N-UCLEEC MD AND PROTEIN METABOLISM
EN THE ZINQDEFICIENT PIG

Thesis for the Degree of Ph. D.
MlCHIGAN STATE UNWERSH’Y
PAD KWEN KU
1970

 

 

0-169

Date

This is to certify that the

thesis entitled

NUCLEIC ACID AND PROTEIN METABOLISM
IN THE ZINC-DEFICIENT PIG

presented by

Pao-Kwen Ku

has been accepted towards fulfillment
of the requirements for

__Eh._11._ degree in AnimL-Hu-sba ndry

Umé

 

 

Major professor

 

ABSTRACT

NUCLEIC ACID AND PROTEIN METABOLISM
IN THE ZINC-DEFICIENT PIG

by

Pao Kwen Ku

   
  
 
   
    
   
  
 
  
 

i,; Four trials, involving a total of 56 baby pigs. were

giroted to investigate the effect of dietary zinc levels

\

7% :iTwelve pigs in trial 1 and 16 pigs in trial 2 were taken
IEfigiiheir dams at 3 days of age, and, after a a day adaptation

. It eemipurified dried diet and environmental conditions,

randomly allotted into two equal groups to a 12 or 90

1{}c diet. The pigs were individually pair-fed so that
9ifi4rumption was equal within pairs. Blood samples were i
A iGTrhm the anterior vena cave on 2 occasions (lhth and l“ i
:_i£ftrial 1, and 20th and 28th day in trial 2). After
Eh trial 1 and 28 days in trial 2 the pigs were killed l:

1“ Bf tissues were taken for RNA. DNA and protein F
12-, _ !

‘(gain. feed intake, feed efficiency. serum zinc

~asa serum alkaline phosphatase activity were

 

 

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Pao Kwen Ku

   
  
  
  
  
  
  
   
 
   
  
   
   
  
  
  
  

Igloantly reduced by inadequate dietary zinc. Weight de-
t;d in zinc-deficient pigs before feed intake was reduced.
~.ugkin lesions that developed due to zinc deficiency were
'éfienly observed by the end of the second week. The reduction
gfleight of the adrenals, thyroid, testes. spleen. thymus.
gist: kidneys. pancreas and pituitary was due to zinc

' aflbfioiency and not to reduced feed intake.

g: _ No treatment effects were noted on RNA. DNA and protein

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Elififigeentrations in brain. kidney and spleen. Zinc deficiency
gggreased the concentration of RNA in liver and pancreas and

fig DNA in the thymus. The protein concentration in liver was

ted in zinc deficiency.

‘v biotin) on tissue nucleic acid and protein concentra-
.; All pigs in this trial were fed ad libitum.

geight gain, feed intake and feed efficiency were signifi-
1"z\.:":l"edueed due to zinc deficiency. The incidence of
thtosis and reductions in feed intake and weight gain
improved by adding biotin to the zinc-deficient diet.
i there was a significant biotin plus zinc interaction,

‘flrn an increase in weight gain and gain/feed from

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_‘;‘;;8npplementation of the diets with biotin did not affect
‘f7ufznfll. DNA and protein concentrations in liver, pancreas.
narun and spleen of the pigs receiving either zinc-deficient
¢‘*«‘linc-edequate diets. Zinc deficiency decreased the con-
_¢Intration of RNA in liver and pancreas and of DNA in thymus
Vandipancreas. Liver protein concentration was elevated in
fiidho-deficient pigs. and protein concentration in the thymus

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A-d:til depressed.

' a .» JIn trial h, sixteen week-old pigs were allotted at
— -Néfaadon to # experimental groups to study the effect of dietary
'V’iine levels and growth hormone administration upon in vitro

_" Iiino acid incorporation into protein and upon pancreatic
{fibdxypeptidase activity. All pigs were individually fed.
~ 1 received a zinc-deficient diet ad libitum containing

dip: zine. Group 2 received the zinc-deficient diet, but

 

tepig was injected subcutaneously on each of 12 days with
dang of porcine growth hormone (0. 7 IU/mg). Group 3 re-
731va a zinc-adequate diet containing 90 ppm zinc. with the
l?”k:3 intake limited to the daily mean intake of the zinc-
afloat pigs. Group # received the zinc-adequate diet 5g
jBlood samples were obtained on 2 occasions (initially
.Sftemination of the experiment). After ill- days the
:23killed and slices of medial lobe of liver. pancreas
‘ 41ers incubated with L-leucine-UL-luc for 60 minutes.

‘_ver. pancreas and thymus were assayed for RNA.

 

 

 

 

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Pao Kwen Ku

Land protein concentrations. Pancreatic carboxypeptidase

-_ .-\ _
:gg‘tvity was also determined.
2'; l

2‘.. ' Height gain, daily feed intake, feed efficiency, serum
:; thine concentration and serum alkaline phosphatase activity
[’iffiire significantly reduced by inadequate dietary zinc. Ad-
;?~:;!inistraticn of growth hormone to zinc-deficient pigs failed
:; F‘te stimulate weight gain and feed intake and resulted in no

c‘ effect on serum zinc concentration, serum alkaline phosphatase
Icttvity and the incidence of parakeratosis.

The rate of incorporation of 1” C-leucine into liver

”lipretein was significantly decreased by zinc deficiency and

*';§nps,not improved by prior growth hormone administration.

 
 

' Efidueine incorporation into pancreatic and thymic protein was
fixrffected by zinc deficiency, treated or untreated with
gmswth hormone. Likewise. the treatments had no effect upon
{aynnereatic carboxypeptidase activity. Liver protein. non-
fiiflufiein.nitrogen and liver free leucine levels were higher
d?‘ ‘331 concentration was lower in zinc-deficient as compared

gins-adequate pigs. The DNA concentration in zinc-

 

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Pao Kwen Ku

    

3 concentrations of thymus and pancreas in trial

' pg this was because the experimental period was not

 

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NUCLEIC ACID AND PROTEIN METABOLISM
IN THE ZINC-DEFICIENT PIG

by

Pao Kwen Ku

A THESIS
Submitted to
Hichigen State University

';;iel fulfillment of the requirements
a for the degree of

DOCTOR OF PHILOSOPHY

~Ii jgrgeent‘of.inina1 Husbandry
1970

 

 

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ACKNOWLEDGEMENTS

   

The author wishes to express his sincere appreciation to
. 3m, D. E. Ullrey and Dr. E. R. Miller for their guidance,
" Vlnderstanding and encouragement throughout his graduate program
i I and for their critical reading of this manuscript. Apprecia-
tion is also extended to Dr. R. W. Luecke and Dr. E. P. Reineke
‘1 for their helpful guidance and interest during the author's

f: '. Eraduate program.

Thanks are due to Dr. W. G. Bergen for his interest and

j, ;as_sistance of this study and Dr. w. T. Magee for his assistance

     
 
     
   

"in the statistical analysis.
2. ° 7 Appreciation is extended to Dr. B. H. Nelson and the
shim Husbandry Department for the use of facilities and

-1 'ggnthIs and for financial support throughout the graduate

’ g) Behoepke and Mrs. Rosemary Covert, and department

.‘ifflvfiItIries for their assistance during the author's stay at

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TABLE OF CONTENTS

mnemcnon
BIVIEU OF LITERATURE
A. ZINC'- An essential trace metal

systems

2. Effect on enzymes

3. Effect on hormones

protein metabolism
Panama PROCEDURE

kj‘fia Introduction

. I» General Conduct of Experiments
Ttrflhslieal Analytical Procedures
f" stunt. ical Analys is

AND DISCUSSION

111

- 3. Factors influencing zinc requirements

e. The essential role of zinc in biological

1. Effect on growth and taste acuity

b. Effect on nucleic acid. amino acid and

'11
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29
36
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53
67
67
67
72

85

85
101
115
123
150
161

 

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We showing possible interrelationships
If time. growth. hormone. nucleic acid
in! protein metabolism in the animal

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LIST OF TABLES

Composition of experimental diet. 68

Effect of dietary zinc level on weights and
feed consumption of zinc-deficient and
control baby pigs - trial 1. 86

 

‘. 3 Effect of dietary zinc level on serum alkaline
phosphatase activity and serum zinc level of
zinc-deficient and control baby pigs -

trial 1. 88

  
   
   
  
    
    
   
    
   
    

, ' 15 Effect of dietary zinc level on organ weights
. of zinc-deficient and control baby pigs -
Iv: trial 1. 90

5 Effect of dietary zinc level on dry matter,
,p . ether extract. protein. RNA and DNA con-
. j~jj centration of liver - trial 1. 92

" ig‘é‘ Effect of dietary zinc level on dry matter.
". ether extract. protein. RNA and DNA concen-
~V§‘ tration of thymus - trial 1. 95

Effect of dietary zinc level on dry matter.
ether extract, protein, RNA and DNA con-
‘centration of pancreas - trial 1. 96

Effect of dietary zinc level on dry matter,
ether extract, protein, RNA and DNA con-
centration of kidney - trial 1. 98

Effect of dietary zinc level on dry matter,
ether extract, protein, RNA and DNA con-
v'eentration of spleen - trial 1. 99

h;, Effect of dietary zinc level on dry matter.
ether extract. protein, RNA and DNA con-
. sentration of brain - trial 1. 100

_ Effect of dietary zinc level on weights and

feed consumption of zinc-deficient and
xefifltrol baby pigs - trial 2. 102

vi

 

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Effect of dietary zinc level on serum alkaline
phosphatase activity and serum zinc level
of zinc-deficient and control baby pigs -
trial 2. 10h

 

. Effect of dietary zinc level on organ weights
; of zinc-deficient and control baby pigs -
3 trial 2. 105

_ 1“» Effect of dietary zinc level on serum proteins
",. of zinc-deficient and control baby pigs -
"" trial 2. 107

; 15 Erfect of dietary zinc level on dry matter.
‘. ether extract, protein. RNA and DNA concen-
3’ tration of liver - trial 2. 108

‘ 16 Effect of dietary zinc level on dry matter,
g' ether extract, protein. RNA and DNA concen-
tration of thymus - trial 2. 109

   
   
 
   
 
   
 
   
 
 
  
 
 
  

1? Effect of dietary zinc level on dry matter,
ether extract. protein, RNA and DNA concen-
tration of pancreas - trial 2. 111

._ 18 Effect of dietary zinc level on dry matter,
ether extract, protein, RNA and DNA concen-
tration of spleen - trial 2. 112

J. 19 Effect of dietary zinc level on dry matter,
ether extract, protein, RNA and DNA concenh
tration of kidney - trial 2. 113

. f;35‘ Effect of dietary zinc level on dry matter.
' ether extract. protein. RNA and DNA concen-
tration of brain - trial 2. 114

.i Effect of dietary zinc and biotin levels on
'If: weights and feed consumption of zinc-deficient
“ and control baby pigs - trial 3. 116

pEffect of dietary zinc and biotin levels on

protein. RNA and DNA concentration of
liver - trial 3. 118

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LIST OF TABLES (Cont.)

Effect of dietary zinc and biotin levels on
protein. RNA and DNA concentration of
thymus - trial 3.

Effect of dietary zinc and biotin levels on
protein, RNA and DNA concentrations of
pancreas - trial 3.

Effect of dietary zinc and biotin levels on
protein, RNA and DNA concentration of
spleen - trial 3.

Effect of dietary zinc and growth hormone
treatments on weights and feed consumption
of zinc-deficient and control baby pigs -
trial 4.

Effect of dietary zinc anc growth hormone
treatments on serum alkaline phosphatase
activity and serum zinc level of zinc-
deficient and control baby pigs - trial 4.

Effect of dietary zinc and growth hormone
treatments on serum proteins - trial 4.

Effect of dietary zinc and growth hormone
treatments on protein. RNA and DNA concen-
tration of liver - trial 4.

Effect of dietary zinc and growth hormone
treatments on protein. RNA and DNA concen-
tration of thymus - trial 4.

Effect of dietary zinc and growth hormone
treatments on protein. RNA and DNA concen-
tration of pancreas - trial 4.

Effect of dietary zinc and growth hormonf
treatments on the incorporation of UL— l"C
leucine into proteins of liver. thymus and
pancreas slices in vitro - trial 4.

Effect of dietary zinc and growth hormone

treatments on pancreatic carboxypeptidase
activity - trial 4.

viii

121

124

127

130

132

134

135

138

142

 

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‘7 3.3. "reflect of dietary zinc and growth hormone
' ’_ ' treatments on liver total nitrogen. protein
nitrogen and non-protein nitrogen of zinc-

' 3,, deficient and control baby pigs - trial 4. 145

Effect of dietar zinc and growth hormone
treatments on iver .4 -amino nitrogen and

free amino acids of zinc-deficient and

control baby pigs - trial 4. 147

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LIST OF APPENDIX TABLES

2252
‘ lineral mixture used in experimental diet. 190
‘sa, Vitamin mixture used in experimental diet. 191

Solutions for—<-amino nitrogen determina-
tion. 192

’ Composition of amino acid in Krebs-Ringer
1. ‘ buffer solution. 193

; ;~jx Gosposition of Krebs-Ringer buffer solu-
. “ tion. pH 7.4. 194

I Liquid scintillation counting solution. 195

 

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I . INTRODUCTION

_ Hetallic zinc was not discovered as early as many of the
J ' Other metals. probably because zinc does not occur in an un-
culbined state in nature. Zinc compounds. and zinc proteins
in Particular. are colorless in biological systems. in con-
SPMt to the highly colored iron and copper containing pro-
1@1113 and enzymes. and therefore. zinc did not attract as

. ‘ 3‘30}: attention from the early research workers as did the

~ P Eiffher metal compounds.

1 However, as early as 1869. Raulin showed zinc to be
.¥“§isessary for the growth of Aspergillus _3523, and in 1921,
3mm and Valdesco reported a relatively large amount of

X «3&0 in the prostate. testes and semen of stallions. They
.h‘: 4* iested a possible role for zinc in cell multiplication.
’Isan:

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conclusive evidence indicating that zinc is a dietary

  

Ontial for mammals was not presented until 1934 by Todd

L
‘*~.greuth of the rat. Later. Tucker and Salmon (i955) ’ A J.‘
‘ a}; that parakeratosis of swine, first described by Kern-

 

 

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The unequivocal requirement of zinc for swine has been

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(5 igsported by Beardsley and Forbes (195?). Lewis 23 2;. (1957).

.. {gesture e_t_ g. (1956), Ritchie £3 9;. (1963). Shanklin gt a_.1_.
if:'(1968) and Miller 33 El. (1968). The essentiality of zinc

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for poultry has also been studied and reported by O'Dell £3

E. g;. (1958). Roberson and Schaible (1958), Kratzer gt El°

". ."(1958) and Fox and Harrison (1964). Zinc has been reported
f to be indispensible for several other species including the
«7 if cal: (Miller and Miller, 1962; Ott 93 §_1_.. 1965; Mills a
3; 31.. 1967), sheep (Ott, a gin 1964). goat (Miller a g”
i ‘1964). dog (Robertson and Burns. 1963). monkey (Macapinlac
..:.322§.§l°' 1967; Barney gt al.. 1967) and man (Prasad gt 31.,
"_,}_i1:§961) .

Ԥ.?fi}: A large amount of work has been done to define the
{Z’Eégnificant biochemical roles played by zinc in mammalian

§f5jg§abolism. Several workers have reported that the effect

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Since zinc-deficient baby pigs usually show a reduction
growth rate before appetite is severely depressed. it
.:£;I- be of interest to investigate whether zinc is related
_:§£§ hormonal function. A growth retardation accompanied by
1‘:9~ptimal function of the anterior pituitary (Prasad 22 al..
| 1963 a.b; Sandstead _e__t a_1.. 1967) related to a deficiency of
-Eéne has been reported. A higher concentration of zinc in
: ¥§!! pituitary. as compared with other organs. has also been
:geported by Hillar g §_1_. (1961).

g; y.., Therefore. a study was undertaken to: (1) collect data
tAgnthe effect of dietary zinc levels on tissue nucleic acid
.protein concentrations in baby pigs; (2) determine the
sieot of dietary zinc levels on the rate of incorporation
w§;.1“c-labelled leucine into tissue proteins in zitgg; (3)
ethe effect of dietary levels of zinc on the response

’3 w9§he baby pig to parenteral growth hormone administration.

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II. REVIEW OF LITERATURE

  

A. ZINC — An essential trace metal.

Since 1869 when Raulin determined that zinc was needed
for growth of the fungus Aspergillug niger. the presence of
zinc in numerous plant and animal tissues has been established
(Lechartier gt gt.. 1877: Bertrand and Vladesco, 1920; Lutz.
1926). However. indisputable evidence that the element zinc
was necessary for growth of the rat was not reported until
1934 by Todd gt gt. Day and Skidmore (1947) later demonstrated
that mice were greatly retarded in their rate of growth and
failed to survive when fed a zinc-deficient diet containing
0.3 ppm of zinc. Nishimura (1953) found that colostrum was
necessary to prevent disorders in newborn mice which were
similar to those reported for zinc deficiency. The adminis-
tration of colostrum or inorganic zinc prevented these dis-
orders. It was suggested than colostrum has a much higher
content of zinc that later milk, and that deprivation of
colostrum leads to manifestation of zinc deficiency in suck-
ling mice.

Since Kernkamp and Ferrin (1953) first described the
characteristic skin lesion of parakeratosis in swine. Tucker

and Salmon (1955) and Luecke gt gt. (1956) reported that zinc

 

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could prevent parakeratosis. and thus was an essential mineral
for the pig. The indispensibility of zinc for normal growth
of baby pigs was also observed by Roberts gt gt. (1962).
Hoekstra gt gt. (1967). Shanklin gt gt. (1968) and Miller gt
gt. (1968). The importance of zinc in many other species has
since been extensively studied by many workers.

O'Dell and Savage (1957a,b) reported zinc was required
for growth and various other functions in chicks. Using
plastic-coated or glass equipment and distilled drinking water.
Roberson and Schaible (1958) fed a purified diet to chicks and
were able to demonstrate a zinc requirement for growth.
feather development and maintenance of normal skin condition.
Other workers have also reported that zinc is required for

the chick (Edwards gt gt.. 1958; Norris gt gt.. 1958; Morrison
and Sarett. 1958). In turkey poults. Supplee gt gt. (1958)
reported that zinc is required for rapid growth, feather
development and prevention of a high incidence of hock dis-
orders. The growth rate of turkey poults was retarded when
fed a zinc-deficient diet over a 3 week period (Vohra and
Kratzer. 1968). ’

That sine is a dietary essential for Japanese quail was
demonstrated by Fox and Harrison (1964) when they omitted

zinc from a purified soybean protein diet, and the young

quail developed a severe deficiency syndrome by 4 weeks of age.

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Blamberg gt gt. (1960). Kienholz gt gt. (1961) and Heth
gt gt. (1966b) all reported that eggs from hens receiving a
low zinc diet had lowered hatchability. Impaired skeletal
development of the embryos and general weakness was observed
in the birds that did hatch.

That congenital malformations of fetuses develop when
adult rats receive a purified diet low in zinc was observed
by Hurley and Swenerton (1966) and Hurley gt gt. (1968).

Reduced growth. food consumption, serum alkaline phospha-
tase activity and development of a non-specific dermatitis
due to zinc deficiency in calves have been reported by Ott
gtg. (1965). Miller and ‘Miller (1962). Miller (1967),
Killer gt gt. (1965) and Mills gt gt. (1967). Similar signs
of zinc deficiency in lambs and goats have been reported by
Ott gt gt. (1964) and Miller gt gt. (1964).

Robertson and Burns (1963) demonstrated that feeding
dogs high calcium diets. low in zinc. resulted in marked
emaciation. keratitis. retardation of growth and lowered
plasma zinc. Macapinlac gt gt. (1967) reported that zinc
deficiency was produced in the squirrel monkel (Saimiri
scitteus) by feeding a low zinc diet. Barney gt gt. (1967)

_ have indicated that parakeratosis of the tongue in the zinc-

deficient squirrel monkey was a unique histopathologic

lesion.

A decreased concentration of zinc in the testes and an -

 

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1 impairment of glucose tolerance was noted in Chinese hamsters
fed a low zinc diet for three weeks prior to IP or IV glucose
injection (Boquist and Lernmark. 1969).

Graham and Telle (1967), in zinc retention studies with
rabbits. found that gains in body weight were 40 percent
less in rabbits fed a low zinc (25 ppm). high calcium diet
than in rabbits fed a high zinc (54 ppm). high calcium diet.
The hair of the zinc deficient rabbits was short and rough as
compared to the control group. They found that the percentage
of orally-administered Zn65 retained by rabbits fed the low
zinc ration was greater than by rabbits fed the high zinc
ration.

A naturally occurring zinc deficiency in human beings
in Iran and Egypt has been reported and reviewed by Prasad
gt gt. (1961) and Prasad (1966).

The importance of zinc to many other species. e.g..
horses. ducks. cats and geese. has not been reported. However.
in view of the essentiality of zinc for the species mentioned
previously. zinc can be assumed to be an essential nutrient

for all animals.

    
   
 
 

 

3. Factors influencing zinc requirements.
There are many factors influencing the zinc requirements
,ef’animals. Included among these are species. age. sex. the

.vghenical form of zinc. combinations in which the metal occurs

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and the nature of the diet.

Smith gt E- (1958) studied the zinc requirement of grow-
1118 swine receiving a Drackett C-1 protein which was prepared
from soybean meal. The basal ration contained 0.66 percent
calo ium. 0.47 percent phosphorus and 16 ppm zinc. They found,
based on growth rate. feed efficiency and freedom from
incidence of parakeratosis. that the zinc requirement was 46
PP“.

In contrast. the zinc requirement for growth of calves
lies somewhere between 3 and 46 ppm in the whole dry ration
as reported by Miller and Miller (1960). Ott e_t_ gt. (1964)
ObserVed that the requirement of lambs lies between 3 and 18
Pm zinc for normal growth. O'Dell gt at. (1957a. 1958) re-
P°rted that the zinc requirement of chicks receiving a washed
8Oybean protein basal diet. which contained 1.6 percent cal-
ci'lul. 0.7 percent phosphorus and 15 ppm zinc, was 35 ppm.
This finding has been substantially confirmed. when chicks
"are fed a soybean protein diet. by Moeller and Scott (1958) ,
ac’1‘1‘1son and Sarrett (1958) , Roberson and Schaible (1958) and
INS g a_1. (1958).

Miller _e__t al. (1958) reported that the minimum dietary

 

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‘h‘d Yohe (1960). in their study of the zinc requirements of

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ppm zinc was the minimum requirement for growth and health
1n,'blnis species. Concerning the requirement of young mice.
Day' sand Skidmore (1947) estimated that a diet containing no
more than 0.3 ppm zinc was necessary to produce zinc deficiency.
This value was much less than for the other species mentioned
nrewriously. but these workers apparently did not consider the
comrbzribution of zinc from the galvanized cages which were
'useui.

Sex differences in the utilization of minerals have also
been observed. Swenerton and Hurley (1968) found that female
rtrts fed diets containing 40 ppm of zinc grew and developed
normally while males receiving 60 ppm of zinc exhibited a
testicular degeneration. More severely reduced growth rates.
Serum zinc concentrations, serum alkaline phosphatase activities.
‘1131 increased incidence and severity of parakeratosis developed
3111 male baby pigs as compared to females (Miller gt gt.. 1969).
"*1°n both sexes were fed diets containing less than 45 ppm
°f Zinc. Liptrap (1969) reported that 26.1 percent of the
b°firs and gilts fed diets containing less than 30 ppm of zinc
dQVOIOped parakeratosis while only 13 percent of the barrows
1‘7010ped parakeratosis. All these data indicate that sex
differences affect the zinc requirement.

Apparently zinc requirement decreases with increasing

‘8‘.

  

Hills fl gt. (196?) reported that the rate of appearance

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of clinical lesions of zinc deficiency was greatly reduced.
and. they developed later in older calves (6 to 9 months old)
than in younger ones. Miller fl a_l_. (1968b), using young
calves. studied zinc absorption. Their results indicated
that Zi-month-old calves absorbed a greater percentage of
ingested zinc from a zinc-adequate diet than did 435-month-old
calves; however, no difference in absorption of zinc from a
zinc—deficient diet was observed. Hoekstra e_t_ gt. (1967)
and Pond and Jones (1964) determined that the only zinc de-
ficiency symptom in gestating gilts fed a low zinc diet was
a. slight reduction of the growth rate. but parakeratosis was
PrOduced if growing pigs were fed this diet. Roberts gt a_l.
(1962) and Henning (1965) , using similar diets for gestating
gilts. found no signs of a deficiency. It has been noticed
at the Michigan State University Experimental Station that
parakeratosis is difficult to produce if the initial weight
°f the pigs is above 15 to 20 kilograms.

The most frequently implicated and pronounced factors
influencing the zinc requirement of animals are other dietary
1t‘igl‘eclients which interfere with the utilization of zinc.
That dietary calcium level affects zinc requirement is the
he‘st known and one of the earliest discovered factors. In
1955- Tucker and Salmon observed that the addition of lime-

3
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creased the incidence and severity of parakeratosis. This
antagonistic relationship between calcium and zinc was con-
firmed by many workers (Luecke g a” 1956, 1957: Hoekstra
e_t_ 21.. 1956; Newland gt a” 1958; Stevenson and Earle.
1956; Bellis and Philip, 1957: Lewis e_t gt.. 1956, 1957;
Hoefer gt g” 1960; and Berry gt fl” 1961). All reported
that raising the dietary calcium level above one percent
significantly increased the incidence and severity of zinc
deficiency as compared to diets containing less than 0.8 per-
cent of calcium. Since these effects were completely over-
come by feeding. or injecting adequate zinc. it was suggested
that high calcium intakes interfere in some way with zinc
utilization and raise zinc requirements. Newland a g. (1958)
and Hansard and Itoh (1968) observed that elevated dietary
calcium increased endogenous fecal zinc excretion in pigs.
This finding was also reported by Heth g gt. (1966a) in the
rat. However. not all workers agree. Forbes and Yohe (1960)
1‘elmrted that the apparent absorption of dietary zinc and
1‘" urinary excretion by rats were not affected by increased
alcium. Berry gt a. (1961) reported that high calcium
‘dded to a low zinc diet for the pig caused reduced blood.
Dlaa'ln and tissue 65Zn activity; however, high calcium
Q"Idea to a zinc-adequate diet increased blood and tissue zinc
"°tiv1ty. That increasing dietary calcium in zinc-adequate

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by IIloyd and Bell (1958). Henning (1965) stated that in
some cases calcium and zinc effects were synergistic and in
other cases antagonistic. Spencer gt gt. (1965) did not find
anqr calcium-zinc antagonism during low and high calcium in-
taJses in man. Kirchgessner gt gt. (1960) examined calcium and
ziJats relations in balance experiments with growing pigs and
demonstrated that zinc retention at a constant zinc level of
the feed was not decreased but increased by the higher dietary
calcium levels. It was suggested by these workers that there
13 inc interaction between calcium and zinc in the gastro-
ithestinal tract. Morgan gt gt. (1969) reported that high
<11etary calcium reduced the zinc concentration of the liver
aumi decreased serum and small intestine alkaline phosphatase
activity in swine; however. increasing dietary calcium did
11°t affect zinc retention.

The inconsistency with which calcium affects zinc
Illetabolism is not understood completely. but the results ob-
talined by Byrd and Matrone (1965) offer a tentative explana-
1510:1. They reported that the detrimental effect of high
‘luletary calcium on zinc absorption was dependent on the
13I'Iiaence of phytate. They further demonstrated that high
‘3“161un enhanced the complexing of zinc with phytate at low
:11“: levels but decreased the complexing at high zinc levels.

