‘ .v.
v .. .3 3.;

 

. :2... . .
.3. V

I

.2. .
f...

\l.I

p L. 9.5.x? .3 V.

.3: «It ; f
.... 1233...»,
. ,. .5,

{5 . .
.ry..k:l.r...f
In. liv‘.$§

. Vang“
.4

3
I 54;...»
n3: v, .
Panama.
ifim‘. 5:31?
7..“ 43“.;

.3131. 5... .
:u -..,,...f9.1...§v

a}. ”N... t.
.. l. .
I «.1

|~§l

I...

is... .1.
.vruifu. .3:

anus).

.5. .
5%»

«.31!!! 1.3".
. z .. {3

I 3

N. .

r If 4 in

.3. AW.»
.
.

“EN.”
.1184” 1.5.1! r.
V org 1 ft.
.Afipfikiwmwa fl

.4

m...
1
rear“

N

.11....nwn‘h.

.

34.3”“ _
.uunh

 

”IL-9: 25

99”)";

(,2 :J' J' 7 ”4‘77

This is to certify that the
dissertation entitled

EFFECTS OF PHARMACOLOGICAL ZINC AND PHYTASE
SUPPLEMENTATION ON METALLOTHIONEIN,
HEPATIC DIFFERENTIAL GENE EXPRESSION,

ZINC TRANSPORTER-1, MINERAL EXCRETION, AND
APPARENT RETENTION IN NEWLY WEANED PIGS

presented by
Michelle Marie Martinez

has been accepted towards fulfillment
of the requirements for the

Doctor of Philosophy degree in Animal Science

 

 

@100. m

a Major Protes’sor’s Signature

September 24, 2004

 

Date

MS U is an Affirmative Action/Equal Opportunity Institution

 

LIBRARY
Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c:/ClRC/DateDue.p65-p.15

 

 

 

EFFECTS OF PHARMACOLOGICAL ZINC AND PHYTASE
SUPPLEMENTATION ON METALLOTHIONEIN, HEPATIC DIFFERENTIAL
GENE EXPRESSION, ZINC TRANSPORTER-1, MINERAL EXCRETION, AND
APPARENT RETENTION IN NEWLY WEANED PIGS

By

Michelle Marie Martinez

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Science

2004

ABSTRACT

EFFECTS OF PHARMACOLOGICAL ZINC AND PHYTASE
SUPPLEMENTATION ON METALLOTHIONEIN, HEPATIC DIFFERENTIAL
GENE EXPRESSION, ZINC TRANSPORTER-1, MINERAL EXCRETION, AND
APPARENT RETENTION IN NEWLY WEANED PIGS

By

Michelle Marie Martinez

The swine industry feeds pharmacological zinc (Zn) as Zn oxide to newly
weaned pigs to improve growth, while exact mechanisms are unknown,
metallothionein (MT) may be involved. Environmental concerns arise due to high
Zn excretion. Exogenous phytase improves mineral availability from swine
feedstuffs. We hypothesized that adding phytase would reduce the amount of Zn
needed to elicit a beneficial response. Experiment 1: pigs (n=96, 5.5kg, 18d)
were fed 150,1000 or 2000 ngn/kg with or without phytase (+P or -P,
Natuphos®, 500 FTU/kg) for 14d post-weaning. Liver and kidney Zn, and relative
MT mRNA abundance and protein were greater (P<0.05) in pigs fed 1000+P,
and 2000-P or 2000+P vs. the remaining treatments. Duodenal MT mRNA
abundance and protein were greatest (P<0.05) in pigs fed 2000+P. Zinc
Transporter-1 (ZnT-1) protein (~50 kDa) was identified. Pigs fed 150 and 1000
had similar ZnT—1 protein expression in the liver, while pigs fed 2000 had
reduced expression (P<0.02). Fifty-two amplicons putatively differentially

