increased linearly with the concen-
tration of the zinc in the diet.

No differences were Observed in the zinc concentration in kid-
neys and liver with the zinc diet. Copper concentration of the liver

decreased significantly with the increase of zinc in the diet.

Egperiment II

ngt A

Forty-nine weanling rats were placed on this experiment and fed
for three weeks. The composition of the basal diet was the same as
used for the Experiment I and it was supplemented with zinc as zinc

sulfate at levels 0 ppm, 5 ppm, 7.5 ppm, 10 ppm, 12.5 ppm and 15 ppm of

55

56
zinc. The results obtained for appetite, growth rate and food efficiency
confirmed the observations of the Experiment I.
Only the analysis of bones for the groups 5 ppm, 7.5 ppm and 12-5
ppm zinc in the diet were done at the end of third week, since the
other three groups were retained for further study. The concentration
of zinc in the bones was significantly related to the zinc contain of

the diet.

Part B

 

The rate of the basal group were interchanged.with the rats on
10 ppm and 15 ppm zinc diets to test the hypothesis that bone plays an
important role in stored and released zinc for use by critical organs
during the depletion and to see if the previous history of the animal
exerts some influence on the appetite and growth rate, as well as the
concentration of zinc in the tissues.

The response of both groups of rats placed on basal diet showed
a similar picture in the rats previously the 15 ppm.zinc diet, the
depression of appetite was observed later and to a lower degree.

The rats from basal diet on repletion fed 10 ppm and 15 ppm zinc
diets showed a very similar reSponse and a very rapid increase in food
intake. The concentration of zinc in bones increased faster in the
animals on 15 ppm zinc diet than on 10 ppm. Also a decrease in copper
concentration was observed with the increase in zinc levels of diet,

but neither liver nor kidney concentration of zinc was affected.

VI. CONCLUSIONS

Supplementation of egg white protein diet with zinc at levels of
10 ppm supplies the minimum requirement by the weanling rats for normal
growth. Inanition accompanying the deficiency plays a significant role
in the growth rate and food efficiency.

Bone zinc concentration is linearly increased with dietary
levels of this element between the limits studied.

Zinc concentration of the liver and kidney showed no significant
alteration in relation to the zinc content of the diet at the levels
used.

Copper content of the liver was decreased significantly with the
first increment of zinc in the diet (5 ppm).

On the repletion with zinc in the deficient group, the zinc con-
tent of bones increased significantly. Also observed was an high
increment in growth rate and appetite.

0n the depletion the zinc content of bones was reduced signifi-
cantly in one week, it was not sufficient to maintain the normal growth

rate and the appetite.

57

LITERATURE CITATIONS

Adelstein, S. J. 1957. Glutamic dehydrogenase, a zinc metalloenzyme.
Ph.D. Massachusetts Institute of Technology, Cambridge, Mass.

Adelstein, S. J. and.B. L. Vallee. 1958. Zinc in beef glutamic
dehydrogenase. J. Biol. Chem. 223:589.

Alexander, G. V. and R. E. Nusbaum. 1962. Zinc in bone. Nature
195:903.

Anonymous. 1957. Ainc deficiency and dietary calcium in swine. Nutr.
Rev. 15:33h.

Anonymous. 1961. Zinc deficiency in chicks. Nutr. Rev. 19:111.
Anonymous. 1960. Zinc metabolism in the chick. Nutr. Rev. 22:309.
Anonymous. 1967. Zinc, calcium and phytate. Nutr. Rev. 25:115.

Barker, G. R. and M. Rieber. 1967. The development of polysomes in
the seed of fiesim‘gzzgnsg, Biochem. J. 105:1195.

Bartholomew, M. E., R. Tupper and A. Wormall. 1959. Incorporation of
65Zn in the subcellular fractions of the liver and Spontaneous
occurring mammary tgmours of mice after the injection of zinc-
glycine containing 52n. Biochem. J. 73:256.

Becker, W. M. and.W. G. Hoekstra. 1966. Effect of vitamin D on 65Zn
absorption, ditribution and turnover in rats. J. Nutr. 90:301.

Bellis, D. B. and J. Philp. 1957. Effect of zinc, calcium and.phos-
phorus on the skin and growth of pigs. J. Sci. Food Agr.
8:5119.

Bertrand, G. et M. Javillier. 1911. Influence du zinc et du manganese
sur la composition minerals de L'Aspergillus niger. Acad. Sci.
152:133.

Bertrand, G. at R. Vladesco. 1920. De la repartition du zinc dans
l'organisme du cheval. Acad. Sci. 171:10h.

Bertrand, G. et R. Vladesco. 1921. Intervention probable du zinc dans

les phenomenes de fecondation chez les animaux vertébrés. Acad.
Sci. 173:176.

Bertrand, G. et B. Benzon. 1922. Sur l'importance du zinc dans
l'alimentation des animaux. Experiences sur la souris. Acad.
Sci. 175:289.