The effect exerted on zinc absorption by phytate is well

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documented. O'Dell and Savage (1960) demonstrated that
phyt ic acid complexed with casein decreased the availability
of zinc to growing chickens. Green gt gt. (1961). using
barrows. studied phytic acid's effect on 65Zn uptake. They
found that the addition of phytic acid to diets caused an
increase of fecal 65Zn excretion. With growing pigs. Oberleas
gt _a._]_.. (1962) showed that the addition of phytic acid to the
diet produced symptoms of parakeratosis and growth depression.
O‘Dell fl a_l. (1964) studied the effect of phytic acid. cal-
cium and ethylenediamine tetraacetate on zinc availability.
They found that the addition of phytic acid to casein-gelatin
diets decreased the availability of zinc, and excess calcium
in the absence of phytate or. in the presence of adequate
zinc. had no deleterious effect on growth; but in the presence
of Phytate it aggravated the zinc deficiency. It was suggested
that a calcium-phytate-zinc interaction existed which de-
°reased zinc availability. Thata decreased zinc availability
3’98u1ts from phytic acid addition to diets containing either
efisein or crystalline amino acids was further demonstrated
by Likuski et al. (1964). Maddaiah fl gt. (1964) observed in
£9 m studies that a more stable zinc-phytic acid complex
he formed in the physiological pH range than was the case
r°r Several other divalent cations. The relative stability

at this cation complex is shown in the following order:

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These observations help explain the results of their
feeding experiments with chicks fed a diet containing 10 ppm
zinc with 0.25 percent added sodium phytate. The weight gain
of the phytate-supplemented birds was significantly lower
than the birds not fed phytate. They suggested that phytate
interfered with the absorption and utilization of the zinc
by forming a zinc-phytate complex. Lease (1967) also using
an _i_n Egg digestion method. found an insoluble, non-dialyzable
Ca-Mg-Zn-phytate complex formed from oil seed meals at chicken
intestinal pH and the zinc in this complex had low availability
for the chick. Lease g E- (1968a) further observed that
Venezuelan sesame meal and soybean meal contained two frac-
tions. an insoluble Ca-Mg-Zn-phytate complex fraction and a
8<7filuble fraction. The inclusion of the soluble fraction from
eilther Venezuelan sesame meal or soybean meal significantly

increased 65Zn uptake. It was postulated that the soluble
I"l'action of Venezuelan sesame meal contained precursors of
substances which formed a more stable bond with zinc during
the digestion process than did phytate; thus zinc was made
a'Vfillable from this complex. Soybean meal also contained a
anbBtance which could render the zinc of an insoluble phytate
°°hp1er available is 11312.
The influence of protein source on zinc requirements has

3°11 reported by many workers in the late 1950's and early

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1960's. O'Dell and Savage (1957) briefly reported that zinc
in soy protein was less available for the chick than that in
animal protein. Morrison and Sarett (1958) showed that when
chicks received either soy. or casein and gelatin. as the
protein source. adding zinc to the soy diet resulted in in-
creased growth rate. That the zinc requirement of growing
pigs fed an isolated soybean protein semipurified ration was
higher than when fed casein was demonstrated by Smith gt gt.
(1961). Smith et al. (1962) later reported that soybean
protein diets not only increased zinc requirement and enhanced
parakeratosis in the growing pigs; but pigs receiving milk
Protein with an even lower zinc content developed no such
Bylllptoms. Zeizler fl gt. (1961) reported that chicks fed a
Purified casein diet had a zinc requirement of 12 to 14 ppm;
however. 2? to 29 ppm of zinc were required when chicks were
fed an isolated soybean protein diet. During that time. they
hYPOthesized that soybean protein appears to possess a
8Petific factor or property increasing the requirement of zinc.
and casein may contain a specific factor or property in-
creasing the efficiency of utilization of zinc. Several
w°rkers have also investigated the effect of soy. casein and
988 white proteins on zinc requirement. other reports of a
hisher zinc requirement with soy protein as compared to
“33m or egg white protein have been presented by Forbes (1964)
in the rat and Neilsen e_t_ a_l_. (1966a) in the chick.

 

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16

  

Edwards (1966) studied the effect of protein source on
the absorption of 65Zn by the chick. It was found that
approximately 16 percent of an oral dose of 652m was absorbed
by chicks receiving a casein-gelatin diet; however, only 9
percent of the dose was absorbed by chicks on an isolated

65

soy diet. Zn was also given intraperitoneally. In this
instance. there was only a slight difference in retention
between chicks fed the different diets. Their results would
appear to indicate that soybean protein is able to reduce the
availability of free zinc for absorption. Shanklin fl gt.
(1968) reported that baby pigs fed a casein diet had a zinc
requirement between 14 and 20 ppm, which was less than one-
half the requirement reported by Smith gt gt. (1961) for pigs
fed soy protein.

It is well established that the source of protein or
alino acids in a zinc-deficient diet for chicks or poults
deBcts zinc availability (Morrison and Sarett. 1958;
110filler and Scott. 1958; and O'Dell and Savage. 1960). More
recently, Nielsen g Q- (1966a) studied the effect of iso-
lated soybean protein, casein hydrolysate and egg white as
dietary amino acid sources in chick diets with varying zinc
°°htent. They found that all diets without supplementary
ting caused severe zinc deficiency as assayed by growth rate.
l"min-11 growth was greater with the soy protein diet than

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the casein hydrolysate or egg white diets, but no effect on
the zinc content of heart, liver or muscle was observed. To
reach a plateau in zinc concentration in the tibias or femurs
of the chick. no ppm more zinc was needed in the soy protein
diet; furthermore. addition of phytic acid in amounts com-
parable to those in soy protein to egg white and casein
hydrolysate diets caused little or no depression in zinc
availability and caused no leg abnormality. They postulated
that soy protein contained a complicating factor other than‘
phytic acid affecting zinc metabolism. Coleman 23 al. (1969)
found that addition of up to 2 percent dietary arginine to
the basal diets (containing 1.2 to 1.66 percent arginine)
hastened the development of leg abnormalities and increased
the severity of zinc deficiency symptoms of chicks fed either
968 White or isolated soy protein as the amino acid source.
It was suggested that high dietary levels of arginine have an
Elm‘asonfmtic effect on zinc metabolism: however, the
metwarden is still unlmown.

III view of these facts, a general statement concerning
th° availability of zinc in different sources of protein or
“MO'acidg can be made. Zinc availability is apparently
“tf‘¢ted.by phytate and/or other complicating factors rather
tn“ the source of protein or amino acids m s3.

Therefore, many compounds have been added to diets to
J ‘7 and overcome the influence of phytate on zinc availability.

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18

Kratzer e_t_ a_l. (1959) observed that turkey poults, fed an iso-
lated soybean protein diet, recovered from zinc deficiency
when small amounts of ethylene diaminetetracetic acid (EDTA)
were fed in the ration. Forbes (1961) using rats and Smith
e_t_ al. (1962) using pigs, studied the effect of EDTA on the
availability of zinc in diets containing phytate. They ob-
served that addition of EDTA to the diet improved the availa-
bility of zinc. Davis Q a_l. (1962) also demonstrated that
chicks receiving an isolated soy protein diet supplemented
with EDTA were superior in daily weight gain. The authors
stated that EDTA is a strong chelating agent and that as
such could form EDTA mineral complexes with zinc which was
bound in the soy protein. The EDTA mineral complexes would
then be available for normal absorption. Other reports of

8 beneficial effect of EDTA upon the availability of zinc in
diets were published by O'Dell _e_t fi° (i964) and Nielsen g
9L1. (1966a.b) in the chick, Vohra and Kratzer (1966) in the
turkey and Oberleas g 5;. (1966) in the rat.

Green g a_l_. (1961) using barrows, studied the influence
°f EDTA on zinc utilization. They found that addition of
EU“ '50 a soy protein diet affected utilization of orally
wmistered 652n by decreasing fecal excretion and increas-
1n; 11‘Ter storage. as well as increasing the urinary excre-

tion 01‘ 652m. Nielsen £1; E- (1966b) reported that in addition

 

 

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19

to EDTA. other synthetic chelating agents, ethylene-N-N'-
diacetic acid (EDDA) , hydroxyethylethylenediamine triacetic
acid (HEDTA) , diethylenediaminepentaacetic acid (DTPA). and a
natural chelating agent, cysteine, alleviated all zinc de-
ficiency symptoms. while N,N-di [Z-hydroxyethyl] glycine Na
salt (DHEG); imino diacetic acid salt monohydrate (IDA),
[ethylenebis(oxyethylenenitrilofl tetraacetic acid (EBONTA).
xanthurenic acid, kynurenic acid, anthranilic acid. glutamic
acid. cystine and tryptophan had no effect on the deficiency
symptoms.

More recently, Suso and Edwards (1968) studied the
effects of feeding different levels of EDTA and other chelat-
ing agents. including DTPA, ethylenediamine [di(o-hydrosyphenyl-
acetic acidfl (EDDHA). [(Z-hydroxyethylimino) diacetic acid]
(HEIDA). 1.2,cyclohexanediaminotriacetio acid (CDTA) or
ethJflenediaminetetrapropionic acid (EDTP) on the absorption
°3 600°. 59Fe, 5am and 65Zn by chickens receiving a corn-soy
diet, They studied levels of EDTA from O to 0.165 percent of
the diet, and found maximal absorption of 65Zn at a level of
°~125 PercentEDTA. Effects of other chelating agents on 65zn
ab8°rpt ion were inconsistent.

V0hra and Gonzales (1969) feeding turkey poults a puri-
tied casein diet, observed EDTA not only improved the availa-
bility of the zinc already present in the diet but also that

o: the supplementary zinc. Simultaneous administration of

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65211. 54pm and EDTA into turkey intestinal segments resulted
in the finding that EDTA preferentially stimulated the ab-
sorption of zinc over manganese.

Powell fl al. (1967) found no benefit of EDTA additions
to the diet of goats or calves: this was probably because
phytate was hydrolyzed by rumen microorganisms which made zinc
more available (Raun _e_t al. , 1956; Tillman fl al.. 1958;
Miller fl al., 1962). Miller (1967) also reported that soy-
bean protein effectively reduced apparent net 65Zn absorption
in calves only when introduced directly into the abomasum; no
significant reduction in 65Zn absorption was observed when
soybean protein went to the rumen. It was suggested that the
Zinc complexing constituent in soybean protein is inactivated
1n the rumen. Hill (1970) studied the effect of EDTA on the
aJDBOI‘ption of zinc from isolated duodenal segments of the
chick E _vi_vg. He found an increase in absorption of zinc
“911 the segment was preincubated with 0.2 ml of 0.1 M EDTA
t°1' 30 minutes and then washed out with 30 ml of deionized r
“1291'- as compared to a control which was incubated with 0.2
III. of deionized water. The resulting 65Zn absorptions in
Dame“? were control 27.? and EDTA preincubated 73.5. It
h“ a“Ssested that EDTA has some role in the intestine other
than 8imply chelating zinc.

sO‘P‘eral workers have examined other compounds or treat-

 

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21

  

merits in an attempt to improve the availability of zinc in
the diet. Forbes (1961, 1961+) demonstrated that the addition
of lactose to the diet has some benefit in overcoming the
zinc-phytate interaction. Kratzer _e_t g. (1959) reported
that autoclaving soy protein at 120°C for 30 minutes sig-
nificantly increased the availability of zinc for the turkey
poult. They suggested that the zinc-complexing factor present
in the soy protein was destroyed. Similar effects from
autoclaving the diet were observed by Smith g E- (1962).
Smith 33 i1, (1960b) and Nielsen g §_1_. (1966a). Recently,
Vohra and Kratzer (1966) studied the effects of phosphate on
Zinc binding. and found that in turkey diets containing 15
Ppm zinc. growth was superior when phosphorus was supplied
by SOdium hexametaphosphate, sodium tripolyphosphate, sodium
3°15. Pyrophosphate or sodium orthophosphate as compared to
dicalcium phosphate and monosodium or monocalcium phosphate.
Moreover. dietary zinc was made more available by adding
EDTA and DTPA than the phosphate compounds.

The effects of other minerals on zinc metabolism have
b9“! investigated by many workers. Some degree of protection
GEM-mt parakeratosis in weanling pigs due to zinc deficiency
"“ °ffered by dietary copper supplementation according to
Boater e_t_ 5;. (1960) and Ritchie e_t_ 13;. (1963). Kirchgessner

‘m weBer (1963) demonstrated that growing pigs receiving a

 

 

 

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diet supplemented with an extremely high level of copper
sulfate had an increased retention of the metal ions Fe3+,
2112+. Mn2+ and C02+. They suggested that the copper ion has
a higher complex association constant with macromolecular
structures than other metal ions. These metal ions are dis-
placed from macromolecular complexes by hydrated copper ions
and thus can be more efficiently absorbed and retained. How-
ever. Gipp Q a. (1967) could find no conclusive evidence
for any copper-zinc interaction. Miller gt fl. (1968) were
unable to demonstrate any benefit of dietary copper supple-
mentation in preventing or alleviating the consequences of
zinc deficiency in weanling pigs. No beneficial effect of
copper upon zinc utilization was reported by Hoekstra (196%
and O'Hara Q g. (1960).

Zinc-copper antagonism does exist; however, excess
°°PPGr does not affect zinc deficiency as severely as an
excess of zinc aggravates copper deficiency. Hill and Matrone
(1962) demonstrated a marked Zn-Cu antagonism when copper was
1"Hum-1'13. Feeding chicks with a low copper (0.66 ppm) diet
°Ontain1n8 24 ppm zinc, they found that the copper deficiency
‘nptoms were aggravated as the level of dietary zinc was
increased. Addition of 0.04 percent zinc to the diet, pro-
dm’“ Severe anemia but in the presence of 10 ppm of copper

th‘huloglobin level was normal. Mortality was decreased.

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23

  

More recently. O'Dell (1967) reported that with chicks fed a
low-copper diet, as the zinc level in the diet was increased
without copper supplementation. the growth rate was depressed,
and the mortality increased. In the presence of added copper
the higher levels of zinc had no detrimental effect. Further-
more, VanCampen (1969) found when both higher levels of copper
and 65Zn were simultaneously administered into ligated rat
intestinal segments that the subsequent absorption of 65Zn was
depressed. No depression in the subsequent absorption of
652n occurred when it was given intraduodenally and copper was
given intraperitoneally. This finding led to the suggestion
that a mutual antagonism apparently took place either in or
on the intestinal epithelium. Vohra and Heil (1969) using
turkey poults, also confirmed an antagonistic relationship
between zinc and copper.
Reports on the influence of cadmium on zinc metabolism

have been published by many workers. Parizek (1960) . Gunn

93 fl. (1961) and Supplee (1963) showed that the element
°5dm1um is antagonistic to zinc for rats and chicks. Supplee 1‘
(1963) reported that cadmium increased the severity of zinc . j
qofi“lancy in chicks. but this could be overcome by large
qmntlties of zinc. Gunn _et a_l. (1961) and Parizek (1960)

°b3°rVed that cadmium produced a rapid necrosis of the testes

 

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and accessory sex glands in rats, which could be prevented

by massive doses of zinc acetate; and Parizek postulated

that cadmium disturbed the physiological function of zinc and
not the absorption and transport of zinc.

In contrast to these previous reports. Powell g al.
(1967) reported that 350 ppm cadmium greatly reduced the ab-
sorption of zinc from the gut of ruminants but did not alter
tissue retention of zinc. Furthermore, Bunn and Matrone
(1966) reported that cadmium treatment caused an increase
in liver and testes zinc concentration of zinc-adequate rats.
Hiers e_t 5;. (1968) did not find any effect of cadmium on
65Zn or dry matter secretion or reabsorption into the small
intestine of calves, and Pond _eg fl- ( 1966) concluded from
work with the pig that cadmium was not an antagonist of zinc.
More recently, Lease (1968b) reported that when chicks

were giVen a basal dose orally of either 18 or 27 ug of 65Zn
BuPPlemented with 5 or 10 mole equivalents of cadmium. ab-
B‘V’I'Pticn of 65Zn was markedly decreased and blood 65Zn levels
were consistently lower over a 2.4 hour period as compared to
chicks receiving 65Zn alone. It was postulated that cadmium
interfered with the absorption of zinc through occupation of
3”“ 0f the binding sites of a transport system in the blood.
F."‘:N‘h¢!1l’.‘lnore. Banis g E- (1969) reported that the addition
a "i190 3 ppm cadmium to the diet of weanling rats caused a

.e ingenulion of weight gain. This effect could be prevented

‘

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25

  

by an addition of 68 ppm of iron and 200 ppm zinc. It is
obvious that cadmium in diet interferes in some way with
zinc utilization and raises zinc requirement.

Working with Japanese quail. Fox e_t_ _a_l. (1970) reported
that with an adequate purified soybean protein diet contain-
ing 75 ppm Zn and 85 to 100 ppm Fe, an addition of 75 ppm Cd
caused an increase in mortality and a decrease in growth. A
reduction of hemocrit by #5 percent and hemoglobin by 60 per-
cent was also observed. No beneficial and protective effect
of adding either Zn or other elements such as Fe. Cu. Co, M0,
N1. Cr(III). Se or folic acid from 1 to 10 times of normal
diet levels upon cadmium toxicity was observed. However.
addition of 0.1 to 1.0 percent ascorbic acid almost completely
Protected against mortality. poor growth and anemia produced
by cadmium. Richardson and Fox (1970) also found that testes
Size and spermatogenes is were impaired when Japanese quail
were fed the same diet used by Fox 3 g. (1970). The detri-
In”ital effect of cadmium upon spermatogenesis could be pre-
Vented by adding 1 percent of ascorbic acid to the diet.
The“ results suggested that ascorbic acid may be important
in Protecting animals against cadmium toxicity.

o‘ther minerals and variables which influence zinc metabo-
11“ have been reported. Kholod (1969) observed that an

.g'dition of molybdenum (3 mg Mo/kg of live weight) to rations
“Qt

311.0p‘produced a decreased zinc concentration in liver,

  

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26

small intestine and heart; in kidney, on the contrary, the
zinc concentration was increased. Thus, molybdenum, is

likely to interfere with the utilization and distribution of
zinc in the animal body. An antagonistic relationship between
lead and zinc was observed by Sporn and Rautu (1969). They
found that adding lead to the diet decreased the zinc re-
tention in liver and kidney of rats.

There is no strong evidence indicating zinc utilization
is interfered with by iron. However. zinc has been shown to
influence iron absorption. Spry and Piper (1969) fed rats an
iron-deficient but zinc-adequate diet. The injection of 150
ug iron intravenously prior to an orally administered dose of
65Zn did not affect oral 65Zn absorption. However, injection
of 240.ng zinc intravenously prior to oral dosing caused a
reduction in the absorption of orally-administered 652h. The
different effects of intravenously injected iron and zinc on
652n retention suggested that iron and zinc are absorbed by
different metabolic pathways. Working with rats, Bunn and
hatrone (1966) found that dietary zinc levels of 200 and 400
ppm increased liver iron levels as compared to a basal level
of 9 ppm zinc. In recent work, however, excess dietary zinc
(0.75 percent) resulted in the formation of an iron-poor
ferritin in rat liver as observed by Coleman and Matrone (1969).

Lease and Williams (1967) reported that the addition of

450 ppm of magnesium to a sesame meal ration containing

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27

strongly bound zinc decreased the availability of added zinc.
This effect was not observed with other meal diets or casein.

Becker and Hoekstra (1966) showed that an addition of
vitamin D caused an increase of zinc absorption in rats. They
concluded that this phenomenon was a homeostatic response to
the increased need for zinc which accompanied enhanced
skeletal growth rather than a direct effect of the vitamin.
However, Chang 33 al. (1969) using rats, observed that the
addition of calciferol to either a 9 percent or 18 percent
casein diet containing 20 to 22 ppm zinc significantly increased
the concentration of zinc in femurs. Their results indicated
that the effect of vitamin D on utilization and deposition of
zinc in bone is not a secondary effect related to calcifica-
tion and bone growth.

Absorption of trace minerals is affected by antibiotics
according to a report by Kirchgessner 23 al. (1960). Working
with growing pigs in eighteen balance trials, they found that
antibiotic supplementation of the diet increased daily re-
tention of cobalt and zinc two-fold and copper three-fold.

Gunn §t_§l. (1960) reported that administration of 0.2
mg per day of interstitial cell-stimulating hormone (ICSH)
to hypophysectomized rats caused a 7-fold increase in 65Zn
uptake by the dorsolateral prostate and a 19-fold increase in

65Zn uptake by the total prostate of control animals. In a

+
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28

subsequent report, Gunn et al. (1961) reported that follicle
stimulating hormone (FSH) was much less effective in pre-
venting the decrease in zinc uptake by the prostate after
hypophysectomy, and growth hormone and prolactin had no
beneficial effect on zinc uptake.

Adrenalectomy caused a reduction in the zinc concentration
of the prostate and blood and a decrease in the uptake of
65Zn by the testes and dorsolateral prostate (Rudzik and
Riedal, 1960a, 1960b). Daily treatment with cortisone re-
sulted in normal zinc concentration and uptake values. Adrenal
corticotrophic hormone (ACTH) injections into hypophysecto-
mized rats partially prevented the decrease in 65Zn uptake by
the prostate due to hypophysectomy. More recently, Cox (1969)

reported that the zinc content of Hela S cells was markedly

3
increased after growth in media containing adrenal gluco-
corticoid hormones.

Leonov and Dubina (196a) showed that in female rats re-
ceiving 2 IU of folliculin daily for 2 weeks, zinc concentra-
tion in the whole blood increased from 580 i opg/ioo ml to
638 i 12.2 jig/100 ml. A supplement of 0.5 mg of testosterone
prOpionate given daily to male rats for 2 weeks increased
whole blood zinc concentration from 600 i 30.5‘pg/1OO ml to
705 i 16.8.pg/1OO ml. It is likely that these hormones
affect zinc utilization.

Corn oil has been shown to have a protective effect

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29

against parakeratosis. Pond 23 al. (1966) fed low-zinc
diets containing 0 or 10 percent corn oil to growing pigs.
They found no parakeratosis produced on the corn oil supple-
ment as compared to 80 percent incidence in the pigs fed a
diet which contained 24 ppm zinc, and 20 percent incidence
in pigs fed a diet containing 35 ppm zinc without added corn
oil.

Working with ten-day-old chicks, Poppe (1968) compared
the retention of zinc from different zinc salts. The com-
pounds used were zinc sulfate, zinc chloride, zinc nitrate
and zinc acetate. He found when chicks were fed a diet
supplemented with 100 ppm zinc from any one of the salts, zinc
was retained equally well by all groups. However, different
zinc salts increased retention of copper in various degrees.
COpper retention was increased by the addition of zinc sul-
fate, zinc nitrate and zinc chloride by 22.7, 45.0 and 19.0
percent, respectively. These salts increased iron retention
by 11 to 25 percent. It should be noted that the zinc supple-
ment was in excess of the daily requirement already supplied

in the feed.

C. The essential role of zinc in biological systems.
1. Effect of growth and taste acuity.
Failure of growth and depression of appetite are two of

the early signs of zinc deficiency in animals. Todd, Elvehjem

.-
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30

and Hart (1934) first described failure of growth of rats fed
a purified diet containing only 0.16 mg zinc per 100 gm.
Since then, the effect of zinc deficiency on growth and
appetite of many other species has been reported. Day and
Skidmore (1947) reported that growth was impaired and death
occurred in 18 percent of zinc-deficient mice before eight
weeks of age. Stevenson and Earle (1956) fed young pigs a
diet low in zinc (32 ppm) and high in calcium (1.03 percent).
They found that after 12 days the zinc-deficient pigs showed
a slight decrease in appetite and rate of gain, but retarda-
tioncfi‘food consumption and gain became more pronounced 2 to
10 days later. Young pigs fed a semi-purified ration con-
taining 9 ppm zinc exhibited reduced weight gains and feed
consumption by the end of the second week on test (Beardsley
and Forbes, 1957). Other reports of the effect of zinc de-
ficiency on growth rate and feed consumption in swine have
been published by Luecke gt gt. (1956, 1957), Lewis gt gt.
(1956, 1957), Bellis and Philip (1957). Newland _e_t a_l_. (1958),
Whiting and Bezeau (1958), Oberleas gt gt. (1962), Smith gt
gt. (1961, 1962), Pond gt gt. (1966), Miller gt gt. (1968)
and Shanklin gt_gl. (1968).

Miller and Miller (1962) fed young calves a low-zinc
purified diet. They found that feed intake and weight gains

of zinc-deficient calves began to decline, relative to the

31

controls, by the tenth week of age. hills gt gt, (1967)

fed a urea, dried egg-white basal diet containing 1.2 ppm

of zinc to 4-week-old calves. A difference in weight gains
during the first 2 weeks between zinc—deficient calves and
controls was observed, although this difference was not
statistically significant. No further gain in weight was
observed in the zinc-deficient calves after the first 2 weeks
of the feeding period. A failure of weight gain after 2 weeks
was also reported by these workers in 5 to 6-week-old lambs
fed the same zinc-deficient diet given to the calves. They
stated that in the young animals, the earliest manifestation
of zinc deficiency was failure to grow, and the other signs
of zinc deficiency developed later. These results were in an
agreement with the findings of Ott gt gt. (1964) in lambs and
of Miller and Miller (1962) in calves.

The effect of zinc deficiency on the rate of growth and
feed intake was demonstrated in chicks by O'Dell 23.2l- (1958),
in turkey poults by Kratzer gt gt. (1958) and in Japanese
quail by Fox gt gt. (1964).

A recent study conducted by Miller gt gt, (1968) revealed
that baby pigs receiving a zinc-deficient soy diet consumed
amounts similar to those consumed by pigs receiving a zinc-
adequate diet during the first week, but their weight gain

during that period was less. Zinc deficiency decreased

.3;
FIV‘
V
P..-
U
u

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.—

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32

growth rate before voluntary food intake was affected in the
second week of the trial. This observation was also con—
firmed by Shanklin gt gt. (1968), using baby pigs to study
zinc requirement on a casein diet. They found that baby

pigs receiving low-zinc casein diets gained weight normally
for only one week; however, the depression of food consump-
tion was not as severe as the growth depression and conse-
quently resulted in less efficient utilization of food (gain/
food).

It is not known whether the decrease in feed consumption,
which occurs during a zinc deficiency, is due to a loss of
taste or is a result of the decreased growth rate, or is an
effect upon the appetite-control center. However, data from
several workers indicate that taste responses of an animal
may be altered by a number of diseases, or when an animal is
deprived of a nutrient to such an extent that a deficiency
state develops. Henkin (1967) reported that adrenal in-
sufficiency increased taste sensitivity to sodium chloride,
and that hypogonadism may result in decreased sensitivity to
urea and hydrochloric acid. Bernard gt gt. (1961) demon-
strated that rats in advanced stages of vitamin A depletion
showed abnormal taste responses. It was suggested that
vitamin A is required for normal taste function. The mechanism
of action of these factors on the acuity of taste is not clear.

Balance studies on patients with Wilson's disease and patients

33

with cystinuria (McCall gt gt.. 1967) showed that administra-
tion of D-penicillamine would increase urinary copper excre-
tion and to a lesser extent, urinary zinc excretion. D-
penicillamine, a natural degradation product of penicillin,
forms relatively stable chelates with all biologically active
trace metals (Lenz and Martell, 1960) and has been used in

the treatment of metal toxicity (Aposhian, 1960). Bostrbm and
Wester (1967) reported that the excretion of copper increased
10 to 40 times when cystinuria patients were treated with D-
penicillamine. Although the increase in zinc excretion was
not as large as that of copper, the excretion of zinc in-
creased 8 to 10 times. Later, Henkin gt gt. (1967) reported
that D-penicillamine treatment of patients with scleroderma,
cystinuria, rheumatoid arthritis and idiopathic fibrosis pro-
duced a decrease in taste sensitivity and/or hypocerulo-
plasminemia. Taste acuity and serum ceruloplasmin could be
restored to normal by either withdrawal of D-penicillamine or
simultaneous treatment of the patients with D-penicillamine
and copper. The data indicate that copper plays a significant
role in the physiology of taste. A decrease in taste acuity
can be produced by raising an animal on a copper-free diet.
Working with weanling rats, Henkin gt gt. (1968) confirmed the
importance of copper in the basic physiology of taste. The

copper concentration in the plasma of rats fed D-penicillamine

f

’-

\-
9“

‘~1
.cy...
v.¢d

9
A

“vs-A‘“.
nc‘fi"“

.
”
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9.".
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.-~
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: 3.4-pg/100 ml), but was

was about one-tenth normal (10.1
not depressed in rats fed both D-penicillamine and copper
(Henkin and Bradley, 1969). Rats were fed 5-mercaptopyridoxal,
an SH-group containing compound, without depressing the copper
concentration in plasma, although a decrease in taste acuity
was observed. Henkin and Bradley (1969) reported that a
patient with multiple myeloma had an impairment of taste

which could be corrected by administering c0pper. They also
found that taste responses returned to normal within 4 days

in a patient treated daily with an oral dose of 60 mg of zinc
as ZnClz. This change was associated with an increase in
serum zinc concentration to normal values. Continuation of
zinc therapy maintained normal taste acuity and serum zinc
concentrations. However, 24 hours after zinc therapy was
discontinued. hypogesuia was observed again.