expressed were identified by differential display RT-PCR in the liver. Five

transcripts with increased expression in pigs fed pharmacological Zn with
sequence similarities to glyoxalase 1 (GLO1), peroxiredoxin 4 (PRDX4),
aminoacylase I (ACY1), orosomucoid 1 (ORM1), and carboxypeptidase U
(CPBZ) were confirmed. Relative GLO1 (P<0.01), PRDX4 (P<0.01) and ACY1
(P<0.01) mRNA abundances were greater in pigs fed 1000 and 2000 than in pigs
fed 150, while relative ORM1 and CPBZ abundances were not affected.
Experiment 2: pigs (n=24, 7.2kg, 20d) were individually fed 100, 1000 or 4000
ngn/kg with or without phytase (+P or -P, Natuphos®, 500 FTU/kg) for 14d post-
weaning, followed by a 100 ngnlkg diet for 7d. Feeding 4000 resulted in greater
(P<0.01) plasma, hepatic, and renal Zn, and hepatic, renal, and jejunal MT
protein than pigs fed 100 or 1000. Duodenal MT was greater (P<0.01) in pigs fed
1000 and 4000 vs. pigs fed 100. Phytase did not reduce Zn excretion. Daily fecal
excretion indicated that feeding pharmacological Zn for 11d vs. 14d reduced
fecal Zn by 50%. Phytase and pharmacological Zn beneficial effects are

independent and not additive, our hypothesis is rejected.

I dedicate this to my dear partner in love and life Carlos M. Lao Dévila and our
growing family. Your constant support, undoubted love, fantastic sense of
humor, steady perspective, persistence at large, fabulous evaluation of life,
questioning everything, uncommonness, passion outdoors, routine nonsense,
outliving star kept me going all these years like essential fluids required for life.
Junta a ti he descubierto muchas cosas. Entre ellas que Ios limites no existen,
aprendi que la Iey es ficcion, a paranne, y que en el mundo de Ios Iocos eI
cuerdo es el loco. Como dice Silvio: “Eres como esos dias en que eres la Vida y
todo lo que tocas se hace pn'ma veia”. I/uminas mis dias y mi Vida con tu
presencia. Te amo. May our future together be full of joy, health and wonderful

moments, m

ACKNOWLEGDEMENTS

My most sincere thank you to Carlos and my family, I appreciate the
support and love throughout the years invested in MSU.

Dr. Catherine W. Ernst for your constant support and for teaching me
many things of science and life, many of which I will further practice in my future
and evolving career. Dr. Gretchen M. Hill, my major professor and Drs. Dale
Rozeboom, Kevin Roberson, Wanda Chenoweth, members of my guidance
committee, for their helpful suggestions and ideas, accessibility and time.

I thank the Michigan Agricultural Experiment Station, the USDA, and EPA
for financial support for my research projects, The Graduate School for the
Competitive Doctoral Enrichment Fellowship, the Department of Animal Science
and the College of Agriculture and Natural Resources for your support
throughout my years at MSU.

The molecular genetics laboratory, I thank for all those special moments of
troubleshooting PCR’s, or oligonucleotide random primer labeling. Nancy Raney,
Valencia Rillington, Megan Closs, Dr. Kwan Suk Kim, Leslie Bach, Katie
Rodriguez, thanks for advise, constructive criticism and help in the lab. Jane
Link, Dana Dvoracek-Driska, Jessica Green, and Allison Meyer in the non-
ruminant nutrition laboratory for your technical assistance, help in the lab and at
the farm. Also, to the MSU Swine Research and Teaching Facility staff for all the

help while I was conducting my field research studies.

Thanks to the Center for Animal Functional Genomics for their real time
RT-PCR technical assistance, especially Dr. Steve Suchyta, Brian Tooker, Chris
Colvin and Dr. Paul Coussens. To the muscle biology laboratory, for your
assistance with the protein assays especially Dr. Matt Doumit, Chuck Allison, and
Emily Helman. Dr. Robert Tempelman, for all our great statistic discussions,
especially those with the dot blot spatial variation. Dr. Jeanne Burton, Dr. Patty
Weber and the lmmunogenetics Lab for your support and troubleshooting
expertise, and to Dr. Steve Bursian, the department of Animal Science Graduate
Coordinator.

Juliana Perez Laspiur, for all the emotional, physical and personal
assistance, you are without a doubt one of the brightest people I know. Thank
you for every single explanation, chickens in good time, advice and friendship.
Guillermo Ortiz Colbn for your exceptional friendship and true values, your
honesty and help are truly appreciated. Thanks for the political discussions,
western blot lectures and RNA troubleshooting days, I wish you the absolute best
in life. Raquel Bastos Tabet for genuinely being you. Thanks for being there and
providing your support.