58

59

Bertrand, G. at R. c. Bhattacherjee. 193a. L'action combine/e du zinc
et des vitamines dans l'alimentation des animaux. Acad. Sci.
198:1823.

Bertrand, G. at R. C. Bhattacherjee. 1935. Recherches sur l'action
combines du zinc et des vitamines dans l'alimentation des
animaux. Ann. Inst. Pasteur 55:265.

Brinegar, M. J. and J. E. Hunter. 1955. Relationship of zinc and
calcium to parakeratosis in swine. Allied. Mills, Inc.
Research Division. Libertyville, 111., March 23.

Bunn, C. R. and G. Matrone. 1966. In vivo interactions of cadmium,
copper, zinc and iron in the mouse and rat. J. Nutr. 90:395.

Byrd, C. A. and G. Matrone. 1965. Investigations of chemical basis
on zinc-calciumbphytate interaction in biological systems.
Proc. Soc. Exptl. Biol. Med. 119:307.

Coble, Y. D., W. O'Dell and W. Bordin. 1966 a. Unpublished data.
C/F Coble 93; 9;. 1966 b.

Coble, Y. D., R. Van Reen, A. R. Schubert, R. P. Koshahji, Z. Farid
and J. T. Davis. 1966 b. Zinc levels and blood enzyme activ-
ities in Egyptian male subjects with retarded growth and
sexual development. J. Clin. Nut. 19:“15.

Coleman, J. E., B. J. Allan and B. J. Vallee. 1960. Protein spheru-
lites. Science 131:350.

Coleman, J. E. and B. L. Vallee. 1960. Metallocarboxypeptidases.
J. Biol. Chem. 235:390.

Coleman, J. E., P. Pulido and B. L. Vallee. 1960. Organic modifica-
tions of metallocarboxypeptidases. Biochem. 5:2019.

Cotzias, G. C., D. C. Borg and B. SelleclicO 1961. Virtual absence of
turnover in cadmium metabolism: 9Cd studies in the mouse.
Am. J. Physiol. 201:927.

Cotzias, G. C., and.P. S. Papavasiliou. 196a. Specificity of zinc
pathway through the body homeostatic considerations. Am. J.
Physiol. 206:787.

Cox, D. H. and D. L. Harris. 1960. Effect of excess dietary zinc on
iron and copper in the rat. J. Nutr. 70:51h.

Curtin. L. V. 1950. Personal communication. C/F Brinegar, M. J. and
J. E. Hunter. 1955.

Dahmer, E. J., C. W. Lin, R. H. Grummer and W. G. Hoekstra. 1966.
Alleviation of zinc deficiency in swine by feeding blood meal.
J. Animal Sci. 25:890.

60

Dahm, K. and H. Breuer. 1964. Concentration of 17-fl-hydroxysteroid:
nicotinamide adenine dinucleotide (phOSphate) (NAD(P))-oxydo-
reductase in rat adrenals. Z. Physiol. Chem. 336:63. Cited
Chem. Abs. 61:2123.

Davis, G. K. 1958. Mechanisms of trace element function. Soil Sci.
85:59.

Dreosti, I. E., S. Tao and L. S. Hurley. 1968. Plasma zinc and leuco-
cyte changes in rats during zinc deficiency. Fed. Proc. 27:484.

Druyan, R. and B. L. Vallee. 1962. Exchangeability of the zinc atoms
in liver alcohol dehydrogenase. Fed. Proc. 21:247.

Edwards, H. M. Jr. 1959. The availability to chicks of zinc in vari-
ous compounds and ores. J. Nutr. 69:306.

Edwards, C., K. B. Olson and J. Glenn. 1961. Intracellular distribu-
tion of trace elements in liver tissue. Proc. Soc. Exptl. Biol.
Med. 107:94.

Follis, R. H. Jr., H. G. Day and E. V. McCollum. 1941. Histological
studies of the tissues of rats fed a diet extremely low in zinc.
J. Nutr. 22:223.

Forbes, R. M. and M. Yohe. 1960. Zinc requirement and balance studies
with the rat. J. Nutr. 70:53.

Forbes, R. M. 1961. Excretory patterns and bone deposition of zinc,
calcium and magnesium in the rat as influenced by zinc deficiency,
EDTA. J. Nutr. 74:194.

Forbes, R. M. 1967. Studies of zinc metabolism. In Newer Methods of
Nutritional Biochemistry (lst ed.). Academic Press Inc., New
York.

Fox, M. R. S. andrB. N. Harrison. 1964. Use of Japanese quail for the
study of zinc deficiency. Proc. Soc. Exptl. Biol. Med. 116:256.

Fujiota, M. and I. Lieberman. 1964. A zinc requirement fer synthesis
of deoxyribonucleic acid by rat liver. J. Biol. Chem. 239:1164.