Interactions of metal ions with sulfhydryl groups of
bovine serum albumin were reported by Klotz gt gt. (1952).
They found that copper, zinc, cadmium and lead ions reacted
with sulfhydryl groups of serum albumin to form albumin
mercaptides. Lorber (1966) noted that serum protein sulfhy-
dryl content was increased by more than 50 percent during
D-penicillamine treatment of rheumatoid patients and decreased
after withdrawal of the medication. The increase of serum
thiol (RSH) concentrations and a lowering of serum copper,

under these conditions, resulted in the deveIOpment of taste

I
‘

v.2)“.
.‘y“.

n

- :3

0

'9
4
U o

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era
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._. as.
3 V.
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nu. \.II
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Yu «\V
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.-
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ta

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35

acuity (hypogeusia) (Henkin gt gt.. 1967). An inverse rela-
tionship between COpper and HSH concentrations in serum was
also noted by Schoenbach gt gt. (1951). Recent work by Hsu
and Anthony (1968) showed that the concentration of liver
non-protein-SH compounds and the reduced form of glutathion
(GSH) was significantly higher in zinc- deficient rats than
in zinc-supplemented controls.

Control of taste acuity is undoubtedly a highly complex
biolOgical process. From the data and clinical observations
by Henkin and Bradley (1969), they postulated that thiols
and metabolic ions are in dynamic equilibrium in the metabolic
network. They suggested that sulfhydryl groups and metal ions
may be involved in maintaining the conformational integrity
of a protein which lines the pores of the taste receptor and
its membrane. An alteration in the equilibrium between thiols
and metal ions results in a change in protein conformation
which alters taste acuity. They concluded that thiol groups
play an inhibitory role in taste.

Osmanski (1969) fed weanling rats a purified diet con-
taining 0.5 ppm zinc. He found that the buccal mucosa was
the first to show structural abnormality. The zinc deficiency
produced a widespread parakeratosis and ultra structural
epithelial changes. The stratum corneum contained structures
which normally disappear during keratinization such as mito-

chondria, endOplasmic reticulum and desmosomes; there was less

 

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‘5' ytavd

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T.

.2“

a?

36

keratohyaline. These changes were evident in all cellular
layers of the buccal mucosa. The author did not speculate
whether these changes would affect feed intake or taste

responses.

2. Effect on enzymes.
Since zinc was shown essential for the growth of

Asperigillus niger (Haulin, 1869), and was found to be

 

abundant in animal tissues (Bernard and Vladesco, 1921), the
involvement of zinc in a number of physiOIOgical roles has
been established. One of the functions of zinc, like many
other essential trace metal ions, is the activation of en-
zymes. Zinc was first observed to be a constituent of car-
bonic anhydrase of the blood cells of cattle by Keilin and
Mann (1940). They found that carbonic anhydrase contained
0.33 percent of zinc which did not exchange freely with ionic
zinc. Observations by Day and McCollum (1940) indicated that
the activity of carbonic anhydrase per unit of erythrocytes
in zinc-deficient rats was unchanged from that of normal
animals. No lowering of the carbonic anhydrase activity of
the erythrocytes in zinc-defiCient rats was observed by
Wachtel gt gt. (1941). However, zinc is an integral part of
the molecule of carbonic anhydrase and the removal of the
metal results in irreversible inactivation (Vallee, 1957).

A highly significant correlation between carbonic

.
-l.-\1P C
.x‘ ~‘-

_,-.ng

 

Ill 1 .
: . ..... .1.
.7 .1 .2 a;
.1 .2 .1
. 3 C .1
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_.._ .. .. m. :4 a. .

 

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«C my 0 » ... a. ..
m» .- : ~ A‘% N; I. u
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. _ nu, .9. e:

 

anhydrase activity and zinc content in plants was reported by
Wood and Sibly (1952). The concentration of protein nitrogen
was higher in plants when carbonic anhydrase activity was
high. They concluded that zinc deficiency limits the forma-
tion of carbonic anhydrase as well as that of protein.

No change in carbonic anhydrase activity in the blood of
zinc-deficient rats was noted by Hove gt gt. (1940b). Work-
ing with young calves, killer and Hiller (1962) observed a
reduced carbonic anhydrase activity in those which were zinc-
deficient. More recently, Lutwak-Mann and McIntosh (1969)
found that the carbonic anhydrase activity in rabbit uterus
roughly paralleled the zinc concentration in the endometrium.

In the last two decades, a number of other enZymes have
been discovered whose activity has been related to zinc
which acts either as an activator or as constituent of the
enzymes. Hove §£.§l° (1940a) reported that the activity of al-
kaline phosphatase decreased in zinc-deficient rat intestine
and kidney. In later studies with the pig, Luecke gt gt.

(1957) observed that zinc deficiency caused a reduction in the
activity of serum alkaline phosphatase. The studies of Killer
gt gt. (1965) with Holstein calves showed that a reduction of
serum alkaline phosphatase activity resulted from zinc-de-
ficiency. The activity of the bone alkaline phosphatase was
decreased in zinc-deficient turkeys (Starcher and Kratzer, 1963).
Working with the alkaline phosphatase from swine kidney, Kathies

(1958) stated that alkaline phosphatase is a zinc metallo-

{3
_v.

A ‘00.
u) . ."’
v8.0“!

 

n s .. :~ A» _\ \
. 3

{K ‘ v a s . a ~ \ . .V
E a Q E t a. . l . ‘
: .. 3 I V. ‘ .5. E T s. ..\
a o .. a .5 .. .... a .. Z .. . i.
:N A... a...“ .a s. ‘ .2 «d x: c ‘
a. T a: T .a .. e... a .. .3. i .. . a.
q . 4 ~ N ~ a: s . 4 ‘ 2A \ e .95 ~.: 2 x

38

enzyme. The activity of this enzyme in various organs and
tissues was considered a potential indicator of the zinc
status of the pig. Reductions in serum and intestinal alka-
line phosphatase activity were observed by Kfoury gt gt.
(1968) and Luecke gt gt. (1968) in zinc-deficient rats.
Luecke stated that the reduction in intestinal phosphatase
activity was due to zinc deficiency but the decrease in serum
alkaline phosphatase activity was caused by inanition, since
restricted-fed control animals also showed a reduced enzyme
activity. Furthermore, zinc was probably involved in the
enzyme synthesis rather than just as an activator. They
found that the addition of zinc ions to duodenal homogenate
of the deficient rats did not increase the enzyme activity,
indicating that the lowered activity was not caused by a
lack of sufficient ionic zinc but a reduced quantity of the
enzyme itself. Recent work by Prasad gt gt. (1969) indicated
that the activity of bone alkaline phosphatase was reduced
in zinc-deficient baby pigs as compared to controls. Lin
and Hoekstra (1969),also working with baby pigs, found that
the serum alkaline phosphatase level of zinc-deficient pigs
was reduced to one-tenth that of controls within 3 weeks. A
significant reduction in alkaline phosphatase due to zinc
deficiency was also observed in thymus, kidney and bone but

not in liver, spleen and intestine. In a histochemical study

A
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.—

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39

of alkaline phosphatase in epiphyseal cartilage of the chick,
Westmoreland and Hoekstra (1969) found that little or no
alkaline phosphatase was present in the proliferating cells
of the zinc-deficient chick epiphysis as compared to the con-
trol groups. Reinhold and Kfoury (1969) observed that zinc
depletion in the rat caused a change of alkaline phosphatase
isoenzyme patterns, a decrease of the affinity of the enzyme
for Zn and Mg, and an enhanced susceptibility to inhibition
by L-phenylalanine. From these results, it was suggested
that zinc depletion in the rat was associated with alterations
in enzyme behavior due to a modification of enzyme structure.
This altered behavior was not rectified by addition of zinc
to an tg ttttg enzyme assay system.

The function of alkaline phosphatase is to cleave the
orthophosphate monoesters in the biological system. The
existence of this enzyme in gt ggtt is well established. This

was initially identified by Horiuchi gt gt (1959) and

Torriani (1960). It was first isolated and characterized by

Garen and Levinthal (1960) and found to be markedly inhibited
by a variety of metal chelating agents. This enzyme contains
from 1600 to 3100‘pg of zinc per gm of protein, equivalent to
2.3 to 4.2 gram atoms of zinc per mole of protein of molecular
weight 89,000 (Plocke gt_gt., 1962; Harris and Coleman, 1968;

Simpson gt gt.. 1968; and Reynolds and Schlesinger, 1969).

ll:

a

u“
.“
C
-
L...
V.“
~¢~.\r
‘
A“:
.u;

q

"Q‘H

nov.‘

3 "'f‘
V

‘r‘

-.‘.. e

F

~
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~

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as.

40

Zinc has been shown to be involved in the function of this
enzyme (Plocke 33 al., 1962), and has been implicated in the
tertiary structure of the protein (Reynolds and Schlesinger,
1969). The studies of Simpson and Vallee (1969) indicated
that zinc also plays a role in the maintenance of the
quarternary structure of the native enzyme molecule. They
further indicated that, although all four gram atoms of zinc
seemed to serve in stabilizing the quarternary structure in
some manner, one pair of atoms primarily functioned in sub-
strate binding and/or hydrolysis. These were referred to as
"catalytic" metal ions and were directly involved in catalytic
functions. In recent work of Csopak (1969), using pH titration
and equilibrium dialysis, the binding strength of zinc ions
to _E_J__._ gp_l_i_ alkaline phosphatase (enzyme protein) was studied.
On the basis of equilibrium dialysis experiments, they
indicated that the binding of the 2 zinc atoms of alkaline
phosphatase may be described as coordination to 2 equivalent
and independent sites. The high values of the binding con-
stants obtained and the fact that denaturation destroyed the
specific binding of zinc to alkaline phosphatase indicated
chelate formation with the protein.

All four zinc atoms of the native alkaline phosphatase
molecule can be replaced by cobalt in in zitrg_studies to

form a new, functionally active, enzymatic species. Thus far,

*4
'v.

 
 

~\b -- . N. A. J ‘ ts.“ 0. . A \ * s1 U .- u «3‘ .
WC .1. «q/ a Du Av.— s v Y“ «EL A“ 0‘ 5k ‘3‘ «V :.
no «I. 1 a M . i. a» .1 .. a 2M 2 . «b M‘ r. J ‘
fl. N. a ‘ fi ( Riv «V a f. «V ‘ . ~
.. u 0 3 .1. at .1 E Z . N C. A: T... E C. 4 ‘ s
t C t ._ r .J J. 1 E T .5 3 E
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. mu m. «.4 Ta .7. ... e: a. a. W. . . a. xix e. L. : a:
s. . a. a... .. . . . 7~ ‘ r k, . . u .2 .3 .. a a: .. . . . I. 5L 4‘

41

cobalt is the only metal other than zinc known to form an
active alkaline phosphatase. However, the maximal activity
of cobalt phosphatase is about 15 percent of the activity of
zinc phosphatase in all instances (Simpson and Vallee, 1969).
The significant amount of zinc in the pancreas and the
role of zinc, along with the several other cations, in the
crystallization of insulin led to speculation by Fisher and
Scott (1935) that zinc might be important to pancreatic
function. The earlier report by Hove E£.§l° (1938) indicated
that the proteolytic activity of the pancreas was reduced in
zinc-deficient rats. Later, carboxypeptidase of bovine
pancreatic juice was shown by Vallee and Neurath (1955) to
be a zinc-containing enzyme. This exopeptidase splits
terminal amino acids from peptides having a free alpha carboxyl
group adjacent to the peptide bond. The enzyme contains one
atom of zinc per molecule and the zinc is indispensible for
its enzymatic activity. The loss of enzymatic activity is
always directly prOportional to the loss of metal even though
there is no alteration in the physicochemical properties of
the protein (Rupley and Neurath, 1960; Vallee 23 al., 1963).
Working with rats, Hsu gt al. (1966) reported that rats fed a
zinc-deficient diet had a decrease in the activity of pan- ’
creatic carboxypeptidase A, but the activity of carboxypepti-
dase E was not affected. Mills gt al. (1967), also working

with rats, found that the carboxypeptidase activity was

 

C .. I .. I. ...,_ .C 3 a. r 1.. n
5 r .3 E u a C S 3 1. g . 3 . c 1 . 4 A ...J‘.
A“ "J "J O «y m. 7 my «d. t. u ( a: he. 2‘ n u ( a: z
. . 3 C 3 a . X l. 2. .5 .l .f. _t .7. T. 8
av .. . 4. . Cy C» e I.“ CU 7L 0 \~ K W.‘ n :L I .3 \ n‘u‘ «RV
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L. ... . .. . r _-_ .5 .3 .. . 0 . L V a: .C s v L y a: k .. be m u.
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.... a; «v a. .. .. a .n... .. . rm L... +9\ 3 .7. ..... 2.. ‘,.\ 5.. 3‘ 6.
r5 .1 .., ~ A: a. . . a Cu 1 . u a. He .1 . . . f .8. «v 7~ .4.

appreciably lowered in zinc-deficient rats and it returned
rapidly to normal on zinc therapy. however, there was no
change detectable in pancreatic trysin-plus—chymotrypsin
activity of zinc-deficient rats. The loss of enzymatic
activity of carboxypeptidase due to the loss of zinc could
be restored in varying degrees by other metals. One gram
atom each of C02+, N12+, En2+ and Fe2+, all metals of the
first transition series, restored activities of one mole of
this enzyme (Coleman and Vallee, 1960).

Alcohol dehydrogenase, whose action has been known
since Pasteur's time, is present not only in mammals but also
in fish, plants, yeast and bacteria. Many reports have shown

that zinc is necessary for activity of this enzyme. Nason

23 al. (1951) found that zinc deficiency in Neurospora crassa

 

caused a diminution in the amount of alcohol dehydrogenase
and tryptophan synthetase. Vallee and Hoch (1955) stated
that yeast alcohol dehydrogenase contained, as an integral
part, four atoms of zinc per molecule of enzyme. Theorell
g3 al. (1955) found that horse liver alcohol dehydrogenase
contained two atoms of zinc per molecule of enzyme and that
zinc is essential for enzymatic activity. In more recent
work, Drum gt_al, (1967, 1969), found that horse liver
alcohol dehydrogenase contained 3 to 4 atoms of zinc per
molecule of protein with a molecular weight of 80,000; two

of the zinc atoms were involved directly in catalytic function.

co.

afl~4v:."7'
DJ.

1

“‘Vé“

.
A
\

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g _V.
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woeiv
.

.-

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unA

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.. . -1
..u h.“
.3

5V 3
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4. i v
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43

Removal of these zinc atoms completely abolished enzymatic
activity. The remainder of the zinc atoms in the molecule
participate in maintaining the sub-unit structure of the
enzyme. The characteristics and properties of human liver
alcohol dehydrOgenase have been studied by Wartburg §£.§l°
(1964). They found that it, too, is a zinc metalloenzyme and
is inhibited characteristically by a variety of chelating
agents. Loss of activity by removal of these zinc atoms,
was also observed. Kfoury gt al. (1968) showed that rats
fed a low zinc diet (1.8 to U.3 ppm zinc) for 61 to 150 days
had a reduced alcohol dehydrOgenase activity in liver in
proportion to the severity of depletion. Working with baby
pigs, Miller et al. (1968) examined the effect of zinc de-
ficiency on liver alcohol dehydrogenase activity. They
found that liver alcohol dehydrogenase activity, but not
glutamic dehydrogenase activity, was positively influenced
by dietary zinc level. Prasad g: al. (1969) reported that
the activities of alcohol dehydrogenase were reduced in the
bone, pancreas, esophagus, pituitary and testes in the zinc-
deficient pigs as compared to the controls. However,
Macapinlac 33 al. (1966) observed that liver alcohol dehydro-
genase activity was not affected when rats were in a zinc-
deficient status.

In 1966, Prasad reviewed the metabolic role of zinc and

:4..,

\

'5...”
“Hr-ll

 

 

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“.g “V .& .- V . W ~.— L s v
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9..” C“ M. a . e a. . .r as» n o . . :9.
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44

reported that zinc was known to be a constituent of the
following enzymes in addition to those previously mentioned:
lactate dehydrogenase, malate dehydrogenase, glutamate dehy-
drogenase, and probably other pyridine nucleotide-dependent
metallodehydrogenases. In addition, zinc increased the
activity of many other enzymes, including arginase, enolase
yeast aldolase, oxalacetic decarboxylase, lecithinase,
histidine deaminase, carnosinase and several peptidases.
Reports on the activities of some zinc metalloenzymes in
zinc-deficient animals have not always been consistent.
Wachtel g: al. (1941) reported that there was no lowering of
the carbonic anhydrase activity of the erythrocytes in rats
deficient in zinc, nor was there a change in concentration
of liver alcohol dehydrogenase and alkaline phosphatase in
liver and brain of rats fed a low-zinc diet for 103 days.
In contrast, the pancreatic carboxypeptidase A and kidney and
tibia alkaline phosphatase concentration were greatly reduced.
The concentration of lactic dehydrogenase was also decreased
(Hsu and Anilane, 1966). Working with rats, Macapinlac 2E.§l°
(1966) also reported that there was no effect of zinc de-
ficiency on liver alcohol dehydrogenase, but there was an
increase in serum glutamic-oxalacetic transaminase activity
instead. Swenerton and Hurley (1968) did not find any sig-

nificant differences in the activities of the liver pyridoxal

 

Var
. r
““

A“

.-

.AVA

;

7—

.va.
Y ACCRA

.AV V

v-”

‘\.
L

fia""‘r"

Vt.
.

AC

A‘Q

. _ *

~ Z-rh
‘U

n.
\
h

 

‘c

45

phosphokinase, glutamic dehydrogenase or lactic dehydrogenase
in rats fed a zinc-deficient purified soybean protein diet.

In another study by Hurley gt_gl. (1968) in which lactic dehy-
drogenase, malic dehydrogenase and carbonic anhydrase were
assayed in 21-day-old rat fetuses of zinc-deficient mothers,
no differences were observed in comparison to their controls.
Working with baby pigs, Miller gt_gt. (1968) found that liver
alcohol dehydrogenase activity was reduced due to zinc de-
ficiency, but activity of liver glutamic dehydrogenase was

not affected. More recently, Cox gt gt. (1969) fed a zinc-
deficient diet (0.75 ppm zinc) to gestating rats gg libitum.
They found that on the 22nd day of gestation, lactic dehy-
drogenase activity was elevated in maternal serum and succinic
dehydrogenase activity was increased in maternal and fetal
heart.

The differences noted previously in the activities of
some zinc metalloenzymes of zinc-deficient animals may be
attributed to the fact that not all tissues are affected to
the same extent as a result of zinc deficiency. Therefore,

a few tissues may be more sensitive than others to zinc
deficiency.

A number of other enzymes whose activities are dependent
upon Zn2+ have been reported. Snaith and Levvy (1968) studied

the aK-D-manosidase from jack bean meal. They found that in-

«D

.71.

Y“
i.-

1"“
VJ.

a...)

q
A—
V

a

.
La;

 

 

00

.A..~
‘VC‘D

1‘"

~

.. “0
v “wave
v.‘v

‘tt

 

 

46

activation of a4-D-manosidase by EDTA could be reversed or
prevented by Zn2+. It was postulated that afi-D-manosidase
is a dissociable Zn2+-protein complex in which Zn2+ is
essential for enzyme activity. Cottam and Ward (1969) using
the 3501 nuclear magnetic resonance (NEH) method, reported
that 4 moles of Zn2+ were bound per mole of rabbit pyruvate
kinase. The divalent metal, Zn2+, cannot be replaced by
Mg2+ and only partially by En2+ for the activation of this
enzyme. Leucine amino peptidase from porcine kidney was
studied by Himmelhoch (1969). He indicated that this enzyme
was a zinc metalloenzyme. Removal of the zinc from the
purified enzyme or its replacement by cadmium resulted in
loss of enzyme activity in proportion to the loss of zinc.
Replacement of the cadmium in the enzyme by zinc restored
native levels of activity. The zinc-to-protein ratio is be-
tween 4 to 6 gram-atoms per 30,000 grams of protein, while
the content of all other metals falls to negligible levels.
Latt gt gt. (1969) studied thermolysine, an enzyme from
Bacillus thermgproteolyticus and reported that this enzyme
is a zinc metalloenzyme. The stimulation of cytochrome C
synthesis in Ustilago sphaerogena by zinc was reported by
Mendiola and Price (1969). Vallee gt gt. (1959) studied the
zinc content of ai-amylase of various origins. They found

that zinc functions solely to link the subunits of the

E 3 a
S . .. v
a C ..
«I. my“ nu
"it 4 . C
I Z .
a 3
. E r...
f" 3 .
. s 7..

.C
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o
.7.

2 2w 7.. .
w.

a. ..

AU 0 0
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m . e

A. l I

~fi u D“ u '5

Pk +v w

M rd . V.o

r a a u A:

 

 

 

I
. . fix .. ~ Q.“ A: A. p ‘ . . . . . . ..
A . ~ A .a . ~ a . .
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.r so“ a r «.7 . (\ + a a e 0
:v 3 rs CV e QiA n‘!‘ 2 O u “~.l\ 0° ‘ PV
8. .‘ C» l W .‘ ( n a . v Q 7M“ Q._ (Is
8 nb 1%.. S . . e; . Vu hi «L
a 2.. ~. y L e i... it e w; r
V K» S RSV O l O . Os .r \g he
Ce .4... x a we. a» 1 ‘ .3 3.“ i Mi ..W .n. A. k;
W... e. u w Lyn .m... ...a u n: M e .T a . . .. u what t. new
.. . . .q‘\ «a -. .s e v... a. . a» x 1
. 4 I s a. .. i. - 1 . -.. . . . .2
.r .. \ e A v. as. NW N»..\ u H. W. M 3.5... HM... 5L; . IN v. .. w‘

47

‘a4-amylase of Bacillus subtilis. The enzymatic activity of

 

the zinc-containing monomer is identical with that of the
zinc-containing dimer. However, Shpak (1969) observed that a
daily dose of 2 mg zinc/kg body weight administered to rabbits
increased the content of glucose and pyruvic acid and lowered
the amylase activity in the blood.

Zinc has been shown not only to be a constituent of a
number of metalloenzymes but also shown to be a cofactor
vital to maintenance of the activities of a number of other
enzymes. In addition ions may act as inhibitors of ribo-
nuclease which may, in part, be responsible for control of
ribonuclease activity in living organisms. The existence
of higher ribonuclease activity in zinc-deficient apple and
citrus leaves than in zinc-sufficient controls was reported
by Kessler and Monselise (1959) and Kessler (1961). Ohtaka

gt_gl. (1963) observed that yeast ribonuclease could be com-

pletely inhibited by zinc at a concentration of 10‘6M. Wojnar

and Roth (1964) investigated the effect of A13+, Ca2+, Cu2+.

Fe3+, Mg2+ and Zn2+ metal ions on rat liver ribonuclease

activity. Of the cations studied, they found that copper and
zinc were among the most inhibitory on ribonuclease activity.
Working with rats, Sommers and Underwood (1969) observed that
the testes of zinc-deficient rats contained significantly

higher ribonuclease activity than the testes of the control

E

1' .. "‘
A-v.A~Du u
. .
9““.39‘5" .’
‘erAfiv‘ 'v
*pHA

D“

(I)

—

 

48

groups. Consequently, zinc at physiological concentrations
appeared to have a protective effect on ribonuclein acid
(RNA) through the inhibition of ribonuclease; otherwise ex-

cessive degradation of RNA would result.

3. Effect on hormones.

Zinc has been implicated in the hormonal function of
animals. The high content of zinc in the pancreas was ob-
served by KOga (1934). Fisher and Scott (1935) reported that
zinc is chemically bound in the crystalline insulin molecule.
The crystallization of insulin as a zinc salt by Fisher and
Scott led to the belief that zinc was an integral part of the
insulin molecule. This belief persisted in Spite of the fact
that the action of zinc was not specific, since cobalt or
cadmium could replace zinc in the crystalline insulin molecule
with no loss in physiological activity (Fisher and Scott, 1935).
Moreover, amorphous insulin is as active physiologically as
crystalline insulin, and there is little convincing evidence
that zinc and insulin must combine in 1319 to form an active
compound (Vallee, 1957). Expressed on a fat-free basis, no
significant difference between the zinc content of diabetic
and normal pancreas has been observed (Scott and Fisher,
1938).

It has been reported (Melhuish and Greenbaum, 1961;

Voya‘ a...“
...«..‘ d v-

T:

A .

o A
l. . 0“
#5 l
.. . . .
m. a.
a. ”.n
AV a:

l

'J

‘A‘
,-
-vdr

A9
V.

v a u .u
L c

O .4 a

a 5

.. . S
«1:. u u
.. a n:

«xv fl .v

:3 Vhfi.“h.
n .—
.- V~_‘~

A
A
u.

‘1
‘Vlb
..L
..

Va
‘ “é~‘-

r:

. .
a“ at
.3 I
4 ‘ n
~73. .1 s
Ni V:
~k~ \1. §
.4. ‘.. 5v
v‘ . Wu!

1 . A: .
a u
2 C T.
u
I“ a . (III
5 1. AV 1...
a. a u r... 2
v.‘ Cy a 30 L u
.‘ Lb 3 a h e . A 3‘ .
Ab a» .. u a» N. u v l
3“ ~f§ Os 4. "i T... u. M
W A ,
Auk uh \“ “H W .WUV N‘Vv H u—Hv

.‘ U
n: «\u
r a
sub .v’

.r ..

I\ U

K V

«A!

 

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A
A

 

 

49

Hunter and Ford, 1955 and Lehninger and Neubert, 1961) that
certain hormones affect their normal target sites by causing
swelling of nitochondrial membranes. Cash 23 al. (1968) com-
pared the effects of amorphous insulin (containing 0.00% of
Zn), Zn-insulin (containing 0.5% Zn) and zinc on the swelling
of rat liver mitochondria. They found that zinc-insulin pro-
duced rapid swelling of mitochondria at much lower concen-
trations with a much shorter lag period than amorphous in-
sulin. It was suggested that the mitochondrial swelling
caused by insulin was attributable to its content of zinc

anc probably of other metal ions. It is of interest that this
finding is somewhat different than the early hypothesis that
insulin attacked the mitochondrial membrane by reactions in-
volving the hormone's disulfide groups. Using light and
electron microscopy, decreased granulation of the pancreatic
islet Af-cells of zinc-deficient hamsters was observed by
Bbquist and Lernmark (1969). They also found that glucose
tolerance was impaired either by intraperitoneal or intra-
venous injection into hampsters fed the zinc-deficient diet.
Heinitz (1969) carried out an experiment to study the in-
fluence of zinc on the blood sugar content in human subjects
with diabetes mellitus. Zinc was administered as Zn-asparaginate
at the rate of 3.18 meg/day/patient. It was found that blood
sugar was significantly decreased after oral administration

of Zn-asparaginate. A combination of Zn-asparaginate and

s

 

Q“
-obb .A—
_
~A

Q

v;% 3?
‘v-
"“~.

0

vv‘

 

 

.v . ..\ o ;A g. ... w. .. a 2“
. _ g . . _ v 2. . .
U r\ . . R a f u: . i . m . t E I a x w.
. ~ . ~ \ .
S r; E .. M1 n... .1 o x e .t H: E .6
A—V .. v C p‘ . .. «M» s‘ v -w ~\~ r «\s ‘.slv fix» F~
.. .1. a ) I u: .m». in en #1. s v «-9 4 .x .b. 2‘
.. u hxg . may... 35* . Maw 5 » 51¢ .f .‘ \H.‘ bk. ‘ Q» \~ .\
o . a. ti 1‘ . H A a» o‘. \ . a. N J \ \ ..
. Wu. H... mm... m. u ...n~ h... 4 ‘ : v 7s 2* ~..~ 1 a .[v 2..