To my cat, my cute Chiqui-Chiqui, for the at home support and snootiness.

“iA TOD@S MUCHAS GRACIASI”

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ xi
LIST OF FIGURES ............................................................................................. xii
CHAPTER ONE
LITERATURE REVIEW ........................................................................................ 1
INTRODUCTION ............................................................................................. 1
Digestive capacity and diet composition .......................................................... 2
Environmental concerns .................................................................................. 4
Phytic acid ....................................................................................................... 5
Phytase ............................................................................................................ 5
Possible phytase sources for pigs ................................................................... 6
Endogenous intestinal phytase ............................................................. 6
Endogenous phytase in feedstuffs ........................................................ 7
Supplemental and resident bacterial phytase ....................................... 7
Phytase supplementation ................................................................................ 9
Zinc essentiality and biochemical properties ................................................. 10
Benefits of zinc supplementation ................................................................... 11
Metallothionein ............................................................................................... 13
Regulation of gene expression by dietary zinc ............................................... 14
Transcriptional regulation of MT .......................................................... 15
Transcriptional regulation of additional genes ..................................... 17
Zinc homeostasis ........................................................................................... 17
Zinc transporters ................................................................................. 18
LITERATURE CITED ..................................................................................... 23
CHAPTER TWO

Martinez, Michelle M., Hill, Gretchen M., Link, Jane E., Raney, Nancy

E., Tempelman, Robert J., Ernst, Catherine W. (2004) Pharmacological

Zinc and Phytase Supplementation Enhance Metallothionein mRNA
Abundance and Protein Concentration in Newly Weaned Pigs. MIMI
mating, 134:538-544 .................................................................................. 38
Pharmacological Zinc and Phytase Supplementation Enhance
Metallothionein mRNA Abundance and Protein Concentration

in Newly Weaned Pigs ...................................................................................... 39
ABSTRACT .................................................................................................... 41
INTRODUCTION ........................................................................................... 42
MATERIALS AND METHODS ....................................................................... 44

Animals and diets ................................................................................ 44
Sample collection and measurements ................................................ 44
Mineral analysis .................................................................................. 45

vii

Metallothionein assay .......................................................................... 46

RNA isolation ...................................................................................... 46
Dot blot and northern blot analysis ...................................................... 47
Statistical analysis ............................................................................... 48
RESULTS ...................................................................................................... 50
Plasma and tissue mineral concentrations .......................................... 50
Metallothionein protein concentration and mRNA abundance ............ 50
Residual correlation ............................................................................ 52
Growth performance ........................................................................... 53
DISCUSSION ................................................................................................ 54
ACKNOWLEDGEMENTS .............................................................................. 59
LITERATURE CITED ..................................................................................... 60
CHAPTER 'IWO Tables ................................................................................ 65
CHAPTER TWO Figures ............................................................................... 68
CHAPTER THREE
Identification of ZnT-1 in Porcine Liver of Newly Weaned Pigs Fed
Pharmacological Zinc and Phytase Supplemented Diets .............................. 75
ABSTRACT .................................................................................................... 76
INTRODUCTION ........................................................................................... 77
MATERIALS AND METHODS ....................................................................... 79
Animals and diets ................................................................................ 79
Sample collection ................................................................................ 79
Total membrane protein preparation ................................................... 79
Production of ZnT-1 antibody .............................................................. 80
Western blot analysis .......................................................................... 80
Statistical analysis ............................................................................... 82
RESULTS ...................................................................................................... 83
Identification of ZnT-1 in porcine liver ................................................. 83
Effect of diet on zinc transporter protein concentration ....................... 83
DISCUSSION ................................................................................................ 84
ACKNOWLEDGEMENTS .............................................................................. 87
LITERATURE CITED ..................................................................................... 88
CHAPTER THREE Figures ............................................................................ 92
CHAPTER FOUR

Dietary Pharmacological or Excess Zinc and Phytase Effects on
Tissue Mineral Concentrations, Metallothionein and Apparent Mineral