Gilbert, I. G. F. and D. M. Taylor. 1956. The behavior of zinc and
radio-zinc in the rat. Biochem. Biophys. Acta 21:545.

Grashuis, J. 1963. Landbouwk. Tijdschr.,'s-Grav. 7521127. C/F Mills
in g. 1967.

Grashuis, J. 1964. Bemesting, Voeding, Gezondheid en Ziekte, Herziene
druk. Arnhem:ILandbouwk. Bereau.Sporenelementen. C/F Mills
25-. 9.1.. 1967.

Hartmans, J. 1965. 'Versl. landbouwk. Onderz. Ned. p. 664. C/F Mills
pt 5;. 1967.

61

Haumont, S. 1961. Distribution of zinc in bone tissue. J. Histochem.
Cytochem. 9:141.

Haumont, S. and F. C. McLean. 1965. Zinc and the physiology of bone.
In Zinc Metabolism, ed., A. S. Prasad, C. C. Thomas, Springfield,
Illinois, p. 169.

Hill, C. H., G. Matrone, W. L. Payne and C. W. Barber. 1963. In vivo
interactions of cadmium with copper, zinc and iron. J. Nutr.
80:227.

Hoefer, J. A., E. R. Miller, D. E. Ulquy, H. D. Ritche and R.‘W.
Luecke. 1960. Interrelationships between calcium, zinc, iron
and copper in swine feeding. J. Animal Sci. 19:249.

Hoekstra, W. G., P. K. Lewis, P. H. Phillips and R. H. Grummer. 1956.
The relationship of parakeratosis, supplemental calcium and zinc
to the zinc content of certain body components of swine. J.
Animal Sci. 15:752.

Hoekstra, W. G. 1964. Recent observations on mineral interrelation-
ships. Fed. Proc. 23:1068.

Hurley, L. S., H. Swenerton and J. T. Eichner. 1964. Reproduction and
bone zinc in zinc deficient rats. Fed. Proc. 23:292.

Johnson, K. E. E., E. I. Linden, W. S. Brammell and E. J. Benne. 1959.
Report on zinc in plants. J. Assn. Official Agr. Chem. 42:363.

Kagi, J. H. R. and B. L. Vallee. 1960. The role of zinc in alcohol
dehydrogenase. V. The effect of metal binding agents on the
structure of the yeast alcohol dehydrogenase melecule. J. Biol.
Chem. 235:3188.

Keilin, D. and T. Mann. 1939. Carbonic anhydrase. Nature 144:442.

Keilin, D. and T. Mann. 1940. Carbonic anhydrase. Purification and
nature of the enzyme. Biochem. J. 34:1163.

Keinholz, E. W., D. E. Turk, M. L. Sunde and.W. G. Hoekstra. 1961.
Effects of zinc deficiency in the diets of hens. J. Nutr.
75:211.

Keinholz, E. W., N. L. Sunde and W. G. Hoekstra. 1964. Influence of
dietary zinc, calcium and vitamin D for hens on zinc content of
tissues and eggs and on bone composition. Poultry Sci. 4:667.

Keleti, T. 1964. Studies on D glyceraldehyde-3-phosphate dehydrogen-
ases. Biochem. Biophys. Acta 89:422.

Kernkamp, H. C. H. and E. F. Ferrin. 1953. Parakeratosis in swine.
J. Amer. Med. Ass. 123:217.

62

Kratzer, F. H., P. Vohra, J. B. Allred and P. N. Davis. 1958. Effect
of zinc upon growth and incidence of perosis in turkey poults.
Proc. Soc. Exptl. Biol. Med. 98:205.

Kratzer, F. H., J. B. Allred, P. N. Davis, B. J. Marshall and P. Vohra.
1959. The effect of autoclaving soybean protein and the
addition of ethylenediaminetetracetic acid on the biological
availability of dietary zinc for turkey poults. J. Nutr. 68:313.

Kratzer, F. H. and.B. Starcher. 1963. Quantitative relation of EDTA
to availability of zinc for turkey poults. Proc. Soc. Exptl.
Biol. Med. 113:424.

Langer, L. J. and L. L. Engel. 1958. Human placental 17-(} -estradiol
dehydrogenase (I). Concentration, characterization and assay.
J. Biol. Chem. 2338583.

Lease, J. G., B. D. Barnett, E. J. Lease and D. E. Turk. 1960. The
biological unavailability to the chick of zinc in a sesame meal.
J. Nutr. 72:66.

Lease, J. G. 1966. The effect of autoclaving sesame meal on its phytic
acid content and on the availability of its zinc to the chick.
Poultry Sci. XLV:237.

Lease, J. G. and W. P.'Williams, Jr. 1966. The effect of added
magnesium on the availability of zinc with some highpprotein
feedstuffs. Poultry Sci. XLVI:242.