50

insulin applied one time only showed no stimulating effect
over insulin alone. Application of a Zn-asparaginate and
insulin combination daily for 6 weeks was more effective in
reducing the blood glucose level than application of insulin
alone. It was postulated that zinc may have a stimulating
effect on glucose utilization.

Failure of growth is one of the most common signs of
animals in zinc deficiency. A higher concentration of zinc
in the pituitary was observed as compared with other organs
in rats (Miller gt_al., 1961) and in baby pigs (Prasad gt al.,
1969). Hove gt_al. (1937) reported that a stimulating effect
on weight gains in zinc-deficient intact rats was produced
by the administration of growth hormone. However, an increase
in weight gains in zinc-deficient intact rats treated with
growth hormone was not observed in similar experiments con-
ducted by Macapinlac et al., (1966). Working with human sub-
jects, Prasad et_al., (1963a,b) reported that a deficiency of
zinc may be one of the factors responsible for growth failure
and retarded genital develOpment. In a further investigation,
Sandstead gt al. (1967) found that pituitary gonadotropin and
17-hydroxysteroids excretions were low. In addition, de-
creased pituitary adrenocorticotropic hormone (ACTH) reserve
and abnormal oral glucose tolerance were also found. Treat-
ment with zinc sulfate rapidly rectified these situations.

From these findings, they suggested that some of the functions

'-

a)‘f\"
V..'v

, ‘
A.,~
V" as.

L:

Ci

EL;

J
.~+~
U

‘

I‘

.-
Ugnv ‘

run: I ' ‘ ¢-

’ 0‘ l

*. I “(J ‘
N -v.‘ LB

11

o

y. J-
U

T

5U

r...

51

of the anterior pituitary gland were suboptimal due to zinc-
deficiency. In view of the above results, zinc may have a
role to play in the function of the anterior pituitary. In
more recent work, Prasad gt gt. (1969) observed that the rate
of growth of either zinc-deficient or zinc-supplemented intact
rats was not affected by growth hormone administration. In
their studies with hypophysectomized rats, they found that
the rate of growth was increased by both zinc and growth
hormone administration and these effects were independent
and without interaction.

In the early report of Bertrand and Vladesco (1921) it
was observed that a high concentration of zinc was present
in the testes and semen of stallions. They also reported that
zinc was present in the reproductive organs of the bull, man
and herring. Mawson and Fischer (1951) stated that in the
rat prostate, the lateral lobe was characterized by the
greatest content of zinc. In their further report on
rabbits, Mawson and Fischer (1952) found that the rabbit
prostate was high in zinc but this concentration was not
closely related to the carbonic anhydrase activity present
in this gland. Working with rats, Gunn and Gould (1957), and
Mann (1964) also observed that the lateral prostate was
characterized by a high content of zinc. In a recent work
with rabbits, Korenman (1968) found that uteri obtained on

the sixth day of pregnancy were an effective source of an

.1 T.

.: o
fix~ .BM G‘I.
by CM .. u
Luv “1. i c
r“ .1

.l A: r.“
a. .ru 0

ma” .t he

rC

u

.p < V. . .8 v I‘I‘

6. O 3

C 4 . .1 C

r 4. o a .5.

9» ad %. ....

C M. AV 2
.. e Z.

a. Z a.

r.. a. h. LC.

.. . flu AJ .5 e

r J
a...

y‘ .3 n .l
~n~ er“ .\a
a» m7
s. 1.
I. 3 o
a.“
sv iv e k
A: ... y .H‘
.p.. Av ad
1‘; V s s J

 

.l .1
A K Q»
«o k v
{1‘ o\ \
sh» . We
.. s
it . .. ..
v.\( \~‘
g :u

52

estradiol-iZ;?-binding protein. It is of interest to note that
at this stage of pregnancy the zinc content of the rabbit
uterus was at a maximum. Furthermore, Lutwak-Hann and
McIntosh (1969) observed that the concentration of zinc in
the rabbit uterine endometrium varied with the endocrine
conditions. The highest zinc concentration was observed in
pregnant and pseudopregnant rabbits during the 4th to 9th day.
The carbonic anhydrase activity roughly paralleled zinc con-
centration with maximum activity at 6 to 7 days after mating
or gonadotropin injection. However, only 1 to 2 percent of
the zinc in the endometrium was associated with carbonic
anhydrase. Working with rats, Tveter (1969) studied the dis-
tribution of 3H-testosterone in the prostate lg 313g. It was
found that the lateral lobe of the rat prostate had the
highest affinity for 3H-testosterone. It was already known
that the greatest content of zinc is present in the lateral
lobe of the prostate (Mawson and Fischer, 1954; Gunn and
Gould, 1957 and Mann, 1964). Emanuel and Oakey (1969) studied
the influence of mono— and divalent ions including Zn2+, Mg2+,
hn2+, Ba2+, Pb2+, Na+, NH4+ and K+, on the binding of 173/?-
estradiol—BH to a macromolecule isolated from the uterine
endometrium of non-pregnant cows. They found that all divalent

cations studied, except Ba2+ and Pb2+

(both of which inhibited
the binding), increased the binding of the hormone, and zinc

exhibited the largest effect. Monovalent cations, except for

Q!-

J.
21310

+59
unsv

53

K+ which increased the binding of the hormone, had little
effect. These results, coupled with those reported for other
species (rabbit uteri, rat prostate) previously, suggested
that zinc may be important in binding steroid hormones to
their target tissues.

It is possible that zinc might be implicated in thyroid
gland secretion. Mel'nik and Skevchuk (1967) found that a
relatively high concentration of zinc was present in the rat
thyroid, especially in the colloid. Inhibition of the thyroid
epithelium by administration of thyroxine caused a decreased
zinc concentration in the colloid, and the concentration of
zinc could be raised by stimulation with 6-methyl thiouracil.

From this brief survey of the literature, the biochemical
interrelationship between zinc and certain hormones is still
not clearly understood but there is evidence that such a re-

lationship exists.

4. Effect on nucleic acid, amino acid and protein
metabolism.
Results obtained with microorganisms have shown that
zinc may play a role in nucleic acid and protein synthesis.

Working with Neurospora, Nason gt gt. (1951, 1953) stated

 

that protein content was markedly lowered in zinc-deficient
mycelia. They further found that protein concentration, or

the activity of alcohol dehydrogenase or tryptophan desmolase

 

a

 

 

54

was not increased when the zinc-deficient medium was supple-
mented with amino acids, vitamins, purines and pyrimidines.

It was suggested that failure of Neurospora to synthesize

 

protein was attributable to zinc deficiency. Foster and
Denison (1950) found that pryuvic carboxylase activity was

absent in extracts of Rhizopus nigiricans but addition of

 

zinc did not increase the activity of this enzyme. It was
concluded that zinc was necessary for synthesis of the enzyme
protein and was not a constituent of the enzyme. In studies
with plants, a lowering of protein content was reported in
zinc-deficient oat leaves (Wood and Sibly, 1952). Possingham
(1956) also reported that zinc-deficient tomato plants had a
decreased protein content and contained about twice the

total amount of amino acids and 10 times the amide content

of normal zinc-supplemented controls. When Rycobacterium

 

smegmatis was grown in zinc- and iron-deficient cultures, a
depression of RNA and an increase of inorganic polyphosphate
was observed by Winder and Denneny (1959). In a later report,
winder and O'Hara (1962) showed that Mycobacterium smegmatis,
when zinc-deficient, had a several-fold increase of insoluble
inorganic polyphosphate and ATP remained high, suggesting
that energy- and phosphate-using reactions were inhibited.

More recently, Harris (1969) working with hycobacterium

 

smegmatis, found a reduction of nicotinamide synthesis and an

 

r.“-
(.5 v.1

-..
D

 

 

 

 

 

-.\

 

55
increase in phosphate content due to zinc deficiency. The

synthesis of RNA and DNA was impaired. Schneider and Price

(1962) and Hacker (1963), using Euglena gracillis, showed a

 

decrease in protein synthesis and RNA content, and a con-
comitant increase in nonprotein nitrogen and amino acids, in
organisms grown in zinc-deficient media. In addition, a
massive accumulation of acid-insoluble polyphosphate was
noted. They concluded that the primary defect in zinc-
deficient organisms was a failure of RNA synthesis and,
subsequently, of protein synthesis. A positive response was
obtained by Wegner and Romano (1963) when they added zinc to

Rhizopus nigricans cells during the course of growth. An

 

immediate increase in RNA, followed by a corresponding in-
crease in protein and cell mass was observed. The DNA con-
tent was affected to a lesser extent. It was postulated
that Zn2+ stimulated growth through a primary effect on RNA
synthesis.

More recently, Kastori gt gt. (1969) observed that the
total amount of free amino acids and amides increased in the
leaves of apple trees when they grew in a zinc-deficient soil.
Treatment with ZnSOu led to the reduction of free amino acids
and amides to normal concentrations. It was suggested that

the content of amino acids, and more especially of the

amides in apple leaves might be a fairly safe indicator of

zinc deficiency.

The metal ions in nucleic acid were examined by Hacker
and Vallee (1959). They reported that many metal ions, in-
cluding zinc were consistantly found in RNA and DNA, regardless
of their origin whether from microorganisms or higher mammals.
The concentration of such metals was less in DNA than in RNA.
The metals found in RNA were firmly bound, as dialysis
against metal-free water did not remove the metals. Some of
the metals were partially or completely removed by dialysis
against various chelating agents. The authors concluded that
certain of these metals may play a role in the maintenance of
the configuration of the RNA molecules, perhaps linking
purine or pyrimidine bases, or both through covalent bonds
possibly involving nitrogen atoms or 7T’electrons of the base.
Furthermore, they indicated that since metals play a role in
RNA, they may bear a functional relationship to protein syn-
thesis and the transmission of genetic information.

An activation analysis method was developed by
Belokobyl'skii gt gt. (1968), which can determine the quantity

'11 g of Au, Cu and Mn, and 10"9 g of Zn

of trace metals to 10
in mg quantities of nucleic acid. The DNA and RNA from
rabbit pancreas were examined by this method after dialysis
treatment. They found that these metal atoms, including

zinc, were firmly bound in DNA. It was suggested that the

s\u .54.

 

 

‘-

I I p < .1 I . . [1| - \ s : P~u
. . . T. _V .J .7. A... *v 0; O. x: M‘ 4‘ {A
w _ z. 2.. n J 1; C; L e L p «u we... 4 a. 2“ a: fin. ...u 3 . u. A»
a |‘ . . . s
“L A. . Cb «\u . v. e o. ‘ ‘~ y .W.‘ M. «\y “J ‘U h v. \.|«\ a
m. at E 3 3 .... no; . u “a .. a e .n. m. e 2.. x; A}... C
a u u .C a .. m . .. i .. ,. 0 a o . t C F r E a d .. 3 z» n... .0
w . I 72 .. . ”i Le + . . . a e A: Ce «o a O y... to A: ab «.0 a.
”.4 r._ a. «L y... .. . a a. «5 al.. :» .: he . u e e «U 2..
. . .. . a u ”i e‘ n: so A: i a a he a. 1» .. ‘ T~ . s
. . 5 ... .9 .q A .2 n . e e a. . v 3. 1.. a. .— .. .2 Eu .. e
. . 2; W. v1“ a: r..\ «a v“ .1 r.. .r.. .1 e . A v i. .r.. .. e a. . 2. 2‘ ,. d
a A a u. 9‘ u». : . a. . 4 . u . s . 4 e s e . .. o . e .. o . 7.5 «a . x ...I. .. x

57

presence of these metal atoms must affect the biolOgical
properties of the DNA: protein synthesis, regulation of
cellular metabolism, and replication of DNA molecules.

In recent years, many studies have been published indi-
cating that zinc is related to nucleic acid metabolism and
protein synthesis in animals. Earlier reports by Lieberman
and Ove (i962) and Lieberman gt gt. (1963) found that zinc
was necessary for the replication of rat kidney cells cultured
tg,ztttg, They also indicated that the lack of zinc prevented
formation of DNA as well as the normally-occurring increase
in activity of DNA polymerase and thymidine kinase, but the
rates of biosynthesis of protein and RNA were not affected by
the lack of zinc in the medium. Fujioka and Lieberman (1964)
in a study of DNA synthesis in the partially hepatectomized
rat, found that continuous perfusion of EDTA inhibited the
post operative rise in DNA synthesis and only Zn2+ was able
to reverse this inhibition. It was suggested that zinc was
necessary for DNA synthesis. These workers reported that
the rate of protein and RNA synthesis was not influenced by
zinc. However, Williams gt gt. (1965) showed that the rate
of incorporation of 32p into both RNA and DNA was reduced at
least 40 percent in the livers of zinc-deficient rats. It

was concluded that zinc deficiency resulted in a defect in

nucleic acid metabolism in this organ. In a more recent re-

~11
v”.
-
A”.
\Lw'

.n A
. .
wr‘
‘5‘ v
1’3““ ‘
Aa'v-
Q2“
«4..

60‘”

n

{-
cc‘a u‘

V‘
v-n
‘gc‘d
Q
‘
”a. ’\

O

a
Z:

s

H
-.. u

{‘c

«VU;
. .
‘v‘

S

58

port, Sandstead and Rinaldi (1969) found that weanling rats
fed an egg albumin semipurified diet containing less than

1 ppm zinc caused a decreased incorporation of 3H-thymidine
into the nuclear DNA of rat liver parenchymal cells. An
immediate increase in labeling of 3R-thymidine of nuclear
DNA was observed after a single intraperitoneal injection of
zinc into zinc-deficient rats. This effect did not depend

on increased feed intake. The authors concluded that dietary

 

zinc deficiency in the rat will impair tg vivo synthesis of
nuclear DNA of liver cells. It has also been reported that
the 12-day-old embryos of rats with a deficiency of zinc

3

showed a reduced uptake of H-thymidine. It was suggested
that the high incidence of gross congenital malformations re-
sulting from zinc deficiency may be caused by impaired DNA
synthesis (Swenerton gt gt.. 1969).

Working with rats, Buchanan and Hsu (1968) conducted a
study to determine the effect of zinc deficiency on the in-

3

corporation rate of R-thymidine into rat liver DNA. They
found that rats fed a low zinc diet exhibited the external
symptoms of zinc deficiency within 3 weeks, and a suppression

of 3

H-thymidine incorporation into zinc-deficient rat liver
DNA was observed; however, this impairment was prevented
when zinc-deficient rats were given.1001pg of ZnCl2 3 hours
before isotopic administration. The authors stated that

zinc deficiency did not affect liver RNA synthesis.

PG“
V-V~“"

‘

 

 

i

«1

59

A study of the role of zinc in DNA and RNA biosynthesis
has also been made by Weser gt gt. (1969). Partially hepa-
tectomized rats receiving an adequate zinc diet were given an
intraperitoneal injection of ZnClz, with a concentration of

3

either 2, 10 or 20 pmoles, 9 hours prior to R-thymidine

administration. They found that injection of Z‘pmoles of
3

zinc had no effect on H—thymidine incorporation into liver
nuclear DNA, but 10 or 20 pmoles of zinc reduced the incorpora-
tion significantly. Thus, it was suggested that either a

lack or an excess of zinc can interfere with DNA synthesis.

The incorporation rate of 6—1uc-orotic acid into liver nuclear
RNA was increased by injection of ZnCl2 when given in the

same levels used in the DNA study. But injection of any one

of these zinc levels had no effect on protein synthesis. The
decrease in DNA synthesis after injection of higher levels of
zinc into the zinc-adequate, partially hepatectomized rats

was suggested to be a result of zinc blocking sulfhydryl

groups in the enzyme systems involved in reduction of ribo-
nucleotide diphosphates to deoxyribonucleotide diphosphates,
and thus the ribonucleotide diphosphates were available for
increased RNA synthesis. In their study with zinc-deficient
rats, they found that an intraperitoneal injection of 1‘pmole
of ZnCl,

2
3H-thymidine administration resulted in a significant in-

into partially hepatectomized rats 9 hours prior to

.ré-
8..»

‘-

I
. ~
. ,W

‘3; «LV

Aa“

he
NH ‘

.7.
.C

.5. v

 

., c

:55 flab .
KV‘ uLC 1
.‘Q.

60

crease in incorporation rate of 3H-thymidine into zinc-
deficient rat liver nuclear DNA. They concluded that the
stimulatory effect of zinc on DNA synthesis in the rats with
a zinc deficiency further supported the suggestion of other
workers that zinc is required in some aspect of DNA synthesis.
The feeding of a purified soybean protein, zinc-deficient
diet to day-old Japanese quail for four weeks has been re-
ported to result in a decrease in plasma proteins as compared
to birds that received zinc. The plasma protein pattern of
the deficient birds, obtained by disc electrophoresis, showed
a marked loss of a major protein component (band 7) during
fasting (Fox and Harrison, 1966). Working with day-old
chicks, Turk (1966) reported that there was no change in
liver content of DNA, RNA, total nitrogen or acid soluble
nitrogen due to zinc deficiency. They concluded that their
study period may not have provided sufficient time for a
change in nucleic acid and protein metabolism to be manifested.
In a more recent study with turkey poults fed zinc-deficient
purified diets over 3 weeks, Vohra and Kratzer (1968) found
no differences in plasma protein pattern by electrOphoresis
as compared to zinc-adequate controls though the birds had
been starved for 24 hours or longer prior to taking the plasma
sample.
Changes in total serum protein concentration and serum

protein pattern of zinc-deficient animals of other species

-Ao“: ‘
“Vt -‘

“H
-..‘u

I
1

Q

2

uad
2.
no A

A‘
V‘

rst-.

{1‘
As A

7‘ a

Q‘s
In

‘C‘
.H-‘vw

is

55‘
. »y u

61

have also been noted. Working with baby pigs, Miller gt gt.
(1968) observed that total serum protein concentration and
the percentages Cd“3'-»and aKZ-globulins were increased in
zinc-deficient pigs; the percentage of serum albumin was
significantly decreased. The authors suggested that the zinc-
deficient pigs had a lesser ability to utilize globulins and
perhaps a diminution in their ability to synthesize serum
albumin. An increase in total serum protein and of globulins
but a decrease in albumin was also observed when lambs were
fed an egg white, zinc-deficient diet (Ott gt gt.. 1964, 1965).
It was suggested that the decrease in serum albumin may be
a first-order effect of the deficiency; however, the increase
in globulin level was probably a response to secondary in-
fections resulting from the open lesions of the skin.

In a study conducted by Nills gt_gt. (1967) it was found

14

that rats fed a UL- C-labelled Chlorella protein basal diet

 

(containing 0.75 ppm zinc) showed no gross evidence of zinc
deficiency, although a reduction in carboxypeptidase activity
due to zinc deficiency was observed, which limited the rate
of protein digestion or absorption.

Riley gt gt. (1969) fed rats a 9 percent casein diet
without supplemental zinc (8 ppm zinc in the basal diet).
They found that plasma threonine, isoleucine and leucine
concentrations increased in zinc-deficient rats as compared

to rats receiving the same diet with 20 ppm zinc. The plasma

7‘

A49”..-

ngngu

f‘i

C

-
a
H

'I‘W
‘.&‘v

 

 

 

. . . . a g
a C .5 .1 m . . ,. .. _ .. . . .. i t
. _ E a c . . i

s i O E C m o i S t: .1.. a 4 ... a... s c .
. . a l . . i .1 re T V. C .. . O .. : Z a .
a. .. S T C a e. .3 u 3 ... a .l. .. . Ir » ..
4n u aw . c a .. . «T. r a t S .1 .N o a a o . mm. .1... AI 6
w.-. H a :9“ M” S c o r. new 7, new . ; a: .. ‘ mm r» W 3 mug

... . a . v . y c s x

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A in . . c v. 0‘ y N i I I. x . Q ¢

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62

lysine level was increased to a lesser extent in zinc-de-
ficient rats as compared to the other amino acids mentioned
previously.

The oxidation of intraperitoneally injected tracer doses
of 14C-labelled amino acids in zinc-deficient rats was
studied by Theuer and Hoekstra (1966). They found that the

1Ll'C-leucine and 14C-lysine was significantly

oxidation rate of
increased in zinc-deficient rats and, as shown with leucine,
this increase was prevented by feeding a zinc-supplemented
diet for 31 hours before tracer dose injection. The oxida-
tion rate of carbohydrates and lipids was essentially un-
affected by zinc deficiency. Based on these observations,
they suggested that zinc deficiency resulted in a defect in
protein synthesis.

Working with rats, a similar result was obtained by Hsu

14

gt al. (1969), when L-methionine-methyl- C was intraperi-

toneally injected in zinc-deficient rats. They found that
significantly higher percentages of L-methionine-methyl-luc
were oxidized by rats deprived of zinc. The increased
oxidation rate of this amino acid was prevented by injection
of 400 pg zinc daily for 3 consecutive days. The oxidation

14C and methionine-Z-luc in zinc-

rate of methionine-1-
deficient rats was not significantly increased as compared

to zinc-supplemented rats. The authors suggested that the

 

 

-
.s--

we.

a:
4 ..

a

 

63

methyl group was specifically involved during zinc deficiency.

Oxidation rates of leucine-l-luC. glycine-l-luc, cystine—l-luc,

1“ 1“C were also significantly

tryptophan-2- C and formaldehyde-
increased during zinc deficiency but to a lesser extent.

In an attempt to determine the effect of zinc on the
incorporation of 1LPG-leucine into testicular protein and
14C-adenine into DNA and RNA of rats, Hacapinlac 33 al. (1968)
found that there was no gross impairment of DNA synthesis
in the testes of rats in early zinc deficiency. The total
protein and RNA content of the testes was reduced in zinc-
deficient rats, but the rate of incorporation of 1LPG-leucine
and 14C-adenine into protein and RNA, respectively, was un-
altered. It was suggested that an increase in protein and
RNA catabolism occurs in zinc-deficient rat testes rather
than a decrease in synthesis.

In contrast to the work of Macapinlac et al. (1968),
data presented by Hsu gt al. (1969) indicated that rats fed
a semipurified diet deficient in zinc incorporated signifi-

L
cantly less DL-methionine-Z-1L

C into liver and kidney pro-
teins. The total radioactivity in the tissues examined was
unaffected by zinc deficiency. They also showed that the
pancreatic cells of zinc-deficient rats were still capable

of synthesizing proteins whenever methionine became available.

In conclusion, they suggested that the effects on the in-

corporation of labeled methionine into tissue proteins were

 

 

 

 

 

.5. . . ' I .
_ . m w . - s a . .. . . c c r
_ - . . .L S. . . . . i
7. as . n». .3 a 9. A . .3 3 E a a . i C M. a.
.r“ . 2 t .h... a e. a» “I. 3.. .2 w-.. v... .2 C C x e.
O 3 .n T. 7: S u _ .f. .. . v . S .1. .. . T. .3 Lg ..
.Tv ..-. .. . A. . .. 4 ...v.. 3 h. u . .. . .3 3. a... .1. Q» ..-. 5v
LI. 1“ v... no a C .2 S. .. . by v .7. Z .,r.. h. C
A: $5. .7. ..« h“ 3-. ah .2 . r... AV “A. a. . 3..
u 4 .C .3 flu .n . «D L . LI. 4 . .n . W. .3 A. . .. . h... 2..
.. . _ _ s u a?. . . ,. a «1 Au .9. a. . .4. «is 54 4 a do w .. é . a...

64

due to zinc deficiency, and were probably unrelated to
methionine uptake.

A possible role for zinc in regulation of sulfur-con-
taining compounds has been reported by Hsu 33 al. (1970) who
found that 60 percent more radioactivity was lost in 24 hours
through urine as well as a decrease of 358 in skin and hair
6 days after isotopic administration in zinc-deficient rats
as compared to controls. An increase in urinary -35S was also
observed in zinc-deficient rats when 35.8-1abeled methionine,
thiamine, thiourea or sulfate were administered. These
findings suggested that zinc may be involved in metabolic
processes of skin and hair.

In a recent study conducted by Somers and Underwood (1969),
young rats were fed a zinc-deficient diet (1.4 ppm zinc) over
a 10-week period. They found that the testes of the zinc-
deficient rats contained significantly lower concentrations
of zinc, RNA, DNA and protein; there was also a significantly
higher concentration of non-protein nitrogen and an increase
in ribonuclease activity in the testes of zinc-deficient rats
as compared to the control groups. They concluded that
nucleic acid and protein metabolism was defective in the
testes of zinc-deficient rats, and it seems that one of the

primary functions of zinc is to regulate ribonuclease activity

at the cellular level. Therefore, the failure to control

q

a

-UdgAVkV ‘I

q Aflhfln

I‘

\ 5\
a.

\N K
.Nk‘
.- us

a:

i. v
I.

.x x
.3.

a C.

65

ribonuclease activity in the testes due to zinc deficiency
caused a consequent increase in protein catabolism in this
tissue.

Research concerned with possible functions of divalent
ions in ribosomal particles were studied by Tal (1968) who
reported an increase in the sedimentation constant as well
as a decrease in the specific viscosity value when Zn2+ or
N12+ were added to the unfolded ribosomal particles of §.ggll.
These values obtained after adding Zn2+ or N12+ were very
close to the values of native ribosome. They stated that the
increased sedimentation constant and decreased specific

2+ can be attributed to con-

viscosity caused by Zn2+ or Ni
formational change and reconstitution of the ribosomal
particles to the normal state. In a subsequent report, Tal
(1969a) found that analysis of the nuclease-free g; coll
ribosomal ash by emission spectrogragnrand X-ray fluorescence
revealed that zinc and nickel were present in significant
amounts and magnesium, calcium and iron in smaller quantities.
More recently, also working with g; 991}, Tal (1969b) re-
ported that the specific viscosity of the robisome solution
increased at elevated temperatures in the presence of EDTA,
and dialysis of EDTA-treated ribosome (unfolded particles)

2+

against 1,uM Zn resulted in a sedimentation profile similar

to that of natural ribosomes. These findings indicated that

'h 11VV9A~

Uni “sb‘ ‘V’

(I!
m
t. 0
{I

 

n.

 

66

the unfolding process caused by EDTA is reversible and
suggested that divalent ions played an important role in the
thermal stability and in maintaining the compact structure

of ribosomes. The implication of such a finding with respect

to protein synthesis is quite evident.

--e

V“

 

 

v

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3-.E£C 9‘
‘kA

fl
v

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Q

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spa»

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1

as.
«he

3.

III. EXPEHIKENTAL PROCEDURE

A. Introduction.

Four trials, involving 56 baby pigs were conducted to
examine the effect of dietary zinc levels on tissue nucleic
acid and protein concentrations, amino acid uptake and protein

synthesis, and growth hormone response.

B. General conduct of experiments.

The diets used were identical except for their zinc
concentrations. The basal diet contained 12 ppm of zinc, and
the zinc-adequate diet was supplemented to a dietary con-
centration of 90 ppm of zinc. Isolated soy protein was used
as the main protein source. The basal diet used in all trials
is shown in table 1. Baby pigs from cross-bred Yorkshire-
Hampshire litters were taken at 3 to 7 days of age. All
baby pigs received the basal diet during a U day adjustment
period before each trial. After the adjustment period, all
pigs were randomly allotted to treatment with an attempt to
equalize for sex, litter and weight. Pigs were housed and
fed in individual wire-bottomed stainless steel rearing
cages equipped with stainless steel feeders and water troughs.

Room temperature was held constant at 300 C during the first

67

68

TABLE 1. COMPOSITION OF EXPERIMENTAL DIsT

 

 

07
Isolated soy1 30.0
D,L-Methionine 0.3
¢’(-Cellulose2 5.0
Lard 5.0
Cerelose3 50.7
mineral mixture“ 6.0
Fat-soluble vitamins in corn 0115 1.0
Water-soluble vitamins in water5 2.0
ISoya Assay Protein, General Biochemicals, Chagrin Falls,
2tho.

polka Floc, Brown Company, Chicago, Illinois.
Cerelose, Corn Products Company, Argo, Illinois.
See Appendix I, Table 1.

5See Appendix I, Table 2.