Retention in the Newly Weaned Pig ................................................................ 97
ABSTRACT .................................................................................................... 98
INTRODUCTION ........................................................................................... 99

viii

MATERIALS AND METHODS ..................................................................... 101

Animals, Diets and Measures ........................................................... 101
Tissue Mineral and Metallothionein Analysis .................................... 102
Fecal and Urine Analysis .................................................................. 103
Statistical analysis ............................................................................. 103
RESULTS AND DISCUSSION .................................................................... 105
Growth Performance ......................................................................... 105
Plasma, Kidney and Liver Mineral Analysis ...................................... 105
Tissue Metallothionein Protein Analysis ............................................ 107
Fecal, Urinary and Apparent Zn Retained ......................................... 108
Daily Zn Fecal Excretion and Retention Patterns .............................. 109
Mineral (Ca, P, Cu, Fe) Apparent Retention ..................................... 110
ACKNOWLEDGEMENTS ............................................................................ 113
LITERATURE CITED ................................................................................... 114
CHAPTER FOUR Tables ............................................................. ' ................ 119
CHAPTER FOUR Figures ............................................................................ 124
CHAPTER FIVE

Identification of Differentially Expressed Genes in Hepatic Tissue of
Newly Weaned Pigs Fed Pharmacological Zinc and Phytase

Supplemented Diets ........................................................................................ 130
ABSTRACT .................................................................................................. 132
INTRODUCTION ......................................................................................... 134
MATERIALS AND METHODS ........ 136

Animals and diets .............................................................................. 136
Sample collection and total RNA isolation ......................................... 136
Differential display reverse transcription polymerase chain reaction
(DDRT-PCR) .................................................................................... 136
Excision and re-amplification of DDRT-PCR products ...................... 138
Cloning and sequencing of DDRT-PCR products ............................. 138
Independent confirmation by relative real time PCR ......................... 139
Independent confirmation by northern blot hybridization ................... 140
Statistical analysis ............................................................................. 140
RESULTS .................................................................................................... 142
Identification of expressed sequences displayed by
DDRT-PCR ....................................................................................... 142
Confirmation of differential expression as affected by dietary
treatment .......................................................................................... 142
DISCUSSION .............................................................................................. 144
ACKNOWLEDGEMENTS ............................................................................ 150
LITERATURE CITED ................................................................................... 151

CHAPTER FIVE Tables ............................................................................... 157

CHAPTER FIVE Figures .............................................................................. 160

LIST OF TABLES

CHAPTER TWO

Table 1. Composition of experimental diets for nursery pigs .............................. 65
Table 2. Concentration of copper, and phosphorus in plasma, kidney and

liver of pigs fed adequate or pharmacological levels of zinc with or without
supplemental phytase ......................................................................................... 66
Table 3. Residual correlation of the copper, iron, zinc, metallothionein mRNA
abundance and metallothionein protein concentrations in liver and kidney of

pigs fed pharmacological levels of zinc with or without supplemental

phytase ............................................................................................................... 67
CHAPTER FOUR

Table 1. Composition of experimental diets for nursery pigs (9 I 100 g diet) 119

Table 2. Effects of adequate, pharmacological or excess zinc with or without
supplemental phytase on plasma and kidney Zn concentrations ...................... 120

Table 3. Effects of adequate, pharmacological or excess zinc with or without
supplemental phytase on plasma, kidney and liver Ca, P, Cu and Fe
concentrations ................................................................................................... 121

Table 4. Effects of feeding adequate, pharmacological or excess zinc with or
without supplemental phytase on Zn balance ................................................... 122

Table 5. Effects of feeding adequate, pharmacological or excess zinc with or

without supplemental phytase on apparent Ca, P, Cu and Fe retention ........... 123
CHAPTER FIVE
Table 1. Anchor and arbitrary primers used in this experiment ......................... 157

Table 2. List of real time PCR primers used for DDRT-PCR confirmation ........ 158

Table 3. List of differentially expressed products and sequence identity
information ........................................................................................................ 1 59

xi

LIST OF FIGURES

CHAPTER TWO

Figure 1A. Concentration of zinc in plasma of pigs fed zinc (150, 1,000
and 2,000 mg Zn/kg) with or without phytase for 14-d post-weaning .................. 68

Figure 1B. Concentration of zinc in liver of pigs fed zinc (150, 1,000 and
2,000 mg Zn/kg) with or without phytase for 14-d post-weaning ......................... 69

Figure 1C. Concentration of zinc in kidney of pigs fed zinc (150, 1,000 and
2,000 mg Zn/kg) with or without phytase for 14-d post-weaning ......................... 70