Lechartier, G. et F. Bellamy. 1877. Sur la présence du zinc dans les
corps des animaux et dans les vegetaux. Acad. Sci. 84:687.

Legg, S. P. and.L. Sears. 1960. Zinc-sulfate treatment of parakeratosis
in cattle. Nature 18621061.

Lewis, P. K. Jr., R. H. Grummer and W} G. Hoekstra. 1957. The effect
of method of feeding upon the susceptibility of the pig to
parakeratosis. J. Animal Sci. 16:927.

Li, T. K. and B. L. Vallee. 1965. Reactivity and function of sulfydryl
groups in horse liver alcohol dehydrogenase. Biochem. 4:1195.

Likuski, H. J. A. and R. M. Forbes. 1964. Effect of phytic acid on
the availability of zinc in amino acid and casein diets fed to
chicks. J. Nutr. 84:145.

Likuski, H. J. A. and R. M. Forbes. 1965. Mineral utilization in the
rat. IV. Effects of calcium and phytic acid on the utilization
of dietary zinc. J. Nutr. 85:230.

LindskOg, S. 1960. Purification and properties of bovine erythrocyte
carbonic anhydrase. Biochem. Biophys. Acta 30:219.

63

Lindskog, S. and B. G. Malstrom. 1962. Metal binding and catalytic
activity on bovine carbonic anhydrase. J. Biol. Chem. 237:1129.

Luecke, R.'W., J. A. Hoefer,'W. S. Brammell and F. Thorp, Jr. 1956.
Mineral interrelationships in parakeratosis of swine. J.
Animal Science 15:347.

Luecke, R. H., J. A. Hoefer, W. S. Brammell and D. A. Schmidt. 1957.
Calcium and zinc in parakeratosis of swine. J. Animal Sci.
16:3.

Luecke, R. W. 1965. The significance of zinc in nutrition. Borden's
Rev. of Nutr. Res. 26:45.

Luecke, R. H., M. E. Olman and B. V. Baltzer. 1968. Zinc deficiency
in the rat. Effect on serum and intestinal alkaline phoSpha-
tase activities. J. Nutr. 94:344.

Lutz, R. E. 1926. The normal occurrence of zinc in biologic materials.
A review of the literature and a study of the normal distribu-
tion of zinc on the rat, cat and man. J. Ind. Hyg. 8:177.

Macapinlac, M. P., N3 N. Pearson and w. J. Darby. 1965. Some charac-
teristics of zinc deficiency in the albino rat. In Zinc
Metabolism, ed., A. S. Prasad, C. C. Thomas, Springfield, III...
p. 142.

Mahler, H. R. and E. H. Cordes. 1966. Biological Chemistry, ed.,
Harper'& Row: New York, N.Y., p. 662.

Mathies, J. C. 1958. Preparation and properties of highly purified
alkaline phOSphatase from kidneys. J. Biol. Chem. 233:1121.

Medina, A. and D. J. D. Nicholas. 1957. Some properties of a zinc

gapendent hexokinase from.Neurospo;a crass§. Biochem. J.
6:573.

Millar, M. J., P. V. Elcoate, C. A. Mawson. 1957. Sex hormone control
of the zinc content of the prostate. Canad. J. Biochem. Physiol.
35:467.

Millar, M. J., M. I. Fischer, P. V. Elcoate and C. A. Mawson. 1958.
The effects of dietary zinc deficiency on the reproductive
system of male rats. Canad. J. Biochem. Physiol. 36:557.

Millar, M. J., P. V. Elcoate, M. I. Fischer and C. A. Mawson. 1960.
Effects of testosterone and gonadotrophin injections on the sex
organ development of zinc-deficient male rats. Canad. J. Biochem.
Physiol. 38:1457.

Miller, J. K. and W. J. Miller. 1962. Experimental zinc deficiency
and recovery of calves. J. Nutr. 76:467.

64

Mills, C. F., A. C. Dalgarno, R. B. Williams and J. Quarterman. 1967.
Zinc deficiency and the zinc requirement of calves and lambs.
British J. Nutr. 21:751.

Morrison, A. B. and H. P. Sarett. 1958. Studies on zinc deficiency in
the chick. J. Nutr. 65:267.

Moses, H. A. and H. E. Parker. 1964. Influence of dietary zinc and
age on the mineral content of rat tissues. Fed. Proc. 23:132.

Nielsen, F. H., M. L. Sunde and'w. G. Hoekstra. 1967. Effect of
histamine, histidine and some related compounds on the zinc
deficiency chick. Proc. Soc. Exptl. Biol. Med. 124:1106.

Nielsen, F. H., M. L. Sunde and'W. G. Hoekstra. 1968 a. Prevention
of leg defects in zinc deficient chicks by antiarthritic agents.
Fed. Proc. 27:484.

Nielsen, F. H., M. L. Sunde and W. G. Hoekstra. 1968 b. Alleviation
of the leg abnormality in zinc deficient chicks by histamine
and by various anti-arthritic agents. J. Nutr. 94:527.