2 weeks of the trial and was decreased to 25° c for the re-
mainder of the trial. Pigs were fed the diet in dry meal
form. The amount of feed supplied to the pigs was varied
according to the experimental design in each trial. This
will be described in more detail under each trial. However,
all feed refusals were weighed out daily and feed consump-

tion was recorded. The pigs were allowed free access to

.
"m", "CM"
av- -‘vné"

..
\r'
.+:r""‘
-uu'v. Id-

*r

L. n;

. 5.
.‘ .4

41¢ a . u...
.:

?u

«C

s:

C. in
T;
.x .
“L. O
-..A
- x Q.
~ \ ac
6.. .. a

.1?

 

69

fresh water (zinc content less than 0.1 ppm) during the ex-
perimental period. Pigs were individually weighed weekly.
Blood was taken from the anterior vena cava at different
intervals during each trial for measures of serum zinc con-
centration and serum alkaline phosphatase activity.
All pigs were killed at the end of each trial to obtain
various tissues for analytical studies. Pigs were rendered

insensible by intravenous administration of sodium penta-

 

barbital and were killed by exsanguination.

1. Trials 1 and 2. Effect of level of zinc on tissue
nuclein acid and protein concentration.

 

 

 

The experimental procedures for trials 1 and 2 were
essentially identical; therefore, they will be presented to-
gether unless otherwise stated. In the two trials, 28 3-day-
old pigs (12 pigs in trial 1 and 16 pigs in trial 2) were
used. After a 4 day adjustment period on the basal diet, the
pigs were allotted into two groups, with half of the pigs re-
ceiving the basal diet containing 12 ppm of zinc (zinc-
deficient) and the other half receiving 90 ppm of zinc (con-
trol). The zinc-deficient pigs were supplied the diet ad
libitum. The control pigs were limited to about the daily
mean intake of the zinc-deficient pigs.

In trial 1, measures of body weight were taken initially,

70

weekly and at the end of the 23-day experimental period.
Blood samples were taken on the 14th and 23rd day.

In trial 2, measures of body weight were taken initially,
weekly and at the 28th day upon completion of the trial.
Blood samples were taken on the 20th and 28th day.

All pigs were killed at the end of each trial. Adrenals,
thyroid, thymus, pancreas, testes, brain, kidneys, liver,
spleen and pituitary were removed by blunt dissection. After
adhering tissue was removed, these organs or glands were
weighed and complete organs or samples of each organ were
placed in a polyethylene bag and frozen on dry ice. They
were stored at -200 C until required for subsequent analysis

of DNA, RNA, protein, dry matter and ether extract.

2. Trial 3. Effect of level of zinc and biotin on tissue
nucleic acid and protein concentration.

A somewhat different design was utilized in trial 3. In
addition to zinc level, a biotin treatment (1 ppm) was intro-
duced based on the suspicion that some of the skin lesions which
had been observed on the basal diet used previously (contain-
ing 50 ppb of biotin) were related to a shortage of this
vitamin.

Therefore, 12 baby pigs were assigned, at 3 days of age,
in a 2 x 2 factorial utilizing a low (12 ppm) or high (90 ppm)
zinc level and 0 or 1 ppm of supplemental dietary biotin;
three pigs were allotted to each treatment. All pigs in this

trial were fed ad libitum.

71

Measures of bodyweight were taken initially, weekly
and at the end of the 3lst day.

The pigs were killed at the end of the trial. Tissue
samples of liver, pancreas, thymus and spleen were collected
and placed in a polyethylene bag and frozen on dry ice. They
were stored at -200 C until assayed for DNA, RNA and protein

concentrations.

3. Trial 4. Effect of level of zinc and growth hormone
injection upon in vitro amino acid incorporation in-
to protein and upon pancreatic carboxypeptidase
activity,

 

 

 

 

Sixteen baby pigs at one week of age were allotted at
random to 4 experimental groups. The pigs in group 1 were
supplied a zinc-deficient diet, containing 12 ppm zinc, for
ad libitum intake. Group 2 received a zinc-adequate diet,
containing 90 ppm zinc, with the daily intake limited to the
daily mean intake of the zinc-deficient pigs. Group 3 re-
ceived the zinc-deficient diet plus 0.5 ml of porcine growth
hormone (0.23 mg) injected subcutaneously into (alternately)
the inguinal or axillary regions of each pig daily, starting
on the second day of the experiment, for 12 days. Porcine
growth hormone1 (0.7 IU/mg) used in this experiment was pre-

pared in the concentration of 0.46 mg per milliliter in saline

solution (Appendix II). The pigs in group 4, receiving a

 

1Sigma Chemical Co., St. Louis, Mo.

zinc-adequate diet, were fed ad libitum. All pigs in this
trial were fed twice daily. Neasures of body weight were
taken initially, at 7 days and at the end of the 14 day ex-
perimental period. Blood samples were taken initially and
at the end of the trial for determination of serum zinc con-
centration, serum alkaline phosphatase activity, total serum
protein and electrophoretic fractions of serum proteins.

At the end of the experiment the pigs were killed and
slices of medial lobe of liver, pancreas and thymus were
incubated with L-leucine-UL-luC for 60 minutes.

Samples of liver, pancreas and thymus were placed in
polyethylene bags, frozen on dry ice, and stored at -200 C
until assayed for DNA, RNA and protein concentrations. Pan-

creatic carboxypeptidase activity was also determined.

C. Chemical Analytical Procedures.
Lass...

A wet ashing procedure was used. A 1 to 4 g sample was
placed in an acid-washed 250 ml Phillips beaker and 30 to 120
ml of concentrated HNO3 were added (the weight of sample and
volume of acid depending on the zinc content of the feed).
This digestion flask was heated on a hot plate to near dry-
ness and cooled. Then 7 ml of 70 percent perohloric acid
were added and the flask was covered with a small watch glass.

The condensation refluxion reaction was continued until the

 

.e ~s~| rt. a: PM

V... a _ r; a .r... .e .. .C 0 .s u
fiv D. :I. u.“ l. .C .C .— v h/n 5 § 0 . «ti
.1 An A. .: . ~ _ C. a .42 o .4. .. .. 5
Lo .7. C n... r. 1-. 2 m. n.
“u 4.. .. _ . ,. .... 4 . «C . e «U
«I. A: . . NJ mu. V“ 5..
ad 7. h” .. .. . .. at a. as a»
Ce 4. . e1 _ s. .2 n1 al.. n v a .

2.

..\~ 5L

73

solution became colorless and near dryness.

After cooling, samples were diluted to volume with deion-
ized distilled water. Standards and blanks were prepared in
an identical manner.

zinc content was determined by atomic absorption spectro-
photometry, using a Jarrell-Ash Nodel 82-516 spectrophotometer
equipped with a Retco total consumption burner. Samples were
aspirated into a air-hydrogen flame. An absorption wavelength

of 2137 X was used.

2. Serum Determinations.

Blood samples from the pigs were collected in acid-washed
centrifuge test tubes and allowed to coagulate. Following
removal of the clot, samples were centrifuged at 550 g for
15 minutes. The serum was then transferred to acid-washed
vials and stored at 50 C.

a. Serum zinc.

Serum samples were diluted to 1:2 or 1:6 with de-
ionized distilled water prior to serum zinc determination
(dilution factors depended on serum-zinc concentration).
No acid digestion was needed. Serum zinc concentration
was then determined by atomic absorption spectrophoto-

metry using the method previously described for feed.

b. Serum alkaline phosphatase activity;

 

Determination of serum alkaline phosphatase activity

was made according to the procedure described in Sigma

74

Technical Bulletin No. 104 (1963). The Sigma 104 phos-
phatase substrate was used in the enzyme activity

determination.

c. Serum total protein.

 

Serum total protein was determined by the modified
Lowry method (Miller, 1959). Ten pl of serum were di-
luted to 5 ml (1:500) with deionized distilled water
prior to total serum protein determination. Bovine al-
bumin was used as the standard reference. A Beckman
model DU spectrOphotometer was used for optical density

determination.

d. Electrpphoretic separation of serum proteins.

 

The serum protein fractions were separated on agar
strips in a modified Beckman-Spinco Durrum cell at room
temperature. The relative staining intensities of the
separated proteins were determined by scanning with a
Spinco Model RB Analytrol equipped with two 500 milli-
micron filters and a 3-5 cam (Cawley and Eberhardt,

3. Tissue Determinations.

 

a. Tissue protein.

 

Homogenates containing 50 mg tissue per ml of de-

 

 

AU C.»

a x t

Wu 0 “.
Pd

 

.~\

Q C
‘Q

a;
«re .2

75

ionized distilled water were prepared using a Virtis
homOgenizer at high speed for 3 minutes. Water was
added to 0.2 ml of the homogenate to make a volume of
10 ml; if necessary, 0.1 N sodium hydroxide solution
was used instead of water to make sure the protein was
completely dissolved. The procedure was then carried

out the same as the total serum protein determination.

b. Tissue dry matter.

Approximately 2 g of sample were weighed into dis-
posable aluminum dishes and dried in a vacuum oven
(approximately 16 hours) at 900 C for dry matter de-

termination.

0. Tissue ether extract.

The dried sample was then extracted with anhydrous
ethyl ether in a Goldfisch Fat Extractor for 6 hours to
determine the fat content. Dry matter and ether extract
were expressed as a percent of the fresh weight.

d. Tissue total nitrogen, tissue protein nitrogen
and tissue non:protein nitrogen.

 

Threetnl homogenates containing 50 mg tissue per ml
were pipetted into Kjeldahl flasks and total N determina-
tions were carried out using the semi-micro Kjeldahl
technique. For tissue protein nitrogen determination, 5

ml of homogenate (5 g/100 ml) were mixed with 2.5 ml of

76

ice-cold 30 percent trichloroacetic acid and held at 50 C
for 10 minutes in order to allow full precipitation be-
fore centrifuging in a refrigerated centrifuge at 32,000
x g for 10 minutes. The supernatant fluid was drained
off and discarded. The precipitate was then washed
twice by resuspension in 5 ml portions of cold 10 per-
cent trichloroacetic acid and centrifugation. The final
supernatant fluid was allowed to drain off thoroughly
by inverting the centrifuge tube. The precipitate was
redissolved in 5 ml of 1 N NaOH solution, using heat if
necessary. Nitrogen determinations were made as pre-
viously outlined for total nitrogen determinations.
Tissue non-protein nitrogen was calculated by
difference from total tissue nitrogen and tissue protein

nitrogen.

e. Tissue a{-amino nitrqgada

The procedure for cK-amino nitrogen determination de-
scribed by Palmer and Peters (1969) was adapted and modi—
fied in this experiment. Two ml tissue homogenates con-
taining 20 mg tissue per ml were mixed with 2 ml of 10
percent sulfon salicylic acid and held at 50 C for 10
minutes. Mixtures were centrifuged in a refrigerated
centrifuge at 10,000 x g for 10 minutes. Then 0.2 ml of

supernatant was placed in a test tube and mixed with 1.6

ml of 0.05 M sodium borate buffer at pH 9.2 and 0.2 ml of

77

0.25 percent 2,4,6-trinitrobenzene sulfonate solution.
Mixtures were incubated at 370 C for 20 minutes. Aluminum
foil was used to cover the sample during incubation be-
cause exposure of the products of the reaction, trinitro-
phenolamino acids, to strong light caused decomposition.

At the end of incubation, 2 ml of 1 N RC1 was quickly added
to the test tubes and mixed well. Standards and blanks
were prepared in an identical manner (Appendix I, table 3).
Optical density was measured at 420 mp using a Beckman

model DU spectrophotometer.

f. Amino acids.

 

Samples for liver free methionine, leucine, and iso-
leucine were prepared essentially according to Clark ad
ad. (1966) using norleucine as an internal standard. The
procedure consisted of adding 1 ml of 1 mm norleucine and
1 g of tissue to 4 ml of deionized distilled water and
homogenizing in a Virtis homogenizer at top speed for 2
minutes. The homogenates were mixed with 5 ml of 10 per-
cent sulfon salicylic acid and centrifuged at 32,000 x g
for 10 minutes. Five ml of the protein-free supernatant
were pipetted into a round bottom flask and concentrated

in a rotary evaporator to near dryness. The residue was

resuspended in 3 ml of lithium citrate buffer at pH 2.0.

78

Amino acid analyses were performed on a 0.4 ml aliquot
of the resultant solution using a Technicon TSN Amino

Acid Analyzer.

g. Pancreatic carboxypeptidase.

 

The procedure for pancreatic carboxypeptidase
activity assay was essentially the same as described by
hills ad ad. (1967). The only modification was that the
protein content of the unactivated homogenates was de-
termined by a modified Lowry method (Miller, 1959). A
0.5 ml aliquot of the final activated homogenate solu-
tion was diluted with 4 ml of deionized distilled water
(1:8) and this solution was used for amino-nitrogen de—
termination by the modified Palmer and Peters method
(1969) described in the tissue cx-amino nitrogen section
of this experiment. (Appendix I, table 3)

h. In.vitro protein synthesis - assay of amino acid
incopporation py tissue slices.

 

The method used for study of amino acid incorpora-
tion into tissue slices was adopted from Ranking and
Roberts (1961) and Campbell and Sargent (1967).

The reasons why the la 31322 tissue slice technique
was used for study of protein synthesis in this study
are as follows: (1) Tissue slices are more representative
of physiological conditions as compared to an acellular

amino acid-incorporating system. (2) Protein synthesis

79

occurs at cell membrane bound ribosomes (Rendler, 1968),
which are larely destroyed in preparing the acellular
system but preserved in the tissue slice. (3) Tissue
slices are more easily prepared than an acellular amino
acid-incorporating system.

There is strong evidence (Rendler, 1968; Rider ap
al., 1969) indicating that extracellular amino acids
seem to have direct access to the sites of protein syn—
thesis. Likewise, Rider a: al. (1969) suggests that
there appears to be no direct relationship between the
total intracellular pool and protein synthesis (i.e.,
extracellular amino acids do ndzhave to pass through the
internal storage pools of amino acids). Protein synthesis
may be limited by either lack of precursors (amino acids)
or the integrate of the protein synthesis machinery, and
zinc deficiency could affect both of these conditions.

If precursors are not limiting, however, the rate of
protein synthesis would be limited solely by the capacity
of the synthesis machinery. In the present work, it is
decided to assess solely the effect of zinc on the
capacity of the protein synthesis system and hence amino
acids were added into the tissue slice incubation media.
Furthermore, the specific activity of the labeled amino
acid in the media can be used to directly calculate the

protein synthesis in that tissue. After the animals

80

were exsanquinated, part of the liver, pancreas and thy-
mus tissues were quickly removed, blotted and placed in
ice-cold saline solution. Tissue slices weighing about
100 to 150 mg were sliced with a Stadie-Riggs microtome2.
They were placed in 50 ml Erlenmeyer flasks with 2.8 ml
ice-cold hrebs-Ringer bicarbonate buffer solution (pH 7.4)
containing 2 mg/ml glucose and cool amino acid mixtures
(nonradioactive amino acids) (Appendix 1, tables 4 and 5).
Each flask was previously gased with 95 percent 02 and 5
percent C02. Then the flasks were placed in a GyrotoryLp
water bath shaker at a rate of 40 cycles/minute for 2
minutes at 370 C to attain temperature equilibrium.
Duplicates of each tissue were incubated. After tempera-
ture equilibrium was established, 2.0 pc of L-leucine-UL-
140 (S.A. = 240 mc/mN)3 was added to each flask and in-
cubated further for 60 minutes. At the end of incubation,
the reactions were stopped by placing the flasks in an

ice bath and adding immediately an equal volume of cold

20 percent trichloroacetic acid containing unlabeled
leucine (14pmoles) to remove unincorporated radioactive

amino acid. The samples were left over night to insure

complete precipitation. The proteins in the control or

 

iArthur H. Thomas Co., Philadelphia, Pa. 19105.
New Brunswick Scientific Co., New Brunswick, N. J.
Tracerlab, Waltham, Mass. 02154.

 

81

the zero time sample were precipitated immediately after
the addition of labeled amino acid.

The trichloroacetic acid-precipitated proteins of
each incubation mixture were then homogenized by hand
with a glass Potter-Elvehjem homogenizer. Homogenates
were centrifuged at 10,000 x g for 10 minutes, and the
supernatant decanted. The protein precipitates were
washed twice with 10 percent trichloroacetic acid (5 ml
each time) and resuspended in 5 ml of 5 percent hot tri-
chloroacetic acid by maintaining the temperature at 900 C
for 15 minutes. After cooling, the samples were re-
centrifuged, dehydrated twice with acetone, and once
with diethyl ether. Then the protein precipitates were
air-dried carefully in a hood at room temperature. The
air-dried protein precipitates were redissolved in 1 ml
of 1 N NaOH solution, and a 0.2 ml aliquot of this solu-
tion was mixed with 10 ml of liquid scintillation count-
ing solution (Appendix I, table 6) in a plastic counting
vial. Samples in the plastic counting vials were kept
in cold storage for 2 hours before counting. The radio-
activity was counted in a Nuclear Chicago liquid
scintillation counter; counting efficiency was about 78

percent.

CD
(‘0

1. Nucleic acid.

 

Tissues for nucleic acid (RNA, DNA) analysis were
placed in polyethylene bags and frozen on dry ice, then
stored at -200 C until analyses could be performed.

Nucleic acid analysis was performed by a modified
(Tucker, 1964) Schmidt and Thannhauser (1945) procedure
with slight changes. A 2 to 4 g sample of the tissue was
weighed and homogenized for 1 minute in 1:20 volume (w/v)
of ice-cold deionized distilled water at top speed in a
Virtis homogenizer.

Duplicate 2 m1 samples of the homogenates were re-
moved into polypropylene tubes and 8 ml of 95 percent
ethanol were added. The samples were incubated at least
12 hours at room temperature with continuous shaking and
were centrifuged in a centrifuge at 0 to 40 C. All
centrifugations were performed at 32.000 x g for 15
minutes. The supernatant fluid was poured off and 9 ml
of methanol—chloroform (2:1) were added to the precipitate.
After incubation at room temperature for at least 24
hours, the samples were centrifuged, supernatant fluid
poured off and 5 ml of anhydrous diethyl ether were
added to the residue. The samples were then incubated
for 4 hours at room temperature, centrifuged, the ether
fraction discarded and the precipitate dried under a
hood.

The residue was extracted twice with 5 ml of 10 per-

83

(went ice-cold trichloroacetic acid which was removed by
vnishing with 5 ml of ice-cold 95 percent ethanol saturated
iqith sodium acetate. The samples were digested with 4 ml
of 0.3 N KOH for 15 hours at 370 c. The digest was
zacidified with 0.3 ml of ice-cold 6 N HCL and 5 ml per-
cfliloric acid. Samples were placed in an ice bath for 10
{minutes to allow for full precipitation. The samples
‘were centrifuged and washed twice with 5 ml each of ice-
cold 5 percent perchloric acid. The combined acid super-
ruitant fluids containing the RNA were adjusted to 20 or
25 ufl.With 5 percent perchloric acid. The amount de-
Eflanded on the content of RNA in the tissue. The RNA-
rifloose was analyzed by the colorimetric orcinol procedure
Of‘ Nejbaum (1939) as described by LePage (1957). This
pIxacedure consisted of mixing a 3 ml aliquot of the
zdbove supernatant fluids with 3 ml of a 1 percent solu-
'tion of orcinol in 0.1 percent FeC12°6H20 dissolved in
Concentrated HCl. The reaction mixture was boiled in a
Water bath for 30 minutes and the optical density was
read in a beckman DU spectrophotometer at 670 mu. The
.RNA content was calculated from a standard curve obtained

from highly purified yeast RNAB.

 

Worthington Biochemical Co.. Freehold, N. J. 07728.

“4
‘\
\J

The DNA was extracted from the residue remaining after
czold perchloric acid treatment with 5 ml of 10 percent
jperchloric acid heated to 700 C for 25 minutes. After
<3ooling and centrifugation, the residue was washed twice
vmith 5 ml of ice-cold 10 percent perchloric acid. Those
IDNA.containing supernatant fluids were combined and ad-
justed to 20 or 25 ml with 10 percent perchloric acid.

'The DNA was analyzed by the colorimetric diphenylamine
Inethod of Burton (1956) as modified by Giles and Myers
(1965). The procedure consisted of mixing a 2 ml aliquot
Of‘ the above supernatant fluids with 2 ml of 4 percent
Cliphenylamine in glacial acetic acid followed by adding
O.1_nfl.of aqueous acetaldehyde (1.6 mg/ml). The reaction
Inixture was incubated at 300 C overnight. The optical den-
Sity was read in a Beckman DU spectrophotometer at 595

Hyl. The DNA content was calculated from a standard curve

Obtained from highly polymerized calf thymus DNAS.

§§atistical Analysis.

The data from trials 1 and 2, where only 2 treatment

grOups were involved, were analyzed for statistical difference

by the t-test. The data from trials 3 and 4 were examined by

analIVsis of variance, and treatment differences were determined

by application of the multiple range test of Duncan (Bliss,

1967).

 

worthington Biochemical Co., Freehold. N.J. 07728.

IV. RESULTS AND DISCUSSION
A. Effect of Level of zinc on Tissue Nucleic Acid and Protein

Concentration.

1. Trial 1.

The weights, feed consumption and efficiency of gain data
are presented in table 2. All the zinc-deficient pigs gained
much less weight than the control pigs by the end of the first
week of the trial and the difference in weight was approach-
ing sigwnidcance (0.05 < P < 0.10). although the zinc-deficient
pigs consumed a quantity of diet equivalent to the unlimited
intake of the control pigs. At the same time, a slight
parakeratotic lesion was observed on one of the zinc-deficient
pigs. During the second week of the trial, the feed intake
of the zinc-deficient pigs was severely depressed, and body
weight gain almost completely ceased, becoming significantly
(P < 0.01) less than that of the controls. Most of these
animals showed moderate to severe skin lesions. In general,
during the first week of the trial, the depression of food
consumption was not as severe as the growth depression and
Consecluently resulted in less efficient utilization of feed
(gain/feed). Similar observations have been reported by
Miller a: elo (1968) and Shanklin a: elo (1968) who reported

that the weight gain of pigs receiving zinc-deficient diets

85

TABLE 2. EFFECT OF DIETARY

ZINC LEVEL 0.

WEIGHTS AND FEED

 

 

CONSUMPTION OF ZINC-DEFICIENT AND CONTROL BABY
Pics.1 TRIAL 1.
Dietary Zinc, pmn 12 90 IS.£
No. of pigs 6 6
Weights, kg
Initial 2.11 2.10 0.10
7 days 2.60 2.98 0.14
lltciays 2.82 3.90: 0.16
21 days 3.69 5.3LL 0.23
23 days 3.76 5.62a 0.25
Ave. deuiy-gain, g 72 153a 7.52
Total gain, kg 1.65 3.5181 0.17
Daily :feed consumption, g
0-7 days 230 225 5052
8-1u days 161 202 7.62
15-23 days 200 215 4.2u
AVG. daily feed intake, g 197 213 b.53
T0tal.:feed intake, kg 4.50 4.91 0.10
Gain/feed 0.36 0.723 0.05
\

 

1
All andeficient pigs were given 100 g of control diet on
the 10th day and 150 g of control diet on the 16th day to

Zstimulate food intake.

aStandard error of the mean.

Significantly (P < 0.01) different from like values on

other zinc level.

87

was ILess than that of control pigs. Pigs receiving 90 ppm
of ziruzixithe diet, even though essentially limited to the
volturtary intake of the zinc-deficient pigs, had excellent
appetfiites and gained quite well throughout the trial.
Effixziency of feed utilization for body weight gain of the
contrwil pigs was about twice that of the zinc-deficient pigs
(153 g; vs 72 g average daily gain and 0.72 vs 0.36 gain/feed),
and tflie difference was significant (P < 0.01). The skin con-
diticni of the pigs receiving 90 ppm of zinc was excellent
tfiumnughout the study. During the second week of the trial,
most of‘the deficient animals exhibited moderate to severe
skin Idesions. It is noteworthy that all of the zinc-deficient
pigs Evere given 100 g of control diet on the 14th day and
150 E: of control diet on the 16th day in order to stimulate
appetite and prevent further deterioration in their condition.
It was found that the zinc-deficient pigs responded rapidly
to Such.treatment by a quick restoration of appetite and by
the let day of the trial all pigs were consuming 300 g of
the diet daily. During the third week, growth resumed and
the Severity of skin lesions diminished somewhat in most of
the Zinc-deficient pigs. During the final two days of the
trial. growth again ceased and appetite again was depressed.
Serum zinc levels and serum alkaline phosphatase activ-
ities are summarized in table 3. Low dietary zinc levels

Caused a significant (P < 0.01) decrease in serum zinc levels

C0
03

TABLE} 3. EFFECT OF DIETARY ZINC LEVEL ON SERUM ALKALINE}
PHOSPHATASE ACTIVITY AND SEfiUI'i ZINC LEVEL OF ZINC-
DEFICIENT AND CONTROL BABY PIGS, TRIAL 1.

 

 

Dietary Zinc, ppm 12 90 iS-E-l
No. of pigs 6 6
Serum alkaline phosphatase,
Sigma units a
14th day 1.8 6.6a 0.73
23rd day 2.2 8.5 0.72
Serum zinc concentration,
Ila/100 ml 3
14th day 26.3 48.8 3.42
23rd day 12.0 62.2(91 1.94

 

 

Standard error of the mean.

Significantly (P < 0.01) different from like values on
C>ther zinc level.

and serum alkaline phosphatase activities after 14 days and

23 days of the study. These findings were consistent with

those of other workers (Miller e_t_ al., 1968; Shanklin e_t_ a_l_.,

1&968). The somewhat higher values observed in three of the

zziiic-deficient pigs on the 23rd day (9 days later) probably

vnezre a result of the small amount of zinc from the control diet
ggixren on the 14th and 16th days. However, serum zinc levels in
efilIL zinc-deficient pigs had declined to a very low level on the

2:31?d day. It is possible that the small quantity of zinc pro-

ificied from the control diet was either rapidly transported to
triee cells or diluted by the increasing body mass of the zinc-

d631?icient pigs. Serum zinc levels and serum alkaline phospha-

t81s;e activities are apparently good indices of the zinc status

Off intact animals.

Other workers, including Luecke et al. (1956, 1957),
beeekstra et al. (1956), Hoefer gt_al, (1960), Ritchie gt al.
(1963), Shanklin £13. a_l_. (1968) and Killer .6215. al- (1968) have re-
PNDIrted similar effects of dietary zinc supplementation on

83?C1wth, feed consumption, feed efficiency, serum alkaline

Eflic>sphatase activity and serum zinc concentration.

Selected organ weights are presented in table 4. Most of

Inlea organs of the zinc-deficient pigs were reduced in size and
lifiéhter in weight on a fresh basis as compared to the controls
(P ‘< 0.01). An exception was the absolute weight of the brain,
Witflq brain weights of the two groups of pigs being almost identi-
Cal- However, the brain, as a percent of body weight, was sig-

niffiicantly (P < 0.01) greater in the zinc-deficient group.

"F1
iheshe findings, coupled with the observation of no significant

90

TABLE 4. r4191?"
2'.

OF DIETARY ZINC LLVL'L OZ“? ORGAN WL‘JIGHTS Or
ILFICIILI‘J l‘ AIJD CONT ‘ROL BABY PIGS, HIAL 1.

 

 

QDiertary zine, ppm 12 90 is.3}

Pk). of pigs 6 6

Initial body weight, kg 2.11 2.10 .10

Firiezl body weight, kg 3.76 5.62 .25

Or6251n weights, g(%)2 ’
Iziver 121.6(3.23) 168. 62(3.0 00)a 8.66(.09)
:Dhymus 4.1(0.11) 10.1a(0.18) 0.59(.01)
I?ancreas 7.4(0.20) 10. 22(0. 18) 0.71(.02)
liidneys 25.3(0.o7) 33. 12(0. 59) 1.85(.02)
ESpleen 6.1(0.16)a 9. 0a(0. 16) 0.57(.01)
IBIain 41.1(1.10) 43.0(0.77) 1.46(.05)

 

 

Lgfiyb51ndard error of the mean.

OI‘Ezsanweights in grams and,
abOdy weight .

in parentheses,

as percentage of

aSiaé‘friificantly (P < 0. 01) different from like values on other

2111c level.