Nishimura, H. 1953. Zinc deficiency in suckling mice deprived of
colostrum. J. Nutr. 49:79.

Oberleas, D., M. E. Muhrer and B. L. O'Dell. 1962. Effects of phytic
acid on zinc availability and parakeratosis in swine. J.
Animal Sci. 21:57.

Oberleas, D., M. E. Muhrer and B. L. O'Dell. 1966. Dietary metal-
compéexing agents and zinc availability in the rat. J. Nutr.
90:5 .

O'Dell, B. L. and J. E. Savage. 1957. Symptoms of zinc deficiency in
the chick. Fed. Proc. 16:394.

O'Dell, B. L., P. M. Newborns and J. E. Savage. 1958. Significance of
dietary zinc for the growing chick. J. Nutr. 65:503.

O'Dell, B. L., J. M. Yohe and J. E. Savage. 1964. Zinc availability
in the chick as affected by phytate, calcium and ethylenediaminete-
tracetate. Poultry Sci. XLIII:415.

Orten, J. M. 1965. Biochemical aspects of zinc metabolism. In Zinc
Metabolism, ed., A. S. Prasad, C. C. Thomas, Springfield,
Illinois, p. 38.

O'Hara, P. J., A. P. Newman and R. Jackson. 1960. Parakeratosis and Cu
poisoning in pigs fed a Cu supplement. Aust. Vet. J. 36:225.

Piras, R. and B. L.'Vallee. 1966. Effect of ultraviolet irradiation
on composition and function of carboxypeptidase A. Biochem.
5:849.

65

Piras, R. and B. L. Vallee. 1967. Procarboxypeptidase A~Carboxypepti-
dase A interrelationship metal substrate binding. Biochem.

6:348.

Piras, R. and B. L. Vallee. 1967. Carboxypeptidase A. Quantum.yields
on ultraviolet irradiation. Biochem. 6:2269.

Plumlee. Ma Pa. De Ra Whitaker. We Ha Smth, Jo Ha Conrad, He Ea Parker
ande. M. Beason. 1960. Phytic acid and unidentified growth
factor reSponse in swine. J. Animal Sci. 19:1285.

Pocker, Y. and J. E. Meany. 1967. The catalytic versatility of erythro-
cyte carbonic anhydrase. Biochem. 6:2269.

Pond,Wé G. 1965. Aspects of zinc nutrition. New York State J. Med.
5:2369.

Prasad, A. S. 1961. Syndrome of iron deficiency anemia, hepatOSphen-
omegaly, hypogonadism, dwarfism and geophagia. In Proceedings
of the Middle East Medical Assembly. American University of
Beirut. May, 1961.

Prasad, A. 3., A. Miale, Jr., Z. Farid, H. H. Sandstead, A. R. Schulert
and W. J. Darby. 1963. Biochemical studies on dwarfism,
hypogonadism and anemia. Arch. of Intern. Med. 111:407.

Prasad, A. S., D. Oberleas, P. wolf, and J. P. Horwitz, with R. Collins
and J. M. Vazquez. Studies on deficiency: changes in trace
elements and enzyme activities in tissues of zinc-deficient
rats. J. Clin. Inv. tg. 46:549.

Quiocho, F. A. and F. M. Richard. 1966. The enzymatic behavior of
carboxypeptidase A in the solid state. Biochem. 5:4062.

Raoult, E.et H. Breton. 1877. Sur la prasence ordinaire du cuvre et
du zinc dans le corps de l'homme. Acad. Sci. 85:40.

Raulin, J. 1869. Etude sur la vegetation. Ann. Sci. Nat. Botan.
11:93.

Reinhold, J. G., G. A. Kfoury and T. A. Thomas. 1967. Zinc, copper
and iron concentration in hair and other tissues. Effect of low
zinc and low protein intake in rats. J. Nutr. 92:173.

Reynolds, J. A. and M. J. Schlesinger. 1967. Conformational state of
ghe subunit of Escherichia coli alkaline phosphatase. Biochem.
33552.

Ritchie, H. D., R. W. Luecke, B. V. Baltzer, E. R. Miller. D. E- Ullrey
and J. A. Hoefer. 1963. Copper and zinc interrelationships in
the pig. J. Nutr. 79:117.

66

Robertson, B. T. and M. J. Burns. 1963. Zinc metabolism and zinc
deficiency syndrome in the dog. Amer. J. Vet. Res. 24:997.

Sampath Kumar, K. S. V., K. A. Walsh, J. P. Bargetz and H. Neurath.
1963. ASpects of protein structure. Proc. Symp. Madras 319.

Savage, J. E., J. M. Yohe, E. E. Pickett and B. L. O'Dell. 1964.
Zinc metabolism in the growing chick. Tissue concentration
and effect of phytate on absorption. Poultry Sci. XLIII:420.

Savage, J. E. 1968. Trace minerals and avian reproduction. Fed.
Proc. 27:927.

Schroeder, H. A., W. H. Vinton, Jr. and J. J. Balassa. 1963. Effect
of chromium, cadmium and other trace metals on the growth and
survival of mice. J. Nutr. 80:39.

Scott, D. A. and A. M. Fisher. 1938. The insulin and zinc content
of normal and diabetic pancreas. J. Clin. Invest. 17:725.

Scott, M. L. and T. R. Zeigler. 1963. Evidence for natural chelates
which aid in the utilization of zinc by chicks. J. Agr. Food
Chem. 1131230

Shikita, M., H. Inano, and B. Tamaoki. 1967. Further studies on
EO-cz-hydroxysteroid dehydrogenase of rat testes. Biochem.
:17 0.

Smith, I. D., R. H. Grummer, W. G. Hoekstra and P. H. Phillips. 1960.
Effects of feeding an autoclaved diet on the development of
parakeratosis in swine. J. Animal Sci. 19:568.

Smith, S. E. and E. J. Larson. 1946. Zinc toxicity in rats. J.
Biol. Chem. 163:29.

Smith, W. H., M. P. Plumlee_and W. M. Beeson. 1962. Effect of source
of protein on zinc requirement of the growing pig. J. Animal
Sci. 21:399.

Snaith, S. M. and G. A. Levvy. 1968. cx-Mannosidase as a zinc depen-
dent enzyme. Nature 218:91.

Spray, C. M. and E. M. Widdowson. 1951. The effect of growth and
development on the composition of mammals. British J. Nutr.
4:332.

Stern, F. E., C. A. Elvehjem and E. B. Hart. 1935. The indesPensabil-
ity of zinc in the nutrition of the rat. J. B101. Chem. 190:347.

Supplee, W. C., G. F. Combs and D. L. Blamberg. 1958. Zinc and
potassium effects on bone formation, feathering and growth of
poults. Poultry Sci. 37:63.

67

Sutton, W. R. and V. E. Nelson. 1937. Studies on zinc. Proc. Soc.
Exptl. Biol. Med. 36:211.

Taylor, D. M. 1961. Retention of zinc65 in the bones of rats. Nature
189:932.

Theorell, H., A. P. Nygaard and R. K. Bonnichsen. 1955. Liver alcohol
dehydrogenase. III. Influence of pH and some anions on reaction
velocity constants. Acta Chim. Scand. 9:1148.

Thiers, R. E. and B. L. Vallee. 1957. Distribution of metals in sub-
cellular fraction of rat liver. J. Biol. Chem. 266:911.

Thomas, G. and A. Eden. 1954. A peculiar nutritional dermatitis in
pigs. Nature 174:187.

Todd, W. R., C. A. Elvehjem and E. B. Hart. 1934. Zinc in the nutri-
tion of the mouse. Amer. J. Physiol. 107:146.

Tucker, H. F. and.W. D. Salmon. 1955. Parakeratosis in swine. J.
Amer. Vet. Med. Assn. 123:217.

Turk, D. E. 1965. Effects of diet on the tissue zinc distribution
and reproduction in the fowl. Poultry Sci. 44:122.

Underwood, E. J. 1962. Trace Elements in Human and Animal Nutrition.
(2nd ed.). Academic Press Inc., New York. Chapters 3 and 6.

Vallee, B. L., H. D. Lewis, M. D. Altschule and J. G. Gibson. 1949.
The relationships between carbonic anhydrase activity and zinc
content of erythrocytes in normal, in anemic and other patho-
logical condition. Blood 4:467.

Vallee, B. L. and H. Neurath. 1954 a. Carboxypeptidase, a zinc metal-
loprotein. J. Amer. Chem. Soc. 76:5006.

Vallee, B. L. and H. Neurath. 1954 b. Carboxypeptidase, a zinc metal-
loenzyme. J. Biol. Chem. 217:253.

Vallee, B. L. and F. L. Hoch. 1955 a. Zinc, a component of yeast
alcohol dehydrogenase. Proc. Nat. Acad. Sci. U.S. 41:619.

Vallee, B. L. and F. L. Hoch. 1955 b. Yeast alcohol dehydrogenase,
a zinc metalloenzyme. J. Amer. Chem. Soc. 77:821.

Vallee, B. L., F3 L. Hoch. S. J. Adelstein and W. E. C. Wacker. 1956.
Pyridine nucleotide dependent metallodehydrogenase. J. Amer.
Chem. Soc. 78:5879.

Vallee, B. L. and W. E. C. Wacker. 1956. Zinc, a component of rabbit
muscle lactic dehydrogenase. J. Amer. Chem. Soc. 78:1771.

Vallee, B. L. 1957. Zinc and its biological significance. A. M. A.
Arch. Industrial Health 16:147.