91

difference in brain nucleic acid and protein concentrations
(table 10) between treatment means would indicate that the syn-
tfliesis of brain tissue was not greatly affected by the zinc
deaficiency. Other organs, with the exception of thymus, in-
cxweased slightly when expressed as a percent of body weight
iri zinc-deficient pigs as compared to control pigs. The weight
of’ the thumus decreased when expressed either in absolute
'tearnm or as a percent of body weight in zinc-deficient pigs
afi; compared to control pigs. This allometric manifestation
twig; also been reported by Miller gt gt. (1968). The signifi-
CELrItly lower organ weights in zinc-deficient pigs emphasize
tYIEB importance of zinc for the growth of these organs.

RNA, DNA and protein concentratiois are often used as
irhfl.ices of cell growth, metabolic activity or of cell size and
nEUnloers. The ratio of RNA to DNA may be used to evaluate the
metabolic activity of the tissue on a per cell basis. There-
1f02?ee, selected organ RNA, DNA and protein concentration deter-
m111£1tions were carried out in both treatment groups in an
‘Efchrt to measure the possible effect of zinc on cellular

act ivity.

Table 5 shows the experimental data obtained from the
livTEr. The RNA content of the liver was significantly reduced

(P <: 0.01) in the deficient animals as compared to their pair-
fed- controls. The protein concentration in liver was elevated
in 2Zinc-deficient pigs and the difference was statistically

Significant (P < 0.01). DNA content showed no significant

92

TAJBLE 5.

EXTRACT, PHOTE H, RNA AND DNA CONCENTB

EFFJCT OF DISTARY ZINC LEVdL ON DRY MATTER, ETHEH

ATION OF

 

 

LIVAA,1 TRIAL 1.

Dietary Zinc, ppm 12 90 "13.3
NC)- of pigs 6 6

Dry matter, 76 27.5 28.751 0.38
Ether extract, i6 1.6a 1.4 0.11
Protein, mg/g 170.0b 153.7 3.55
111-1A, Lug/g 17.4 29.8b 1.95
DNA, lug/g 1.99 2.10 0.14
RNA/DNA 9.0 1.4.4b 1.27
Protein/311A 9.8a 5.3 0.35
Protein/DNA 88.0 74.3 5.39

 

 

2

Concentration expressed on a fresh basis.
Standard error of the mean.

as ignifioantly (P < 0.05) different from like values on

other 2 inc level.

Significantly (P < 0.01) different from like values on

O”Cher zinc level.

93

differences between treatments. RNA/DNA ratio was higher

(P < 0.01) in zinc-adequate pigs, indicating that there was
more RNA per cell and suggesting; a higher level of liver cell
activity. winick and Noble (1967) reported that relative
cell size may be indicated by the ratio of protein/DNA or
gland weight/DNA. Liver protein/DNA ratios were higher in
zinc-deficient pigs than in control ones, although the
difference was not significant. These findings would suggest
that liver cell size of the deficient animals tended to in-
crease, perhaps as a result of hypertrophy. Since RNA is
I‘63]..eited to protein synthesis, it was expected that protein
content would be a reflection of changes in RNA content of
the liver cells. This was not observed under these condi-
tions of experimentation. The inverse relationship of the
liver protein and RNA concentrations observed in zinc-de-
f1Client pigs is not explainable at the present time. It is
DOSsible that liver RNA of the zinc-deficient animal, even
thOugh in a reduced amount, functioned normally in promoting
protein synthesis, or perhaps protein catabolism lagged
Substantially behind synthesis in the liver cell of the zinc-
deficient animal. However, it has been shown by Yatvin and
watl’lrlemacher (1965) that adrenocortical hormones will stimulate
protein synthesis in liver and diminish it in muscle, and in

the liver this increased rate of protein biosynthesis was

94

accompanied by a decreased cellular RNA level. Thus, this
observation may be related to a general stress effect and not
to zinc deficiency peg; §_e_.

A significantly higher (P < 0.01) liver dry matter con-
tent was observed in zinc-adequate animals; however, ether
extract was higher in zinc-deficient pigs than in the con-
trol group.

The data on thymus dry matter, ether extract, RNA, DNA
and protein concentrations are summarized in table 6. Thymus
DNA content in zinc-deficient pigs was significantly (P < 0.01)
decreased. Essentially no changes in thymus protein and Bl-IA
concentrations were observed. The significant decline in
thymus DNA was consistent with histological observations of
Whitenack (1970) that indicated a reduction in the relative
Concentration of cortical thymocytes. The remaining cells
were less closely packed in the cortex, and there was a de-
crease in size of the medulla. The ratios of protein/DNA
(P < 0.05) and RNA/DNA (P < 0.01) were significantly higher
in Zinc-deficient pigs as compared to control ones. Based
on these findings, it would appear that thymus tissue of
Zinc ~deficient pigs had fewer cells per unit of weight. No
Sigl’lificant differences were observed in dry matter or ether

extract.

Results of the pancreas dry matter, ether extract, RN ,

DNA and protein analyses are presented in table 7. Pancreatic

95

TABLE 6. EFFECT OF DIETARY ZINC LEVEL ON DRY KATTER, ETKER
L.TRACT PROTEIN REA AND DNA CONCENTRATION OF
YI‘lUS , TR IAL 1 .

 

 

Dietary zinc, ppm 12 90 iS.E.
No. of pigs 6 6

Dry matter, % 20.83 21.1 0.53
Ether extract, % 2.64 2.1 0.23
Protein, mg/g 121.9 126.2 2.19
RNA, mg/g 9.3 9.0 0.51
DNA, mg/s 22.0 30.2b 1.70
RNA/DNA 0.02b 0.31 0.02
Protein/RNA 13.3 1U.1 0.69
Protein/DNA 5.6a 4.3 0.33

 

:Concentration expressed on a fresh basis.

Standard error of the mean.
“Lean of 4 values.

Mean of 3 values.
aSignificantly (P < 0.05) different from like values on
bother zinc level.

Significantly (P < 0.01) different from like values on

other zinc level.

 

 

 

TABLE 7. EFFSCT 0F DIETARY ZINC LEVEL 0N DRY MATTER, ETHER
EXTAACT, PROTEIN, RNA AND DAN CONCENTRATION or
DANCRAASH TRIAL 1.
Dietary zinc, ppm 12 90 :3.m
No. of pigs 6 6
Dry matter, % 23.8 22.6 0.42
Ether extract, % 1.8 1.9 0.11
Protein, mg/g 165.2 156.4 7.55
RNA, mg/g 18.0 22.7 0.60
DNA,mg/g 0.28 1.26a 0.12
RNA/DNA 130 19 45.93
Protein/RNA 9.2a 6.9 0.30
Protein/DNA 485a 125 125.00
in

,concentration expressed on a fresh basis.

Standard error of the mean.

aSignificantly (P < 0.01) different from like values on
other zinc level.

97

RNA was not significantly influenced by the zinc deficiency
although there was a trend indicating pancreatic RNA con-
centration of zinc-deficient pigs was depressed. Significant
(P < 0.01) changes were observed in pancreatic DNA content in
zinc-deficient pigs, but not much change in protein concen-
tration was observed. However, the protein/DNA ratio was
significantly (P < 0.01) greater for zinc-deficient pigs than
for controls, indicating an increase in the size of the pan-
creatic cells.

As can be seen from table 8, essentially no compositional
differences were produced in the kidneys by the two treatments.
The DNA content of the kidneys in zinc-deficient animals
tended to increase, but differences were relatively small.

The dry matter content of the kidneys in the deficient animals
was significantly (P < 0.05) greater than that of controls.
These findings would suggest that, at least in this ex-
periment and for the parameters checked, zinc deficiency had
no effect on kidney nuclekzacid and/or protein metabolism.

Table 9 shows the results obtained in the spleen. Once
again, no differences in dry matter, ether extract, protein,
RNA and DNA content were observed between zinc-deficient and
zinc-adequate pigs.

Analytical values obtained on brain dry matter, ether
extract, protein, RNA and DNA are summarized in table 10. No

effects of treatment on these parameters were observed.

*3
p»
U.)
h
tel
CI)
[*1
f 1;]
3*11
Lt" H
0
JH

OF DIJIALZY ZINC LDV; L 011 DR i‘ATT ETHL‘R
{112‘— Bi PRO‘iml-.. HI IA AiJD Dr A COITCA’L‘RATIL” CF
KIDH:;, TRIAI11.

 

PG

 

Dietary zinc, ppm 12 90 fS.£.
No. of pigs 6 6

Dry matter, % 20.6a 19.7 0.26
Ether extract, % 2.3 2.2 0.07
Protein, mg/g 117.0 119.0 1.21
RNA, mg/g 7.2 7.2 0.15
DNA, mg/g 5.2 4.9 0.17
RNA/DNA 1.39 1.48 0.05
Protein/RN 16.2 16.5 0.29
Protein/SKA 22.5 24.6 0.79

 

}Concentration expressed on a fresh basis.

Standard error of the mean.

aSignificantly (P < 0.05) different from like values on
other zinc level.

 

 

TABLE 9. EFFECT OF DIETARY ZINC LEVEL ON DRY RATTER, ETHEI
DETRACTI PEOTDIN, RNA AND DNA CONCENTRATION OF
SleEn, lRIAL 1.
Dietary zinc, ppm 12 9O tS.E.
No. or pigs 6 6
Dry matter, % 21.1 20.7 0.11
Ether extract, % 1.73 1.86 0.08
Protein, mg/g 153.4 154.1 1.37
RNA, mg/g 9.8 9.8 0.29
DNA, mg/g 6.8 7.1 0.38
RNA/DNA 1.44 1.39 0.07
Protein/RNA 16.2 15.8 0.45
Protein/DR: 23.5 21.9 1.20

 

1Concentration expressed on a fresh basis.

2Standard error of the mean.

100

TA

(11

LE 10. EFFECT OF DIETARY zINC LEVEL ON DRY MATTE}, ETRER
EXTRACT, PROTEIN, RNA AND DNA CONCENTRATION OF
BRAIN,I TRIAL 1.

 

 

 

Dietary zinc, ppm 12 90 1”5.33.2
No. of pigs 6 6

Dry matter, % 18.7 19.4 0.30
Ether extract, % 7.1 7.4 0.22
Protein, mg/g 98.7 98.8 1.94
RNA, mg/g 3.6 3.6 0.11
DNA, mg/g 0.54 0.50 0.04
RNA/DNA 6.6 7.4 0.50
Protein/RNA 27.9 27.7 0.60
Protein/DNA 184.0 205.3 14.91
1

2Concentration expressed on a fresh basis.
Standard error of the mean.

101

Studies with rats (Mandel gt al., 1964) have indicated that
by 16 days of age the rat brain has its full adult complement
of DNA and RNA. Moreover, Rajalakshim gt_al. (1967) found
that undernutrition from birth until 28 days of age did not
affect rat brain size or its DNA, RNA or protein content. It
is apparent that the brain is relatively more resistant to

undernutrition than the other organs studied.

2. Trial 2.

 

The experimental design and conditions were essentially
identical with trial 1, but, the numbers of experimental
animals were increased from twelve to sixteen, and they were
divided into 2 groups, eight to each group. The weights,
feed consumption and efficiency of gain data are presented
in table 11. A decrease in weight gain was observed in
zinc-deficient pigs at the end of the first week of the
trial. However, the weight gain difference between the two
treatment means, even by the end of the second week of this
study, was not nearly as great as that observed in trial one.
This may be due to the fact that one of the pigs in the
control group developed diarrhea and gained very little in
the first week of the trial; consequently, this poor per-
formance influenced the subsequent weight gain difference
between the two treatments.

The feed intake was once again severely depressed during

102

 

 

TABLE 11. EFFECT OF DILTARY ZIKC LEVEL ON HEIGETS AND FEED
CONSUYPTION 0F ZINC-DEF CIEUT AND CONTROL BATE
1.133.3- TXJILD 2.
Dietary zinc, ppm 12 90 tS.E.
No. of pigs 8 8
Weights, kg
Initial 2.36 2.36 0.13
7 days 3.37 3.61 0.30
14 days 3.77 4.63 0.29
21 days 4.20 5.28: 0.28
28 days 4.57 6.56 0.31
Ave. daily gain, g 79 150b 8.51
Total gain, kg 2.21 4.20b 0.25
Daily feed consumption, g
0-7 days 225 225 25.50
8-14 days 225 225 26.25
15-21 days 180 185 4.53
22-28 days 190 215 6.67
Ave. daily feed intake, g 205 212 11.18
Total feed intake, kg 5.74 5.95 0.32
Gain/feed 0.38 0.70b 0.02

 

1All Zn-deficient pigs were given 100 g of control diet on

2the 16th day to maintain feed intake.

Standard error of the mean.

aSignificantly (P < 0.05) different from like values on
bother zinc level.

Significantly (P < 0.01) different from like values on
other zinc level.

103

the second week of the trial, and 100 g of control diet were
given to all zinc-deficient pigs on the 16th day to maintain
feed intake. Skin lesions appeared in most of the pigs after
they had been fed the zinc-deficient diet for 2 weeks. Sig-
nificant (P < 0.01) differences in daily gain and feed
utilization were observed in zinc-deficient pigs as compared
to controls.

Serum zinc level and serum alkaline phosphatase activity
determinations on day 20 and day 28 are summarized in table
12. Zinc supplementation of the diet resulted in significantly
(P < 0.01) higher serum zinc levels and serum alkaline phos-
phatase activities in control pigs at both sampling times.
The preceding observations are in agreement with those on
trial one and similar observations have been reported by
Miller gt_al. (1968) and Shanklin gt al. (1968).

Selected organ weights, expressed either in absolute
terms or as a percent of body weight are presented in table
13. With the exception of brain, all of the organ weights
from deficient animals were significantly (P < 0.01) lower
than from pigs receiving the zinc-adequate diet. when ex-
pressed as a percent of body weight, only thymus and pancreas
were significantly (P < 0.01) decreased by zinc deficiency.
The decrease in selected organ weights in zinc deficiency
during this trial was similar to that observed in trial one

and was in agreement with the data reported by Miller gt al.

104

TABLE 12. EFFECT OF DIETARY ZINC LEVEL ON SERUM ALKALINE
PHOSPHATASE ACTIVITY AND SERUM ZINC LEVEL OF ZINC-

 

 

DEFICIENT AND CONTROL BABY PIGS, TRIAL 2.
Dietary zinc, ppm 12 90 13.13.1
No. of pigs 8 8

Serum alkaline phosphatase,
Sigma units

20th day 2.9
28th day 0.4
Serum zinc concentration,
.Mg/lOO ml
20th day 17.4
28th day 14.0

8.6: 1.1a
3.7 0.36
52.68 2.93
60.3a 2.15

 

1Standard error of the mean.
aSignificantly (P < 0.01) different from
other zinc level.

like values on

105

 

 

TABLE 13. EFFdCT OE DIETARY ZINC LEVEL ON ORGAN WEIGHTS OF
ZINC-DEFICIENT AND CONTROL EASY PIGS, TRIAL 2.

Dietary zinc, ppm 12 90 i3,;.1

No. of pigs 8 8

Initial body weight, kg 2.36 2.36 0.13

Final body weight, kg 4.57 6.56 0.31

Organ weights, g(%)2 a
Liver 117(2.6) 171 (2.6) a 9.75(.oe)
Thymus 4.6(.10) 14.43(.21) 1.65(.03)
Pancreas 6.0(.i4) 13.5a(.20)a 0.71(.02)
Kidneys 32(.70) 4ia(.62) 1.98(.03)
Spleen 8.2(.17) 11.53(.17) 1.13(.02)
Brain 47.0(1.0) 48.0(O.7) 1.12(.05)

 

1Standard error of the mean.
*Organ weights in grams and,

body weight.

in parentheses, as percentage of

aSignificantly (P < 0.01) different from like values on other

zinc level.

106

(1968) and Shanklin gt gt. (1968).

Data from the serum protein analyses are presented in
table 14. No significant differences were observed in serum
protein concentration either on day 20 or on day 28. This
finding was in contrast to the observations in the study by
Miller gt_gl. (1968) who reported that total serum protein
and the percentage of 3'- and ¢%é-globulins was increased in
zinc-deficient pigs, along with a significant reduction in
the percentage of serum albumin.

Liver dry matter, ether extract, RNA, DNA and protein
analytical values are presented in table 15. No significant
treatment differences were observed in liver analyses, but
liver RNA concentrations tended to be decreased and protein
concentration elevated in zinc-deficient pigs. DNA content
in zinc-deficient liver was comparable to the controls. De-
spite the lack of statistical significance, trends in the
data were similar to the observations in trial one.

Table 16 summarized the data with respect to the thymus.
Thymus protein concentration was slightly depressed. A sig-
nificant (P < 0.01) decrease in thymus DNA was observed in
deficient animals. Both protein/BYA and protein/DNA ratios
were significantly (P < 0.01) higher in zinc-deficient
thymus, indicating that the thymus had undergone involution
with possibly fewer cells per unit of weight but increased

cell size. The effects of zinc deficiency on the thymus

107

 

 

14. EFFECT OF DIETARY ZINC LEVEL ON SERUM PROTEINS OF
LINC-DiFICIENT AND CONTROL BABY PIGS, TRIAL 2.
Dietary zinc, ppm 12 90 Is.g.
No. of pigs 8 8
Total serum protein, g/100 ml
serum 0
20th day“ --- --- -_-
28th day 6.0 6.1 0.36
Serum albumin, % of total
serum protein
20th day 49.7 47.5 2.37
28th day 43.0 45.2 3.05
Serum ak-globulin, % of total
serum protein
20th day 31.0 31.0 1.54
28th day 33.6 29.8 1.84
Serum/Q-globulin, % of total
serum protein
20th day 10.3 10.6 0.42
28th day 11. 12.0 0.56
Serum r-globulin, % of total
serum protein
20th day 10.4 11.2 0.87
28th day 10.8 13.0 1.47

 

1Standard error of the mean.

Total serum protein values were not determined on day 20.

108

 

 

TABLE 15. EFFECT OF DIETARY ’lINC LEVEL ON DRY IZATTER, LTEER
CKTRACT, PROTEIN, RNA AND DIIA COIICSII‘LRATION OF
LIVER, TRIAL 2.

Dietary zinc, ppm 12 90 1.5.13.2

No. of pigs 8 8

Dry matter, 78 27.0 27.3

Ether extract, 75 2.0 2.0 0.09

Protein, mg/g 185.3 178.0 5.45

RNA, mg/g 17.0 20.0 1.01

DNA, mg/g 2.36 2.56 0.12

RNA/DNA 7.7 7.8 0.71

Protein/RNA 10.8 9.2 0.76

Protein/DNA 79.8 70.1 3.32

k

:Concentration expressed on a fresh basis.
Standard error of the mean.

109

 

 

‘TAJBLE 16. EerCT 0F DIETARY zINC LTVAL 0N DRY AATTAA, ETHER
Axia CT 1 PROTEIN, AAA AND DAA COACAATAATIOA OF
‘1‘- -.1~. US, TRIAL 2.
Dixetary zinc, ppm 12 90 is.A.2
No. of pigs 8 8
Dry matter, '77. 20.2 20.3 0.28
Ether extract, % 2.3 2.2 0.20
Prwatein, ms/s 120.1 128.3 3.59
RNA, Ins/s 7.2 9.0 0.42
DNA, mg/g 21.3 28.281 1.97
RNA/DNA 0.37 0.32 0.03
Prcnzein/AAA 16.9a 14.4 0.61
Prot ein/DNA 6. 3a 4. 5 0 . 80

_1

:Concentration expressed on a fresh basis.

aStandard error of

other z inc level .

the mean.

Significantly (P < 0.01) different from like values on

110

observed in this trial were similar to those noted in the first
trial. The percent dry matter and the percent ether extract
did not differ significantly between the groups. I

Data from the pancreatic tissue analyses are presented
in table 17. Pancreatic ”RI-IA concentration tended to decrease,
but not much change in protein concentration was observed.
No other significant treatment differences were observed in
deficient animals as compared to the control group.

The analytical data from the spleen are shown in table
18. The dry matter, ether extract, protein, RNA and DNA
content of spleens from pigs receiving the zinc-deficient
diet did not differ significantly from that of controls.
Only the protein/DNA ratio was significantly (P < 0.01)
greater in the control group.

The data presented in table 19 indicate that the kidneys
were not markedly affected by zinc deficiency. The results
Obtained in this study were very comparable to those noted
in the first trial. The only difference found was the ratio
of Protein/RNA which was significantly (P < 0.01) greater in
deficient animals as compared to the control group.

The dry matter, ether extract, protein, RNA and DNA
content of brain (table 20) as in trial 1, exhibited no sig-
nificant treatment differences between zinc-deficient and

2 m0 ‘Supplemented pigs .

111

TABLE 17. EFFECT OF DIETARY ZINC LEVEL ON DRY PLATTER, ETHER
EXTRA ‘1‘,1PROT3I1‘-l, 313A AND DNA COI‘I LEI‘I'I'RATION OF
PANCRAZAS , TRIAL 2.

 

Dietary zinc, ppm 12 90 13.3.2
No. of pigs 8 8

Dry matter, 56 23.3 24.3 0.41
Ether extract, % 1.9 2.0 0.05
Protein, mg/g 150.0 158.0 3.69
RNA, mg/g 19.8 21.9 1.32
DNA, mg/g 0.91 0.65 0.30
RI‘IA/DI‘IA 170 103 69. 78
Protein/RNA 7.8 7.4 0.52
Protein/DNA 1511 842 631.71
1‘

 

2Concentration expressed on a fresh basis.
Standard error of the mean.

112

 

 

TABLE 18. AFAACT OE‘ DIATAAY ZIIIC LAI/ AL 011’ DAY r-LATTAA, ATAAA
ATAACTi PAOTAIA, RNA Al‘ D DI. A COACAATAATIOA 0F
8 LAD *I‘I TAIAL 2.
jDiJetary zinc, ppm 12 90 :S.E.2
Ht). of pigs 8 8
Dry matter, 76 21.0 21.0 0.13
.Etflier extract, % 1.74 1.85 0.05
Protein, nag/g 153.0 156.4 2.09
RNA, HIS/85 9.8 9.4 0.26
DNA, rug/A 9.2 8.4 0.30
ANA/DNA 1.07 1.13 0.03
Prcrtein/RNA 15.7 16.7 0.46
Protein/DNA 15.8 1.8.9a 0.60

—_.__

'Coruzentra tion expiessed

aStalidard error of

on a fres
the ean.

h basis.

aSignificantly (P < 0. 01) different from like values on

other z inc level.

113

TABLE 19. EFFECT OF DIETARY ZINC LEVEL OI‘I DRY IHEATTER, ETHAR
flTRACTi PRO‘I‘EII‘J, Rl-IA AND DITA COI‘ICEI‘CTRATION OF
KIDI‘JEY, TRIAL 2.

 

 

Dietary zinc, ppm 12 90 i'S.E.2
NO. of pigs 8 8

Dry matter. % 20.4 21.2 0.28
Ether extract, 3% 2.8 2.9 0.09
Protein, mg/g 137.3 135.2 3.27
RNA, nag/g 7.7 8.2 0.22
DNA. mg/g 5.0 4.7 0.16
RNA/DNA 1 . 57 1 . 73 0. O7
Protein/RNA 17.881 16.6 0.39
Protein/DNA 27.8 28.8 1.02
K

2\fiaoncentration expressed on a fresh basis.

Dtandard error of the mean.

SléA‘Ilfoicantly (P < 0.01.) different from like values on
Other zinc level.

11D.

 

 

TABLE 20. 23:? :JCT OF DIPJTARY ZIN" .1 L23 TEL 013 DRY PLATTER, EDIE}!
quEIACi I’DOTEII‘ , RITA AND DNA C“-‘C"3TRATIOI‘T OI‘
1*.1AI'1‘3 TRIAL 2.
Dietary zinc, ppm 12 90 iS.E.2
NO. of pigs 8 8
Dry matter, 75 20.1 20.02 0.36
Ether extract, 36 8.1 8.0 0.18
Protein, mpg/g 101.5 103.0 1.93
RNA, mg/g 2.7 2.9 0.06
DNA, mg/g 0.45 0.41 0.05
RNA/DNA 7 . 1 7 . 5 0 . 86
Protein/813A 37.0 36.6 0.85
Protein/DNA 264.2 265.2 32.15

¥

1
2Concentration expressed on a fresh basis.
Standard error of the mean.

115

5. Effect of Level of Zinc and Biotin on Tissue Nucleic Acid
and Protein Concentration.

1. Trial 3.

In this experiment, a 2 x 2 factorial involving 2 levels
of‘ zinc (0 and 78 ppm), and 2 levels of biotin (0 or 1 ppm)
eadried to the basal diet (which contained 12 ppm zinc and 50
‘pgfb biotin) was used. This design was used in an effort to
irrvestigate the validity of the suspicion that some of the
Sliin.lesions observed in previous zinc experiments were due,
iri part, to biotin deficiency. These pigs were fed ad
3;ilaitum, and the experiment was terminated after 31 days.
‘Tkue weight, feed consumption and efficiency of gain data are
skuawn in table 21. Weight gain differences due to zinc de-
fixziency were evident after 7 days of the experiment, and
:realched significance (P < 0.05) at 21 days. Feed intake was
:recruced to a great extent on the zinc-deficient diets, and
tflie ciifference was significant (P < 0.05) after only 7 days
Of 'tIie trial and continued throughout the experiment. In!
adQQJlate dietary zinc also caused a significant (P < 0.01)
Taiuflrtion in the gain/feed ratios of the deficient animals
as COmpared to the zinc-adequate animals.

EJhen.the data from pigs fed the basal diet or the basal
diet Eilus zinc were combined, no effects of supplemental
biotin. upon the incidence of parakeratosis, feed intake or

m1 o o c
weiuflt .gain vere noted. However, there was a Significant

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117

(P < 0.05) biotin plus zinc interaction, resulting in an in,
crease in weight gain and gain/feed from biotin supplementa-
tion when zinc was adequate, as compared to no increase from
Supplemental biotin when zinc was deficient.

The analytical values of RNA, DNA and protein in liver
are summarized in table 22. The relative changes in RNA and
protein concentrations were similar to those in trials 1 and
2, The protein concentration of the liver was significantly
(P < 0.05) elevated in zinc-deficient animals, while liver
RNA concentration was significantly (P < 0.01) reduced. No
Significant difference in liver DNA concentration was ob-
served. The liver RNA/DNA ratio in zinc-supplemented pigs
was significantly (P < 0.01) higher than in zinc-deficient
animals, indicating that synthetic activity per cell was
greater in zinc-supplemented than in zinc-deficient pigs.
'The analytical data indicated that there were no differences
in.liver nucleic acid and protein concentrations between
animals receiving the basal diet and those receiving
supplemental biotin. Similar observations have been reported
by Caldarera (1967) who found that rats fed a biotin-free
diet had normal hepatic RNA and DNA concentrations and a
normal rate of 32F incorporation into hepatic RNA and DNA.

Table 23 shows the analytical data obtained in the

thYmus. The DNA and protein concentrations of the thymus

\vfi hi‘ m
1“ Fuc .1.

TABLE 22.

¥

118

3 OF DIETARY ZINC AND BIOTIN
TEI‘, RNA AND DNA CONCENTRATION OF LIvsH,1 TRIAL
3.

EVELS ON PRO-

 

 

Dietary zinc. ppm 12 9O f8.3.2
Biotin added, ppm 0 1 O 1

No. of pigs 3 3 3 3

Protein. ms/a 137.3a 1A0.8a 112.2 108.5 9,49

RNA, mg/g 18.00 22.na 34.95aa 35.73aa4,19

DNA, mg/g 2.70 2.09 1.90 2,13 0.36

RNA/DNA 7.2 11.0 21.82aa 16.74332.37

Protein/RNA 7.62aa 6.69aa 3,65 3.15 0.97

Protein/DNA 54.9 69.6 63.20 50.99 8.06

 

1Concentration expressed on a fresh basis.
2Standard error of the mean.
aSignificantly greater than least two values

aa(P < 0.01).

(P < 0-05);

119

 

 

 

”ABLE 33. EFFECT OF DIETARY ZINC AND BIDTIH LEVELS on P30-
TAIN, RNA AUD DNA CONCENTRATION 03 THYMUS,1
TRIAL 3.

. ‘5, g-v". , ppm 12 90

chtJlJ 2110 . is.E.