68

Vallee, B. L. 1959. Biochemistry, physiology and pathology of zinc.
Physiol. Rev. 39:433.

Van Campen, D. R. and P. U. Scaife. 1967. Zinc interference with
copper absorption in rats. J. Nutr. 91:473.

Vincent, J. 1963-1965. Microscopic aspects of mineral metabolism in
bone tissue with special reference to calcium, lead and zinc.
Clin. Orthp. 26:161 (1963). Cited Chem. Abst. 62:2081 (1965).

Vikbladh, I. 1951. Studies on zinc in blood. Scand. J. Clin. and
Lab. Invest. Suppl. 2.

Von Wartburg, J. P., J. L. Bethine and B. L. Van .., 1964. Human
liver alcohol dehydrogenase, kinetic and physzlco—chemical
properties. Biochem. 3:1775.

Vohra, P. and F. H. Kratzer. 1964. Influence of various chelating
agents on the availability of zinc. J. Nutr. 82:249.

Wacker. W. E. C. 1962. Nucleic acids and metals. III. Changes in
nucleic acid, protein and metal content as a consequence of
zinc deficiency in Euglena gracilis. Biochem. 1:859.

wallace, H. D., J. T. McCall, B. Bass and.G. E. Combs. 1960. High
level copper for growing-finishing swine. J. Animal Sci.
19:1155.

Widdowson, E. M. 1950. Chemical composition of newly born mammals.
Nature 166:626.

Wintersberger, E. 1965. Isolation and structure of an active center
peptide of bovine carboxypeptidase B containing the zinc-
binding sulfhydryl group. Biochem. 4:1533.

Worker, N. A. and Magicovsky, B. B. 1961. Effect of vitamin D on
the utilization of beryllum, magnesium, calcium, strontium
and barium in the chick. J. Nutr. 74:490.

Young, R. J., H. M. Edwards, Jr., and M. B. Gillis. 1958. Studies
on zinc in poultry nutrition. 2. Zinc requirement and
deficiency symptoms of chicks. Poultry Sci. XXXVII:1100.

APPENDIX TABLE 1

Zinc Content of Diets by Analysis
Experiment I

w

Level of Zinc in o 5 10 15 20
the Diet (ppm)

 

 

 

 

 

 

 

 

By analysis 0.80 5.08 10.83 15.69 18.91
Experiment II
. Level of Zinc in
the met (ppm) 0 5 7.5 10 12.5 15
By analysis 0.78 4.50 6.80 9.29 12.30 14.30

 

69

APPENDIX TABLE 2

Exp. I-dGain Weight, Food Intake, Bone Ash, Zinc in Bones, Livers and

Kidney, and Copper in.Livers and Kidneys

 

 

 

 

Rat Weight Food Bone Zn in Zn in Cu in Zn in Cu in
No. gain, g. intake ash % bones liver liver kidney kidney
PPm' PPm- PPm PPm PPm
Lot I - Basal diet - 0 ppm Zn
1 11 103 58.7 87.2 78.1 12.8 86.7 24.1
2 10 102 56.3 68.6 90.5 13.4 88.7 26.3
3 24 108 58.5 75.3 79.2 12.6 -- --
4 19 101 57.5 59.3 79.1 10.4 -- --
5 13 113 55.3 75.1 143.7 23.7 88.2 18.7
6 9 100 5701 66.2 8900 1301 II- --

7 23 117 58.3 56.9 75.5 14.3 -- --
8 20 122 54.9 77.4 113.3 21.4 -- --
Mean (16.1) (108.3) (57.1) (70.8) (93.6) (15.2) (87.9) (23.0)
SE 2.1 2.9 0.5 3.5 8.4 1.7 0.6 2.3

Lot II - 5 PPm.Zn
9 84 195 60.3 84.0 88.0 8.6 80.6 16.0
10 65 182 57.3 82.0 86.0 11.8 83.4 20.8
11 68 203 60.7 84.3 76.7 8.5 74.9 20.6
12 95 215 60.0 87.0 66.8 11.6 -- --
13 57 215 59.7 81.9 71.4 9.4 -- --
14 65 194 61.9 90.6 79.2 8.1 -- --
15 62 166 62.6 106.1 81.5 9.9 -- --
16 104 240 59.6 84.1 73.9 9.7 -- --
Mean (75) (201.2) (60.3) (87.5) (77.9) (9.7 (79.6) (19.1)
6.1 8.0 0.6 2.8 2.6 0.5 2.5 1.6

 

70

71
APPENDIX TABLE 2--CONTINUED

 

 

 

 

 

 

 

Rat Weight Food Bone Zn in Zn in Cu in Zn in Cu in
No. gain, g. intake ash % bones liver liver kidney kidney
ppm ppm Ppm ppm ppm
Lot III - 10 ppm Zn
17 134 295 62.5 208.4 71.4 4.3 86.8 17.4
18 161 338 60.6 174.0 69.5 6.9 85.0 16.9
19 141 284 61.3 172.9 69.6 9.2 89.8 16.7
20 123 307 57.9 185.8 79.7 3.9 -- --
21 144 314 59.9 185.2 84.4 6.8 -- --
22 135 282 59.8 180.8 83.4 2.5 -- --
23 160 330 59.2 191.8 89.0 2.6 -- --
24 124 271 59.4 193.3 73.9 8.5 -- --
Mean (140. 3) (302. 6) (60.1) (186.5) (77.6) (5.6) (87.2) (17.0)
SE 5.1 8.4 0.5 4.1 2.7 0.9 1.4 0.2
Lot IV - 15 ppm Zn ‘
25 116 244 59.6 232.4 87.9 7.0 92.3 15.5
26 125 290 59.2 224.7 80.8 5.3 95.6 17.1
27 159 313 59.6 225.5 87.1 10.9 89.6 15.6
28 144 301 60.7 243.4 91.8 7.1 -- --
29 116 273 60.6 256.8 86.7 7.1 -- --
30 147 324 59.9 250.3 76.2 5.5 -- --
31 124 268 61. 7 234. 6 77.0 5.4 -- --
32158 316 609 258 7 80. 7 6.5 -- ..
Mean (135 9) (291.1) (60 3) (238 2) (83 5) (6.8) (92.5) (16.1)
SE 6 9.8 0.3 4. 4 2.0 0.6 1.7 0.5
33 140 289 60.1 254.2 76.3 5.5 80.8 16.7
34 114 261 60.1 249.9 92.2 4.4 89.3 19.4
35 119 271 60.5 279.0 87.5 4.7 88.1 15.1
36 135 284 60.4 253.6 83.1 4.5 -- --
37 118 251 60.7 242.5 76.3 4.3 -- --
38 155 347 61.3 257.3 90.6 8.1 -- --
39 131 279 61. 3 261.5 93. 2 6.6 .. --
40 128 281 61.2 252. 8 79. 9 5.1 -- --
Mean (130.0) (282.9) (60. 7) (256. 4) (84. 9) (5.4) (86.1) (17.1)
SE 4 810.2 0. 2 3. 8 2. 5 0.5 2.7 1.3

 

APPENDIX TABLE 3

Exp. II--Gain Weight, Food Intake, Bone Ash, Zinc in Bones

 

 

 

 

 

Rat 'Weight Food Bone Zn in
No. gain, g Intake, g Ash % Bones
Ppm

 

Lot I - Basal diet - 0 ppm Zn

 

1 14 91

2 19 97

3 24 121

4 22 103

5 17 90

6 14 98

7 17 95

8 20 103

9 24 104
Mean (19.0) (100.2)
SE 1.3 3.1

 

Lot 11 - 5 PP!!! zn

 

1o 73 185 54.6 88.0
11 79 175 57.7 88.1
12 69 166 55.9 86.7
13 84 212 58.5 87.4
14 63 155 57.7 67.3
15 96 208 57.9 69.9
16 86 193 58.6 75.8
17 71 176 55.9 89.5
Mean (77.6) (183.8) (57.1) (81.6)
SE 3.8 7.0 0.5 3.2

 

72

73
APPENDIX TABLE 3--CONTINUED

 

Rat Weight Food Bone Zn in
No. gain, g Intake, g Ash % Bones
PPm

 

Lot III - 7.5 ppm Zn

 

 

 

 

 

18 127 269 58.3 126.7
19 116 253 58.7 140.9
20 125 246 58.6 114.6
21 127 249 58.4 116.2
22 114 240 58.1 134.9
23 136 255 58.5 110.1
24 128 258 56.9 110.5
25 112 235 56.4 148.9
Mean (123.1) (250.6) (58.0) (125.5)
SE 209 308 003 5'2
Lot IV - 10 ppm Zn
26 135 264
27 128 247
28 129 251
29 120 228
30 127 231
31 112 219
32 - --
33 114 230
Mean (123.6) (238.6)
SE 3.2 6.0
Lot V - 12.5 ppm Zn
34 123 247 56.8 232.5
35 136 254 57.8 250.9
36 116 231 54.5 227.1
37 105 211 57.9 251.8
38 121 235 57.9 248.2
39 122 233 56.9 237.0
40 108 212 56.6 221.8
41 131 239 56.3 219.0
Mean (120.3) (232.8) (57.1) (236.0)
SE 3.7 5.4 0.4 .

 

74

APPENDIX TABLE 3--CONTINUED

Rat 'Weight Food Bone Zn in
No. gain, g Intake, g Ash % Bones
PPm

 

Lot VI - 15 ppm Zn

 

42 132 260

43 118 238
44 137 263
45 129 257
46 137 267
47 139 269
48 125 257
49 128 261
Mean (130.6) (259.0)

SE 2.5

 

N

 

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