Biotin added. ppm 0 1 0 1

No. of pigs 3 3 3 3

Protein. mg/g 65°C 89-1 lluooaa 115-5aa 5.40

DNA. ms/s 5-74 ”-80 18-31aa 19.25aa 0.96

RNA/DNA 1.72eta 2.25“Eta 0.116 0313 0,39

Protein/RNA 9.76 9.13 13.62 14.70 2.21

aa
Protein/DNA 13.625181 19.44 6.24 6.02 2.32

 

}Concentration expressed on a fresh basis.
2Standard error of the mean.

aSignificantly greater than least two values (P < 0.05);
aa(P < 0.01).

120

were significantly (P < 0.01) reduced in the zinc-deficient

animals as compared to their controls, whereas RNA content

showed no significant changes. The ratio of protein/DNA
and RNA/DNA in zinc-deficient animals was significantly
(P < 0.01) increased. These observations are in agreement

with those data obtained in trials 1 and 2. The decrease

in DNA Content of thymus repr*sents at least two possibilities.
The thymus exhibitis either reduced mitotic activity or in-

creased DNA turn-over rate due to zinc deficiency. Ninick

and Noble (1965) reported that total thymus DNA of the
normal rat decreased after the 39th day of post-natal life

and observed no incorporation of thymidine into thymus DNA

by the buth day. Therefore, the decrease of DEA content in

zinc-deficient pig thymus during early post-natal life is

Inore likely a result of a decrease in the rate of DNA synthesis

due to zinc deficiency. The thymus DNA turn-over rate was not

determined; hence, this explanation can neither be sub-

stantiated nor repudiated at the moment. No treatment

differences, due to biotin supplementation were observed in

thymus RNA, DNA or protein concentrations.
The pancreatic RNA, DNA and protein data of the pigs
used in trial 3 are presented in table 24. Both pancreatic

RNA and DNA concentrations were significantly depressed

 

 

 

TABLE 24. AFFACT 09 DIATARY LING AND BIOTIN LEVELS N PRO-
TEIN, AAA AND DNA CDNCANTAATICN OF PANCREAS',
TRIAL 3.

Dietary zine: PPm 12 9O is.A.

Biotin added. ppm 0 1 o 1

No. of pigs 3 3 3 3

Protein. mg/g 139-0 138-0 143.2 151.0 8.04

RNA. ms/s 20~55 20-37 25-64aa 26-52aa 1.59

DNA, mg/s 1-58 1-55 3.76a 3.30a 0.79

RNA/DNA 14.14 21.36 7.26 11.15 5.12

aa , -- a
frotein/BNA 6.89 0.76 a 5.44 5,72 0.34
I a ,
Protein/DNA 93.978 142.38 39.02 02.34 30.4

}concentration expressed on a fresh basis.

2Standard error of the mean.

aSignificantly greater than least two values (P < 0.05);
83(9 < 0.01).

 

122

(P < 0.01 and P < 0.05, respectively) in zinc-deficient pigs.
Also pancreatic protein concentration tended to be depressed
in zinc-deficient pigs. The highly significant effect on
pancreatic RNA due to zinc-deficiency in trial 3 was not ob-
served in previous trials 1 and 2, although pancreatic RNA

in both trials 1 and 2 tended to be depressed in zinc-
deficient animals. This difference may be attributed, in
part, to the fact that the experimental periods were of
different duration. Trial 3 was terminated after 31 days
while trials 1 and 2 lasted 23 and 28 days, respectively.
The different feeding regimes could have influenced this
also, since the zinc-supplemented pigs in trial 3 were
allowed an unlimited feed intake while in trials 1 and 2
they were pair-fed with the zinc-deficient pigs. An in-
crease in volume of secretion and protein output of the
pancreas caused by external stimuli or sham-feeding of dogs
has been reported (Christoduolopoulos gt gl., 1961; and
Preshaw gt gt.. 1966). The total exocrine volume and en-
zyme output by the pancreas were found to be proportional
to the amount of food ingested in dogs and pigs (Magee and
Hong, 1959; Pekas gt gl.. 1960. 1966). Therefore, the in-
creased feed consumption of the zinc-supplemented pigs in

this study would, in turn, stimulate the synthetic activity

of the pancreas and result in more pancreatic RNA. The de-

crease in DNA content of the pancreas in zinc-deficient pigs
indicated that cell division was affected. Once again, no
biotin treatment differences in pancreatic RNA, DNA and
protein concentrations were observed.

Spleen RNA, DNA and protein values from trial 3 are pre-
sented in table 25. Essentially no treatment differences
were observed and results obtained in this study were
similar to those noted in previous trials 1 and 2. Supple-
mentation of the diets with biotin did not affect the spleen
RNA, DNA and protein content.

This experiment established that the parakeratotic
lesions which developed on the zinc-deficient diet could not
be prevented by 1 ppm of supplemental biotin. No skin
lesions were observed on the zinc-adequate diet. Nevertheless,
1 ppm of supplemental biotin significantly (P < 0.05) im.
proved weight gain of pigs receiving the Zinc-adequate diet,
but did not increase the weight gain of zinc-deficient pigs.
It would appear that a weight gain response to biotin supple-
mentation is limited by the level of zinc present, and that
the high zinc diet used in this study may have been deficient

in biotin.

C. Effect of Level of Zinc and Growth Hormone Injections Upon
lg Vitro Amino Acid Incorporation Into Protein and Upon
rancreatic Carboxypeptidase Activity.

1. Trial LL.

TABLE. 25. 1:98.503 05‘ DISTABY ZIZ‘C‘C AND EIO‘I‘IN LL‘VELS ON 30-
. ‘r._-~ A

no DNA CDACANTAATIDN 09 sBLAsw,

 

 

 

Dietary zinc, ppm 12 90 is : 2

Biotin added, ppm 0 1 0 1 on“
ISO. of pigs 3 3 3 3

Protein, mg/g 149.8 153.5 150.4 160.7 8.80
IRIAA, mg/g 9.46 9.44 10.76 9.60 0.69
DNA, mg/g 4.90 5.28 4.31 4.94 1.22
RI‘JA/DI-IA 2.78 1.95 2.65 2.17 0.75
lgrotein/RI‘JA 15.87 16.23 14.11 17.32 1.71
Protein, DNA 42.91 30.89 37.40 40.14 12.74

¥

Concentration expressed on a fresh basis.

2Standard error of the mean.

In trials 1 and 2, it was necessary to periodically pro-
vide supplemental zinc to pigs on the zinc-deficient diet to
avoid severe depression of appetite and early death. When
the deficiency state was maintained for 3 or more weeks, second-
ary problems related to decreased intake of a number of nu-
trients and to development of bacterial infections may have
Inasked the effects of a simple zinc deficiency. Therefore,
‘trial 4 was of a somewhat different design. First, the ex-
13eriment was terminated after 14 days. This period of time
tvas found sufficient for the zinc-deficient pigs to develop
cilinical lesions, and it has been well established that bio-
cliemical lesions always occur before clinical lesions are
CDtxserved. Second, no zinc-deficient animals were given any

CDf‘ the control diet during this study in order to simplify
‘thee interpretation of the results. Thus, tissues from the
‘Zzizic-deficient animals were adequate for the examination of
Gee]_lular functions and secondary factors did not complicate
iritzerpretation. Sixteen baby pigs at the age of one week
VHBITS included in this study, and they were divided into
fcruar groups (for details see Experimental Procedure 3).

Several researchers have presented evidence or specu-
latned that zinc may play a role in the function of endocrine
horrundes (Hove gt gl.. 1937; Prasad gt gl., 1963; Sanstead
2: al. . 1967). Since zinc-deficient baby pigs usually show

a reducdiion of growth rate before appetite is severely de-

126

pressed, it would be of interest to investigate whether zinc
is related to growth hormone function. Therefore, purified
porcine growth hormone, with a potency of 0.7 IU/mg was
given subcutaneously to the pigs from one of the two zinc-
deficient groups. A comparison then could be made between
growth hormone treated and untreated zinc-deficient pigs.
Pvrthermore, one group of pigs receiving the zinc-adequate
ciiet gg libitum was included in this study, along with
zanother group of pigs receiving the zinc-adequate diet pair-
I‘ed to the daily mean intake of the zinc-deficient pigs.
CDhus, the effects of inanition and of zinc deficiency per se
C(Juld be differentiated. All dietary conditions were the
£35tme for this experiment as for the previous trials 1 and 2.
Data relating to the weights, feed intakes and the feed
thzilization of the pigs in the four groups are presented in
'tsalole 26. Difference in weight gains between zinc-deficient
aldri restricted-fed control pigs was observed at the end of
tiles first week of the trial and the difference approached
£31{§nificance (0.05 < P < 0.01). However, the difference in
weeiéght gains due to dietary treatment between zinc-deficient
'Piéfls, including both growth hormone treated and untreated
grcnips, and gg libitum-fed control pigs was slight at the end
of tflie first week of the trial. This may be attributed to

the Isuyt that one of the pigs in the gg libitum-fed control

127

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128

group had diarrhea during the first week of the trial. It
gained very little weight and negatively influenced the first
week's average gain of this group. The feed consumption of
the pigs in the four groups was essentially the same at the
end of the first week of the trial. Weight and feed efficiency
data indicated no difference in performance between the zinc-
cieficient pigs and zinc-deficient pigs administered growth
Iqormone. Similar observations have been reported by hacapin-
lac g: al. (1966) and Prasad gt al. (1969) who found no sig-
Iiificant response in weight gains to growth hormone administered
1:0 zinc-deficient intact rats, although bovine growth hormone,
iiistead of porcine growth hormone was used in their studies.
Differences in weight gains and feed utilization between
ZZiJJc-deiicient and zinc-adequate pigs were highly significant
( < 0.01) after 14 days of the trial. No significant differ-
eldrzes in weight gains between zinc-adequate restricted-fed
CCDIitrol and ad_libitum control groups were observed. It is
aipzparent that the pigs receiving unlimited zinc-adequate
<1113t consumed more feed but made no better use of it when
ccmnpared to the zinc-adequate restricted-fed control group
IH1dJar these conditions of the experimental period. Thus, not
surjxrisingly, a significant (P < 0.05) difference of feed
utifllization.(gain/feed) between these two groups was
observed.

Eflcin.lesions due to zinc deficiency as described in the

 

129

‘previous studies were observed in both growth hormone treated

sand untreated zinc-deficient pigs during the second week of

'the trial.

Serum alkaline phosphatase activities and serum zinc

ZLevels of the pigs in the four groups ar“ summarized in table

237. ko significant differences in initial serum alkaline
gflqosphatase activity and serum zinc concentrations were ob-

sserved. Serum alkaline phosphatase activity was signifi-

cerntly (P < 0.01) reduced by inadequate dietary zinc at the

texrminal sampling period (14 days later). No significant

tureatment differences were observed in serum alkaline phos—
pfiuatase activity between zinc-deficient growth hormone
trffiated and untreated pigs, although the serum alkaline
phcmiphatase activity in zinc-deficient pigs treated with

The maximum

8rourth.hormone tended to be slightly increased.

Seruni alkaline phosphatase activity was observed on the ad

libitILm-fed control pigs. The difference in enzyme activity

betweneri zinc-adequate restricted-fed control and ad libitum:
fed ocbritrol groups was approaching significance (t=2.436.
While E) < 0.05 t=2.uu7). The present data indicate that
feed I‘enstriction affected the serum alkaline phosphatase
actingtsf. The reduction in serum alkaline phosphatase
astiVj5537 due to feed restriction was not as serious as that

due tC’ fihe inanition of zinc deficiency. A similar observa-

AIJ

m gpfiz mosmofiyfigwfim mmgomOHmam mmSHm> How mama 03p oSp Sooepop mosohmgmfl b mg.

.m::.m u p .mo.o V m Hoe .om:.m u p p2m©5pm

 

 

 

 

.Afio. 0 V 9v mmSHm> 03p pmmma cmSp Hopmmam mapsmoflmma win
.moSHm> mmhxp go :mo:m
.Smoa ogp go Hoaam ohm:mmp w
.ososhon xpzoat n .m.0m
01.0 mmm.om moo.mm m.fim mm.bfi amp Spdfi
em.m mmm.mm mm.fim om.mm mm.&m HmeHQH3
Ha oofi\u1
.SoHpmeQoocoo oQHN Edam
mm.o p.mao.m :0 mm.“ No.0 see spefi
oc.m mmw.cm imm om mm.om ms.em HQHPHQH
mpfiss mawflm .mmmp
logomogg ocfiamxam ESHmm
: a a : mmfia mo .02
@m% we% .m.w meHOHmmU
.q.o- -QHH e< .empofiwpmma +.wme-en -QN msopu
w r c+ 00 NH and .oQfiN msmpofla
o ‘3 1H¢H.-.WC

._ :HoHaannzm o qa>m H olH\ gasnn a.< N5H>Heo< .m<rq <m$
amom :Bso.w oz¢ quN :m<

....5mflm :0 much... 981me .420

sum amen goaaer g:<

n2: EHQ<:

nHm mo Booze: .mm nqm<e

'tion has been reported by Luecke gt_al. (1968) who reported

'that rats fed a limited-intake of a zinc-adequate diet had a
ssignificant decrease in serum alkaline phosphatase activity.

ESerum zinc concentrations were significantly reduced by

Zlowered dietary zinc (P < 0.01). No significant effect due

1:0 growth hormone treatment was observed in serum zinc con-

czentration of zinc-deficient pigs. The difference in serum

zsinc concentration between restricted-fed and ad_libitum-fed

c>ontrol pigs was not significant. Pigs fed a zinc-adequate

ciiet ad_libitum did not have any higher serum.zinc concentra-
t:ion.than pigs fed this same diet in restricted amounts.
Ihavertheless, a parallel between serum zinc concentrations
aIKl serum alkaline phosphatase activity was consistently ob-
serwred in this study and in previous trials 1 and 2. Thus,
botki serum zinc concentration and alkaline phosphatase
actixrity are sensitive indicators of dietary zinc adequacy.
fiesults of the serum protein analyses are presented in
table; 228. Decreasing dietary zinc levels resulted in in-
creas:iddg total serum protein concentrations (P < 0.05).
GrOthl ihormone treated zinc-deficient pigs showed an even
highel? total serum protein level than deficient pigs re-
ceiVine; no growth hormone. No significant treatment differ-
ences ‘Mnere observed in the serum albumin level of pigs in
the fCNJI‘ groups. Serum 3’-globulin levels were not signifi-

cantly Eiltered by zinc deficiency, although there was a

132

.Amo.o v my moSHm> ooHSp ammofl szp HopMon aapsmoawfiswfio

CD

.Afio.o v mvmm “30.0 V my mosam> 03p pmmma soap powwoam mapsoofiafiswamm

.Qmos mSp wo Hosao oamwcmpmm

 

 

 

 

 

 

 

.ososho: SQSOHM n .m.vm
so.fi m.ea H.ma o.oa m.oa Henna
ea.m e.mm N.em m.mm m.mm HoneazH

:Hopoaa ESHom Hopop wo

R .QHHSDoamI.« asaom
no.0 H.mfi :.ma m.afi H.HH Hosea
Hm.o b.mfi m.mfi b.afi N.HH HmeHQH

aflopoha asamm HmpOp mo

E .Qflazpoamumx azaom
om.a 3.0m o.mm e.mm o.mm Hosaa
mm.fi m.ofi m.om H.0N 0.0m HmeHsH

Campoaa BSHmm Hmpop mo

m“ .QHHSQOHwn x» 859mm
as.a m.o: H.me s.oe “.0: Hosam
mfi.m m.om m.od 0.0m m.o: HmeHsH

saopoaa asaom HmpOp
mo R .aaesnam 859mm
em.e ee.m me.n e.eema.e emm.o Henna
om.o No.0 Hfi.© om.b mm.b HmeHQH
azhmm He oofi\w
.sfiopoaa asaom Hmpoe
d 3 d 3 mmfim go .02
now top .fim .0 psofiofipoo .
.a.o- -QHH ea neopoflapmom +.eoouow uow macaw
m a :+ on ma HQ. . .
Q OLHN keepeaa
I. . a ,1. .; {.113 .1 3 11:. 4 4a 4 4.1 . .. 0.3 wHMNHxFH .mz. .443
om; aprem ; mezaaeaeaa .zoaeoe at. ee nee ozHN ameeeHo no eoemea .em momee

133

trend towards an increase in pigs receiving the zinc-deficient
diet whether or not they were growth hormone treated. Appar-
ently, the zinc—deficiency effect on serum protein components
in this experiment was not nearly as great as that observed

in earlier studies (miller it al., 1968) and does not appear

 

to be a very sensitive measure of dietary zinc levels.

As indicated in studies 1, 2 and 3, RNA, DNA and protein
concentrations of liver, thymus and pancreas are more sensi-
tive to zinc deficiency than other tissues. Therefore, only
'these three tissues were taken from the pigs when they were
liilled. Data from the liver RNA, DNA and protein analyses
axre presented in table 29. RNA content of liver was reduced
331 zinc-deficient pigs, both growth hormone treated and un-
txreated, as compared to the other zinc-adequate groups, and
tile differences were significant (P < 0.05). The protein
ccndcentration in liver, as before, was elevated in zinc de-
Ificiency. No significant effect due to growth hormone treat-
Iment was observed in liver protein and RNA concentration on
Zinc-deficient pigs. Liver DN concentrations of the pigs
311 all four groups showed no significant differences. The
I"BESults obtained in this study are essentially similar to
those observed in previous trials. Table 30 shows the
analytical data obtained from the thymus. No differences of
ByUX, DNA and protein content were observed in the thymus of

tYNB pigs in the four groups. Neither zinc nor growth hormone

134

 

 

 

.Aao.o V mvpn "Amo.o V my mosam> ozp pmmoa soap Monmoaw zapsmofiwfizwfimn
.Amo.o V mv odamb ummma smzp Hopmmhm mapSModafianmm
.zmos man no Hoaho oamosmpmm
.onoaaos saxopm n .m .wm
.mflmmn smohg m so ommmohmxm Sofipmapsoosoofi
mm.e m.eo m.mo we.fim m.os emo\:aooowm
mm o m n e.m no.m oom.m <2m\naoeowm
00.0 m.HH an.ma 3.0H H.m ¢2Q\<zm
mfi.o m.m m.m o.m N.m m\wa .dzm
Hm.fi mo.om no.om 3.0m m.ma w\ma .«zm
ma.m m.aefi o.mefi oa.aoa om.moa w\me .eaoooea
: 3 d 3 meQ ho .oz
.Umw Umrw NJMPU PC®HOH%®U QSOHO

.3. I nQHH fig noopoflapmom + moelsN ISN
m h m+ om Na Baa .oafim mampoam

 

.3 A<Hm9 .mm>Hq b0 ZOHEdeZMUZOO <29 02¢

¢zm .ZHmeomm ZO mBZfl£B<HmH mzozmom $.20m0 QZ¢ OZHN NS<BBHQ m0 Hommmm .mN mamfle

135

.amms oSp mo popao oamosmpmm
.ozoaaos SpBOHm u .m .UN

.mHme Smog“ o no Ummmoaaxo Sofipmapsoosoofi

 

 

 

Hm.o w.m o.m H.o o.o <zo\nfioooam
we.fi o.afi e.ofi m.afi o.HH <zm\oaoeowm
no.0 mm.o mm.o Hm.o em.o <2o\<zm
mn.o m.mfi 0.0m o.mfi o.mfi m\ma .«zm
mm.o m.0a m.oa m.o o.ofi m\me .ezm
em.m m.HHfi m.maa m.mfia H.ofifi m\we .caopoam
: d d 3 mmHQ a0 .02
wow wow .m.w pSoHonoo asoau

.n.m- noaa e< -ooeoanemom + oeusm new
m r + om NH and .0:HN hampmfim

 

<Zm .ZHmBOmm ZO mezm

.3 ddeB H.mDENmB m0 ZOHBfimBZMUZOU <ZQ Qz¢

2e mme mzoemom mezomo m2< ozHN mmeemHm ac Bowman .om mamas

136

treatment significantly altered the parameters checked at
this stage of the two-week experimental period.

The results of RNA, DNA and protein analysis of the
pancreas are presented in table 31. No significant treatment
differences were observed in pancreatic DNA and protein con-
centrations. However, pancreatic RNA concentrations were
higher (P < 0.05) in zinc-adequate pigs fed ad libitum as
compared to the deficient pigs. Similar observations were
noted in trial 3.

Since RNA is related to protein synthesis, one might ex-
pect that the protein content of an organ would reflect
cihanges in RNA content of that organ and vice versa. But
tfliis is not what was found in the liver of zinc-deficient
EDigs. In general, liver RNA concentrations decreased with an
Eilevation of protein concentration, therefore, an attempt was
Imade to use a radioactive amino acid as a tracer to determine
if zinc were affecting protein synthesis and RNA metabolism.
Al: the end of the experiment, the pigs were killed and
ESlices of the medial lobe of the liver and of pancreas and
tflfiymus were incubated with L-leucine-UL-luc for 60 minutes.
3318 results obtained in this study are summarized in table 32.
Irlczorporation of amino acids into liver TCA-precipitable
EHNDtein was significantly (P < 0.01) reduced in zinc-

def‘icient pigs as compared to zinc-adequate pigs. A de-

14

(IPEased rate of incorporation of D,L-methionine-2- G into

137

.Amo.o V my moSHmb 03p pmmoa QmSp Hopmmaw hapsmofiwaswfimm

.Qmoa map mo Homao Uhmosmpmm

 

 

 

 

.mcoeaos szohw u .m .Um
.mfimmn smoaa m so oommoaaxo QofipmhpsoocooH
0m.ea H.0m 0.00 m.0o m.ee eao\maooowm
05.0 m.0 m.0 em.0 s.0 <zm\gaoooam
Hm.a 0.HH m.0 m.e m.m <20\<em
em.0 m.m 0.m s.m m.m m\ma .«zo
mm.a om.em e.mm 0.0a 0.0a m\ma .<am
No.0 e.mmfi m.eefi 0.5mfi 0.omfi m\me .gaopoam
: d 3 : mwam mo .02
mom ooh .m.u pzmHOHmoU @5090

.1. . Iowa ea neopoaapmom + moenom new
m a m+ 00 NH sag .osHN hampofia

.3 qume .mammozem m0 oneamezmQZQU «an amm

«am .sza0mm 20 magmzeemme mzoam-H meeomo Qz< osz wmeamHm m0 sommmm .fim mqmwe

.Afio.o V mv moSHmb 03p ammofi Smnp Hopmmaw mapcmofiaanwfimw
.mopscaa ow .oafip nofimeSQQHm

.Qmoa ozp mo Honao osmosmpmm

.ososao: :pBoyw n .m .m.

 

 

 

m
H.H00H wmoaa Heme 0mme omao mmowosom
e.mmem osfisfi mmooa mmo0a mmmafl manage
mfi m.mmm mmHHm mmmfim Mbfifi omfifi ao>flq
1 mwsfimpoaa mo ma\aaov
osfiozofi U and:
mo QoHpmHo HoosH
e e e a mafia ao .oz
60% 69% Hom.U meHOHhmfi QSOHO
.u.nu upfla o< loopofiapmom + .aoous usN
m r -+ 00 NH Eda .oQHN mhmpmam

 

.: A<Hm8 .O$9H> ZH mmoHdm
m<mmoz<@ 02¢ mzfiwmfi .mm>Hq m0 mszBOmm OEZH MZHUDMQ Uh IA: mo ZOHB
IdmommoozH Mme ZO MBZMSB<EmB mzozmom mezomo mad UZHN Nm¢eemo mo Hommmm .Nm Mdm¢e

139

liver protein of rats has been demonstrated in 3132 (Hsu et
al., 1969b). Specific activities in liver protein of the
zinc-deficient pigs treated with growth hormone were not
significantly different from the deficient group which did

not receive growth hormone. These findings are somewhat
different from the observations in hypophysectomized rats,
where stimulation of amino acid incorporation into hepatic
protein after growth hormone administration has been demon-
strated (Korner, 1960). The data in table 32 also show that
.pigs receiving the restricted-fed control diet incorporated
ssignificantly (P < 0.01) more L-leucine-UL-luc into liver
Lxrotein than zinc-deficienspigs. This indicates that the de-
cxreased 14C-leucine incorporation was due to a specific effect
(Df zinc deficiency rather than due to a general reduction in
fNeed consumption. Moreover, no significant treatment differ-
Enices were observed in specific activities between restricted-
Ifed.control and ad libitum-fed control pigs. It is apparent
'that a reduction in feed consumption did not affect the pro-
'teixisynthetic capacity under these conditions of experimenta-
tion. Increased oxidation of amino acids in zinc-deficient
1%rts has been reported by Theuer and Hoekstra (1966) and Hsu
it a_l_. (19693:). With these findings, the question arose
thEther increased oxidation of amino acids in zinc-deficient

raizs is due to a decrease in the availability of protein pre-

Clursors (other amino acid sources). Recent studies of

140

Nacapinlac gt al. (1968), using the testes of zinc-deficient
rats, reported that 14C-leucine and 1LLC-adenine incorporation

into protein and RNA was unaltered. Thus, nonlabeled amino

acid mixtures (see Appendix 1, table 5) which were comprised l
of twice the plasma amino acid concentrations found in the

young pig receiving a complete soybean meal diet (Puchal at

al,, 1962) were provided in the incubation medium. In general,

 

 

liver amino acid concentrations are two to three fold higher
than in blood plasma. Thus, the addition of those concentra-
‘tions of amino acids to the incubation medium would more
<3losely approximate the liver levels. However, the experi-
Inental data showed that the addition of these precursors had
110 promoting effect on protein synthesis in zinc-deficient
Sliver slices. The decreased amino acid incorporation into
lgiver protein in zinc deficiency is apparently not a conse—
QJience of the shortage of precursors.

Results of the effect of zinc deficiency and growth hor-
Iflone treatment on the uptake of labeled leucine by the thymus
éire shown in table 32. No significant treatment differences
Vfiere observed in protein synthesis between thymus of zinc-
<iefdcient and zinc-adequate pigs. Growth hormone treatment
Of‘ the zinc-deficient pigs did not produce any response in
tkwfmus protein synthesis as compared to the other deficient
$1T>up receiving no growth hormone injection. Despite the

laxxk of statistical significance, trends were apparent toward

141

decreased incorporation of amino acid into protein in zinc-
deficient thymus.

Experimental data (table 32) obtained from the study of
leucine incorporation into pancreas did not show any signifi-
cant differences between treatments. Cnly the zinc-adequate
ad libitum-fed control group had slightly higher incorporation
rate than the remaining treatment groups. It has been
mentioned before that increased feed intake may stimulate the
metabolic activity of pancreatic cells, and the pancreas is
known to synthesize more protein per gram of gland than any
other body tissue (Kukral 33 al., 1965). Zinc-deficient pan-
creatic cells still capable of synthesizing proteins have
been reported in rats (Hsu 33 al., 1969b). No significant

14

growth hormone treatment effects on C-leucine incorporation
into the protein of zinc-deficient pancreas were observed.
Results of the pancreatic carboxypeptidase assay in trial
4 (table 33) established no significant difference in activity
due to zinc deficiency under these experimental conditions.
Growth hormone treatment of zinc-deficient pigs as compared
to the zinc-deficient pigs receiving no growth hormone treat-
ment also produced no response. This is somewhat different
than the results reported by Hsu at al. (1966) and kills e_
al. (1967). Using weanling rats fed zinc-deficient diets for

either at least 35 days (diet contained 0.75 ppm Zn (Mills et

al,, 1967)) or 103 days (diet contained 0.5 ppm Zn (Hsu §£_§lg.

 

142

 

 

 

 

E51”... u n. ...... -Lriphr
.Qmos ogp mo Hoaao oamosmpm
.osoeaos SpZoaw u .m .03
mm.m 0.mm e.em m.am 0.mm .sas 0m\saoeoaa
wa\oom5 mpmhmeSm
moaous apabflpom mammsm
3 3 3 3 mmfla mo .02
tom tom w.m.o psofloflmmo QSOHD
.a.o- uoaa ea -eoeoaaonom +.q e-sm usN c
m I c+ om. NH Eda .ocflw mampoflm

 

.3 Q¢Hme .MBH>HBL4 mm<QHBmmmwmomm<o 0HE<flmo
mHEHEB<fimB Hzosmom mflzomw QZ< UZHN deEmHQ mo Boummm .mm Mdm<e

143

1966)), they found that pancreatic carboxypeptidase A, not
carboxypeptidase B (Hsu gt al,, 1966) or gross carboxypepti-
dase activity (hills gt al., 1967) decreased significantly

as compared to the control group. One possible explanation

 

for this discrepancy is that the experimental period in this
study was not long enough to manifest such an effect due to

zinc deficiency. Likewise, no significant treatment differ- 3_

 

ences were observed in 1LPG-leucine incorporation into pan- éj
creatic protein between zinc-deficient and zinc-adequate pigs.
These observations, at least in part, may explain why zinc
deficiency at this stage had little effect on pancreatic
function. Evidence to date indicates that notall tissues
show changes to the same extent as a result of zinc deficiency.
A few tissues are more sensitive than others (Prasad gt al.,
1969). Therefore, one would not be surprised that pancreatic
carboxypeptidase activity had not changed at this early stage
of zinc deficiency. It is also possible that in the presence
of a subcritical level of zinc, the pancreatic cells are
still able to maintain quite normal functions.

Chemical analysis of the three selected tissues of the
zinc-deficient pigs of this study (trial 4) showed that
liver was apparently more severely impaired than thymus and
pancreas. Total nitrogen, protein nitrogen and non-protein

nitrogen values of the liver of pigs in the four groups are

um

presented in table 34. Significant (P < 0.05) treatment
differences were observed in liver non-protein nitrOgen con-
centrations between zinc-deficient and zinc-adequate pigs.

Such an effect of zinc deficiency on rat testes non-protein

 

F-
nitrogen has been reported by Somers and Underwood (1969). E
Similar observations of increasing non-protein nitrogen and I
acid-soluble amino acids in plants and microorganisms caused
by zinc deficiency have also been reported (Possingham, 1956; g,

Schneider and Price, 1962; Hacker, 1962; Kastori, 1969).
Growth hormone treated, zinc-deficient pigs showed no signifi-
'cant differences in liver non-protein nitrogen concentrations
as compared to the zinc-deficient pigs without growth hormone
treatment, although liver non-protein nitrOgen concentrations
in growth hormone treated zinc-deficient pigs tended to be
decreased. The difference in liver non-protein nitrogen con-
centration between growth hormone treated zinc-deficient and
restricted-fed control pigs was significant (P < 0.05); how-
ever, the difference between growth hormone treated zinc-
deficient and dd libitum-fed control pigs was not. The con-
centration of protein nitrogen in the liver of the zinc-
deficient, growth hormone treated or untreated pigs tended

to be higher than in either of the control groups, although
there was no statistically significant difference. This

finding was in contrast to the observation in rat testes

145

.Amo.o V my mmsfiw> 03p pmmma camp Hmpmmwm mapsmoawflsmflmp

.Aao.o V mvwm "30.0 V mv msam> pmmma smzp Hopdohm mapmmOHgfimemm
.Qwoa esp mo HOHHo chmcsmpmm

 

 

 

.msoaaos SpBOHw n .m .ow
.mHme Smowm m :o pmmmmhmxo Goapmhpsmosoow
mm.o oa.m mm.m mam.m now.m w\ms .2 sampowassoz
om.o mm.mm fifi.mm mm.mN om.hm ,mw\mE .2 Qflmpohm
ms.o mo.mm we.sm mqfi.am p.mmom.wm m\me .2 Hmpoe
: 3 3 : mwfid mo .02

U09 pow .m.o pnmfioammp
.J.o- -QHH we -empoflppmmm +mcmeusm new dsowo
k C+ 0% NH and .oQHN mwmpmfim

 

IULHN mo ZHUOmBHZ EH38
q

.: q<Hma H.mon momezoo mze ezmHonmo
62¢ :meomaHz szeomm .zmcomaH: neeoa
mam mezome 62¢ ozHN MmeamHo mo Hommrm .em mamas

mmnaoz
a a

O H

1u6

where non-protein nitrogen concentration increased and the
concentration of protein nitrogen significantly decreased in
zinc-deficiency (Somers and Underwood, 1969). No explanation
for this discrepancy was available. It is possible that the
different results obtained may be due to different tissues
analyzed, species and period of experimentation between these
two studies.

The liver free ¢<-amino nitrogen, liver free methionine,
isoleucine and leucine concentrations of the pigs in the
four groups, expressed as‘y moles of either ¢(-amino nitrogen
or amino acid per g of liver, are presented in table 35.
Total liver free cK-amino nitrogen tended to be increased in
zinc-deficient pigs as compared to zinc-adequate, restricted-
fed control pigs. The difference in livercd-amino nitrogen
concentration between the zinc-deficient pigs administered
growth hormone and the zinc-adequate restricted-fed control
pigs was significant (P < 0.05). However, liver c‘-amino
nitrOgen level was also higher (P < 0.05) in zinc-adequate,
aa libitum-fed pigs as compared to the zinc-adequate
restricted-fed group. This difference was probably due to
the fact that zinc-adequate aa libitum-fed pigs consumed more
feed; consequently, greater amounts of protein were ingested
and resulted in an increase in liver free v‘-amino nitrogen.

An increase in free amino acids in swine plasma after feeding

147

.Afio.o v mama “30.0 v m0 msam> pmmma gasp ampmmpm gapsmoapfizmamw
.smoa esp wo Howam waQUQMQmm
.mmoaaos Spaoaw n .m .wm

.mHme Smohm m :0 pmmmohaxo SOHpMHmeoSoo

 

 

 

H
00.0 mm.0 03.0 00.0 mmm0.0 w\mHoa 1..maaosmq
No.0 mm.0 mm.0 mm.0 mm.0 m\maoe 1..oafiosmaomH
no.0 :H.o ofi.o NH.o om.o m\maoa a .msaqOfigpwa
mm.m www.mm 00.00 mefi.fim 00.00 0\oHoB a.

.smmoapH: oQHEman
e 0 0 0 mmfld mo .02
:00 00 «.3 .0 psmfiofimmp macaw
.e.ol IQHH pd Ifimpofihpmmm + w®UICN ISN
m x c+ 00 NH and .oQHN AHonHQ

 

.3 Q¢Hme .mUHm dem QOmB
IZOD 9.3. BamHonmQIOH/HHN mo mQHU< OZHFQ mmmm 02¢ ZML mBHZ 025,2... V‘s
mmmm mm>HQ ZO mBszB¢mmB mzozmom mezomw QZ¢ UZHN Mm<BMHD mo Hommmm .mm mqm¢9

1&8

high protein diets has been reported (Richardson a: al., 1965;
Krysciak ag al., 1966). The observation that the free amino
nitrogen content of the liver increased was also reported by
Van Slyke and Meyer (1913) when dogs were fed a high protein
diet.

No significant treatment differences were observed in
liver free methionine and isoleucine concentrations of pigs
in the four groups. Only liver free leucine concentration,
the amino acid of most interest in the present study, tended
to be higher in the pigs fed zinc-deficient diets, either
treated or untreated with growth hormone, as compared to the
zinc-adequate restricted-fed pigs. And the differences of
liver free leucine concentration between zinc-deficient and
zinc-adequate restricted-fed pigs was significantly (P < 0.01)
different. It has been reported that plasma isoleucine and
leucine increased in rats receiving diets alike in protein
but low in zinc as compared to rats supplemented with zinc
(Riley a; al., 1969). These findings may indicate that zinc
has an effect on amino acid utilization, although no plasma
free amino acids and not all liver free amino acid level de-
terminations were made.

From the observations of decreased 1LPG-leucine incorpora-
tion into liver protein, and the increase in liver non-protein
nitrogen fraction, free a‘-amino nitrogen and free leucine

concentrations of zinc-deficient pigs, it would seem that

149

zinc deficiency may also change the amino acid pattern in the
body of the zinc-deficient animals. Almquist (1954) and
Kellinkoff (1957) have suggested that changes in blood amino
acid pattern, especially an accumulation of amino acids which
cannot be diverted into protein synthesis may serve as a
satiety signal for an appetite-regulating mechanism and
thereby result in depressed food intake. More recently, an
inverse relationship between the concentrations and patterns
of plasma and liver amino acids and food intake in rats has
also been reported (Peng and Harper, 1969). If amino acids
did accumulate in zinc-deficient pigs as has been reported

in plant leaves (hastori a; al., 1969; Possingham, 1956) and
in microorganisms (Schneider and Price,1962; Wacker, 1962) and
the suggestions of Almquist (1954) and Hellinkoff (1957) were
correct, then the depression in feed intake observed in zinc-
deficient animals may be, at least in part, explained this

way .

150

General Discussion

Many reports relating dietary zinc deficiency to nucleic
acid and protein metabolism in rats have been published
(Theuer and Hoekstra, 1966; Kacapinlac a; al., 1968; Hsu a2
al., 1968a,b; Riley a; al., 1969; and Somers and Underwood,
1969).

From data obtained in the present experiments, it would
appear that zinc deficiency had effects on nucleic acid metabo-
lism and protein synthesis in certain tissues of baby pigs.

Linc deficiency seemed to have a more detrimental effect
on pig liver than other selected tissues studied. Liver RNA
concentration was depressed and the protein concentration was
elevated in zinc deficiency in all trials. Since RNA is re-
lated to protein synthesis, decreased RNA would indicate the
metabolic activity of the cells was impaired. In the 14C-

f 14C-leucine

leucine incorporation study 12.X2E£2: the rate 0
incorporation into liver protein was significantly depressed
in zinc deficiency. These findings are in agreement with
those of Hsu a2 al. (1969b) who reported that rats, fed a
semi-purified diet deficient in zinc, incorporated signifi-

_14

cantly less DL-methionine-2 C into liver and kidney proteins

la vivo. However, liver protein concentration does not
parallel the decrease in liver dNA or the significantly

14

(P < 0.01) lowered incorporation of C-leucine into liver

protein in zinc deficiency. No explanation can be given for

151

the inverse relationship between liver protein concentration
and RNA level or protein synthesis rate observed in the pre-
sent experiment. But increased total serum protein concen-
tration and changes in serum protein pattern of zinc-deficient
pigs and lambs have been reported (Miller a; al., 1968; Ott
a_ al., 1964). A significantly (P < 0.01) higher level of
nonprotein nitrogen, cK-amino nitrOgen and leucine in zinc-
deficient pig liver was observed in trial 4. Similar obser-
vations of higher non-protein nitrOgen in zinc-deficient rat
testes and zinc-deficient rat plasma amino acids have been
reported (Somers and Underwood, 1969; Riley at_al,, 1969).
The findings in the present study may imply that zinc de-
ficiency resulted in not only an effect on protein synthesis
but also in a substantial lag in protein utilization. Liver
NA concentration was not affected in any of the four trials.
Based on these observations, there seemed to be neither de-
creased DKA synthesis or increased DNA catabolism due to
zinc deficiency. Similar observations have been made by
Macapinlac at al. (1968) who reported that there was no gross
impairment of DNA synthesis in the testes of zinc-deficient
rats. However, conflicting evidence on the effect of zinc
deficiency on rat liver DNA synthesis has been reported

(Fujioka and Lieberman, 1964; Sandstead and Rinaldi, 1969;

Buchanan and Hsu, 1968; Weser a: al., 1969).

152

The effect of zinc deficiency on thymus nucleic acid
and protein was quite different from the effect on liver.
In trials 1, 2 and 3, thymus DNA concentration was depressed
in zinc deficiency. On the contrary, no change in RNA con-
centration was observed. It is apparent that the effect of
zinc deficiency is different on different tissues. The thymus
is a lymphoid organ that plays a vital role in the development
of the entire lymphoid system and immunological competence
(Clark, 1964). It is well known that thymus cells are rich
in DNA content. The great decline in thymus DNA concentra-
tion in zinc deficiency indicates the thymus gland has under-
gone a significant structural and metabolic change. This
could have been either a change in predominant cell type with
fewer but larger cells being present, or else the cells norm-
ally present have undergone hypertrophy. As can be seen
from tables 4 and 13, thymus weights in zinc deficiency were
severely depressed. Consequently, it appears that under the
conditions of these xperiments thymus NA synthesis was
seriously impaired or DNA catabolism was exaggerated by zinc
deficiency. One may also have to take into consideration that
it is possible that zinc has another important role, namely,
in influencing cell division. Thymus protein was depressed
in the first three trials. But, no treatment differences were
observed in thymus protein in trial 4. This difference may

be attributed to the experimental period which was not long

153

enough to produce such results in trial 4.

Pancreatic RNA concentration tended to decrease, but not
much change in protein concentration was observed in zinc de-
ficiency in the first three trials. Pancreatic DNA concentra-
tion in two out of three trials was decreased. These observa-
tions imply that the zinc-deficient pancreas has fewer but
larger cells (lower concentration of DNA) which are less
active metabolically (lower concentration of RNA). No sig-
nificant treatment differences were observed in these para-
meters in trial 4 when pigs were receiving a zinc-deficient
diet for two weeks only.

At any rate, data obtained from these experiments would
suggest that there is little doubt that zinc deficiency re-
sulted in an effect on nucleic acid metabolism and protein
synthesis in the pig, and that this species is similar in
this respect to the rat (Hacapinlac 23 al., 1968; Hsu a: al.,
1969a,b; Somers and Underwood, 1969).

However, precisely how zinc is involved in nucleic acid
metabolism and protein synthesis is not completely under-
stood. Zinc has been found in RNA and DNA, and it has been
demonstrated that zinc is firmly bound to the nuclein acids
(Hacker and Vallee, 1959; Belokobylskii a: al., 1968). It
has been suggested that zinc has a role in determining and

stabilizing the configuration of these nucleic acids and in

154

maintaining their biological activity (Schneider and Price,
1962; Hacker, 1962; Hegner and Romano, 1963). Kore recently,
zinc has been shown necessary to maintain the ribosome part-
icles in their normal state (Tal, 1968; 1969a,b). A molar
relationship between RNA and zinc in the liver of the normal
rat fed a zinc-adequate diet was observed (Holt €3,2l-' 1970).
Many investigators have found that ribonuclease is a
protein inhibitor of the cell-free protein synthesizing sys-
tems isolated from many different organisms (Allen and Schweet,
1962; Hardesty et al., 1963; Korner, 1961; Talal, 1966). In-
corporation of amino acids into TCA-precipitable protein was
inhibited to the extent of 90 percent by as little as 0.1,ug
of pancreatic ribonuclease in a cell-free system derived from
rabbit reticulocytes (Allen and Schweet, 1962). Thus, the
effect of ribonuclease on protein synthesis could have been
at the ribosomal level. Later experiments with E; aall and
rat liver ribosomes indicated that this nuclease destroyed
the intact messenger RNA causing a degradation of polyribo—
somes to monomeric ribosomes (Hangiarotto and Schlessinger,
1966; Franklin and Godfrey, 1966). Beitz a: a_l_. (1969) found
that no 1[‘LC-leucine was incorporated into protein when only
1 pg of pancreatic ribonuclease was added to a cell-free
system isolated from lactating bovine mammary tissue due to

the destruction by ribonuclease of the intact messenger RNA

 

155

causing polyribosome breakdown. Because polyribosomes
(aggregates of ribonucleoprotein particles held together by
strands of messenger RNA (mRHA)) are the major synthetic form
of ribosomes, they are essential functional units in protein
biosynthesis (Schweet and Heintz, 1966; Warner, EB al., 1963;
Wettstein a3 al-: 1963); and degradation of the mRHA of the
polyribosome caused inhibition of protein synthesis.

On the other hand, zinc acts as an effective inhibitor
of ribonuclease which, may, in part, be responsible for con-
trol of ribonuclease activity in living organisms (Ohtaka a:
al., 1963). That the ribonuclease activity of apple and
citrus leaves increased sharply in zinc deficiency has been
demonstrated (hessler and Honselise, 1959; Kessler, 1961).
Wojnar and Roth (1964) reported that RNA molecules containing
zinc inhibited ribonuclease activity. Wore recently, Somers
and Underwood (1969) observed that the testes of zinc-
deficient rats contained significantly higher ribonuclease
activity and decreased RNA, DHA and protein concentrations.

From these findings, a tentative conclusion may be
reached. It seems that zinc is important for the maintenance
of normal nucleic acids synthesis via the inhibition of
ribonuclease activity and the stabilization of nucleic acid
configuration structurally and functionally so that normal
protein synthesis in mammalian tissues can proceed. Enzymes

are protein in nature, furthermore, and zinc is quite evi-

 

156

dently involved in enzyme function as an activator (Plocke a:
al., 1962; Simpson and Vallee, 1969; Vallee a3 al., 1963;
Drum a3 al., 1967, 1969; Himmelhoch, 1969). Therefore, when
animals are zinc deficient, the balanced relationship of the
metabolic reactions involving zinc and ribonuclease activity
and nucleic acid synthesis would be deranged, with a conse-
quent effect on protein metabolism in the tissues.

It is well documented that hypophysectomy of young rats

leads to a retardation or c ssation of the growth of the

(D

liver and other tissues. The administration of growth hor-
mone to hypophysectomized rats has been shown to stimulate
liver growth (Simpson a3 al., 1949) and to increase the con-
centration of liver RNA and protein (Geschwind a£,al.. 1950).
Stimulation of amino acid incorporation into protein after
growth hormone administration to rats has been demonstrated
;a alga (korner, 1960), and a stimulation of the incorporation
of RNA precursors in rat liver RNA has been reported (Talwar
a3 al., 1962; Iwamoto a: al., 1963; Talwar a: al., 1964;
Korner, 1964). In more recent work, Nidnell and Tata (1966)
reported that stimulation of RNA synthesis followed by that
of protein synthesis resulted from the action of growth hor-
mone on liver. Korner (1964) reported that growth hormone
was able to exert its stimulatory effects on the protein-

synthetic ability of ribosomes, and on a number of polysomes

in rat liver. Therefore, this stimulatory effect of growth

 

157

hormone was largely localized in the ribosomes of the target
cells (Hilliams-Ashman a2 al., 1964; Korner, 1965; Muller,

1965). From these reports, the hormonal control of protein
synthesis can be explained in terms of its effect on the F1
ability of ribosomes to assemble activated amino acids into I

polypeptide chains.

No significant effects of growth hormone treatment on

 

zinc-deficient pigs were observed in the present study. 5g
Similar observations in zinc-deficient rats have been reported
(Hacapinlac 33 al., 1966; Prasad a: al., 1969). What the
relationship between growth hormone and zinc is and how

their interaction affects growth is not known. But, based

on the observations of the effect of zinc on ribosomal con-
figuration integrity, ribonuclease activity inhibition and

the stimulatory effect of growth hormone on ribosomal func-
tions, one might speculate on some possible explanations.
First, zinc may be necessary for growth hormone production

or release by the pituitary, but these factors were not
measured in this study. In addition, its stimulatory effect
on cell growth may be mediated through zinc; therefore, a

lack of zinc may block the action of growth hormone upon the
target cells. Secondly, since there is strong evidence that

zinc deficiency has a serious effect on nucleic acid metabolism,

158

this may explain why growth hormone administration to zinc-
deficient animals did not produce a significant response. It
is apparent that growth hormone cannot exert any stimulatory
effect on ribosomes if their functions were impaired by

zinc deficiency. Likewise, one would not expect growth hor-
mone administration to stimulate protein synthesis. Under
these circumstances amino acid utilization would be decreased
and an elevation of amino acid concentration would occur,
which in turn would depress feed intake.

If these speculations are correct, then the relationships
of zinc, growth hormone, nucleic acid and protein synthesis
may be illustrated by the following scheme (Fig. 1).

This scheme is a hypothetical supposition outlining
possible interrelationships of zinc, growth hormone, nucleic
acids and protein metabolism in the animal body. Clearly,
further work must be done before conclusive statements can be
drawn. In future experiments, measures of growth hormone
blood levels, cellular ribosomal integrity, ribonuclease
activity, and amino acid concentration and pattern in zinc-
deficient pigs should be determined. When these results are
obtained, they will provide more information on zinc's action
at the cellular level and will further elucidate what rela-
tionships exist between zinc and the failure of growth and
the depression of feed intake in pigs suffering from zinc

deficiency.

 

159

   

 

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Scheme showing possible interrelationships of
zinc, growth hormone, nucleic acids and protein
metabolism in the animal body.

a. +Zn = zinc adequacy. -Zn = zinc deficiency.

b. Solid line represents the reactions occurring
in the zinc-adequate condition.

0. Dashed line represents the reactions which
will either be increased or decreased in
the zinc-deficient condition.

 

 

 

V . CONC LUS ION

Within the limits of the experimental conditions employed,
the following conclusions can be made:

1. Low levels of dietary zinc caused severe reductions
in growth rate, feed consumption, gain/feed ratio, serum

alkaline phosphatase activity and serum zinc concentration.

 

2. Weight gain decreased in zinc-deficient baby pigs
before feed intake was reduced. The skin lesions that de-
veloped due to zinc deficiency were consistently observed by
the end of the second week of the experiments.

3. Serum alkaline phosphatase activity and serum zinc
concentration were sensitive measures of dietary zinc ade-
quacy in intact animals.

4. Selected organs (liver, thymus, pancreas, kidneys
and spleen) of zinc-deficient pigs were reduced in size and
‘weight on a fresh basis, an exception being the absolute
‘weight of the brain, which was almost identical to the brain
‘weight of pigs receiving zinc-adequate diets.

5. Liver RNA concentration was depressed in zinc de-
ficiency. Protein concentration, non-protein nitrogen and
:flree leucine in liver were higher in zinc deficiency. DNA
<3ontent in zinc-deficient liver was comparable to the con-
trols.

161

162

6. Thymus DNA content was significantly decreased in
zinc-deficient pigs, and protein concentration tended to be

depressed. Essentially no change in RNA concentration was

observed in zinc-deficient pigs.

7. Pancreatic RNA concentration tended to decrease in
zinc-deficient pigs, but there was not much change in protein
concentration. DNA concentration tended to be depressed.

8. Essentially no changes in RNA, DNA and protein con-
tent were noted in the brain, kidney and spleen of zinc-
deficient pigs.

9. The incidence of parakeratosis, and depressions of
feed intake and weight gain were not improved by additions

of biotin to the zinc-deficient diet.

10.- Supplementation with biotin did not affect the RNA,
IHLA and protein concentrations in liver, pancreas, thymus and
:spleen.of the pigs receiving either zinc-deficient or zinc-
zwdequate diets.

11. Administration of growth hormone to zinc-deficient
piggs failed to stimulate weight gain or feed intake and had
IN) effect on tissue nucleic acid and protein concentrations.

12. 1LPG-leucine incorporation into liver protein was
siguiificantly decreased by zinc deficiency and was not im-

pmnyved.by prior growth hormone administration.

 

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"\

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APPEE‘ID Di

190

APPENDIX I. TA LE 1. LINERAL EIXTURE USED IN EKPERINENTAL

 

 

DIET.

%
KCl 10.0
KI 0.002
FeSOQ°2H20 0.7
CuSOu 0.1
CoCO3 0.1
HnSOQ-HZO 0.1
ZnSOu-HZO o.oa(o.4)b
figCOB 2.0
NaHCO3 25.0
CaHPOu‘ZHZO 36.0
CaCO3 12.5
Cerelose 13.098

 

aZinc deficient diet.
bControl diet.

191

APPENDIX I. TABLE 2. VITAMIN MIXTURE USED IN EXPERIMENTAL

 

 

 

 

DIET.

_ppm in diet [a
Thiamine mononitrate 3 7”“
Riboflavin 6
Nicotinaminde 4O 5'»
Calcium pantothenate 30 SJ
Pyridoxine hydrochloride 2
Para-amino benzoic acid 13
Ascorbic acid 80
d-¢*-Tocopheryl acetate 10
Inositol 130
Choline chloride 1300

ppb in diet
Pteroylglutamic acid 260
Biotin 5O
Cyanocobalamin 100
2-Hethyl-1,4-naphthoquinone 40
Vitamin A palmitate 1500

Vitamin D2 12.5

 

 

APPEI‘JDIX I. TABLE 3. SOLUTIONS FOR d—AEINO NITROGEN ‘ "
~'.‘1."I‘L:'I;i1'1INA'TION

 

0.25% 2,4,6-trinitrobenzene 2.5 g TNBS/liter H20, made
sulfonate (TNBS) fresh weekly, stored in a
brown bottle and refriger-
ated
0.05 sodium borate buffer, 19.07 g NaZBgo '10H,O/liter
pH 9.2 H20, pH adgustgd wiéh NaOH
1 N HCl
DL-Citrulline standards 1.7520 g citrulline/liter

H 0 = 10 p.moles/ml
aépropriate dilutions made
to obtain standards of
lesser concentration.

 

APPENDIX I. TABLE 4.

193

ARINO ACID

COMPOSITION OF KHEBS-

HINGEH BUFFER SOLUTION

 

L-Aspartic acid
L-Threonine
L-Serine
L-Glutamic acid
L-Proline
Glycine
L-Alanine
L-Cystine
DL-Valine
L-Nethionine
L-Isoleucine
L-Leucine
L-Tyrosine
L-Phenylalanine
L-Lysine-HCI
L-Histidine°HCl
L-Arginine°HCl

L-Tryptophan

mg/500 ml

 

2.80
22.50
40.05
23.55
33.20
57.85
44.70

5.45
30.10
16.05
18.45
22.30
18.00
10.95
57.10
14.80
22.50
16.05

 

 

“1 {E
Pm. .7

194

APPENDIX I. TABLE 5. COMPOSITION OF KREBS-BINGER BUFFER
SOLUTION BSFOfiE AMINO ACID ADDITION,

 

 

 

pH 7.4
Concentration
0.9% NaCl 100 ml
1.15% KCl 4 ml
0.6% CaCl2 3 ml
3.8% MgSOu-7H20 1 ml
2.11% KHZPOQ 1 ml

2 for 1 hour) 21 ml

Glucose 0.268 gm

1.3% NaHCO3 (gassed with CO

 

195

APPENDIX I. TABLE 6. LIQUID SCINTILLATION COUNTING SOLUTION

 

p-Dioxane

Xylene

Absolute ethanol

PPO (2.5-Diphenyloxazole)

Naphthalene

385
385
230
5
80

13012019 (1,4-813 C2-(5-phenyloxazolle-

benzyne, phenmS-oxazolylphenyl-

oxazolylphenyl

Thixotropic gel powder
(Cab-O-Sil)

100

25

ml
ml
ml

gm

gm

ms

gm

 

 

AEPENDIK II

The disage for administration of growth hormone was cal-
culated from the following information presented by hachlin
et al. (1968).

1. The overnight fasting values of plasma growth hor-
mone (GE) for young pigs (9 to 12 kg) were 10.7i
4.8 ng/ml.

2. GH disappearance from plasma was linear, with a
half-life varying between 20 to 30 minutes.

3. Blood plasma GH concentration was in equilibrium
with extravascular, extracellular fluid GH con-
centration.

4. Total extracellular fluid (including blood plasma)
comprised 15 to 18 percent of body weight.

5. Plasma GH turn-over rate was 2.3 to 2.8 percent
per minute.

Calculations:

Assume pigs weigh approximately 2.5 kg and the plasma
growth hormone (GH) level is 10 ng/ml and GH is distributed
in 16 percent of the body weight. Then:

a) Total ug GH in the extracellular space will be (A)

A _ (Body Weight) (Distribution percentage) (CH level)
_ 1000

b) Total GH turn-over per day will be (GH in the extra—
cellular space) (Turn over rate) (24 hour) (00 minute)
= Total GH (mg/day) when GH activity is 1.1 IU/mg.

c) If the GH activity is 0.7 IU/mg then the dose for a
2.5 kg weight pig will be (X)

(Total GH mg/day)_(1.1 IU/mg)

X = 0.7 IU/mg

Based on this information, 0.23 mg GB at an activity of

of 0.7 lU/mg was used for each pig per day on trial 4.

 

2a) _(2.5) (0.16) (10) = 4

.0 ug GH in the extracellular
1000 spa

C

(D

b) (4.0) (€33) (24) (60) = 0.144 mg/day (1.1 IU/mg 011)

)I
c) X = (0.144) (1'1) = 0.2262 mg GH/day

0.7