Figure 2. Dot blot analysis of liver MT mRNA of pigs fed zinc (150, 1,000
and 2,000 mg Zn/kg) with or without phytase for 14-d post-weaning .................. 71

Figure 3. Relative MT mRNA abundance and protein concentration in the
liver of pigs fed zinc (150, 1,000 and 2,000 mg Zn/kg) with or without
phytase for 14-d post-weaning ............................................................................ 72

Figure 4. Relative MT mRNA abundance and protein concentration in the
kidney of pigs fed zinc (150, 1,000 and 2,000 mg ankg) with or without
phytase for 14-d post-weaning ............................................................................ 73

Figure 5. Relative MT mRNA abundance and protein concentration in the
Intestinal mucosa of pigs fed zinc (150, 1,000 and 2,000 mg ankg) with

or without phytase for 14-d post-weaning ........................................................... 74
CHAPTER THREE

Figure 1. Detection of ZnT-1 in two protein fractions .......................................... 92
Figure 2. Specificity of rabbit anti-rat ZnT-1 antibody ......................................... 93

Figure 3A. Expression of ZnT-1 protein in liver homogenates of pigs fed
dietary Zn (150, 1000 or 2000 mg Zn/kg) with or without supplemental
phytase (P) and in liver of an adult rat ................................................................. 94

Figure 38. Expression of ZnT-1 and B-actin proteins in liver homogenates
of pigs fed dietary Zn (150, 1000 or 2000 mg Zn/kg) with or without
supplemental phytase (P) and in liver of an adult rat .......................................... 95

xii

Figure 3C. Relative ZnT-1 protein abundance in liver homogenates of pigs
dietary Zn (150, 1000 or 2000 mg Zn/kg) with or without supplemental
phytase (P) .......................................................................................................... 96

CHAPTER FOUR

Figure 1. Concentration of zinc in liver of pigs fed 100, 1,000 or 4,000 mg

Zn/kg as zinc oxide, with or without 500 FTU/kg of phytase for 14 d

post-weaning and a common diet (100 mg ankg) for the subsequent

7 d ..................................................................................................................... 124

Figure 2A. Concentration of metallothionein in duodenum of pigs fed 100,

1,000 or 4,000 mg Zn/kg as zinc oxide, with or without 500 FT U/kg of

phytase for 14 d post-weaning and a common diet (100 mg Zn/kg) for the
subsequent 7 d .................................................................................................. 125

Figure 28. Concentration of metallothionein in jejunum of pigs fed 100,

1,000 or 4,000 mg Zn/kg as zinc oxide, with or without 500 FTU/kg of

phytase for 14 d post-weaning and a common diet (100 mg ankg) for the
subsequent 7 d .................................................................................................. 126

Figure 2C. Concentration of metallothionein in kidney of pigs fed 100,

1,000 or 4,000 mg Zn/kg as zinc oxide, with or without 500 FTU/kg of

phytase for 14 d post-weaning and a common diet (100 mg ankg) for the
subsequent 7 d .................................................................................................. 127

Figure 20. Concentration of metallothionein in liver of pigs fed 100, 1,000

or 4,000 mg ankg as zinc oxide, with or without 500 FT mm of phytase

for 14 d post-weaning and a common diet (100 mg Zn/kg) for the

subsequent 7 d .................................................................................................. 128

Figure 3. Daily fecal zinc excretion patterns of pigs fed 100, 1,000 or

4,000 mg Zn/kg as zinc oxide, with or without 500 FTU/kg of phytase

for 14 d post-weaning and a common diet (100 mg Zn/kg) for the

subsequent 7 d .................................................................................................. 129

CHAPTER FIVE
Figure 1. Northern blot analysis of liver GLO1, ACY1, and PRDX4 mRNA of

pigs fed 150, 1000 and 2000 mg Zn/kg (with or without phytase) for 14 d
post-weaning ..................................................................................................... 160

xiii

Figure 2. Relative PRDX4 mRNA abundance in the liver of pigs fed 150,
1000 and 2000 mg ankg (with or without phytase) for 14 d post-weaning ....... 161

Figure 3. Relative GLO1 mRNA abundance in the liver of pigs fed 150,
1000 and 2000 mg ankg (with or without phytase) for 14 d
post-weaning ..................................................................................................... 162

Figure 4. Relative ACY1 mRNA abundance in the liver of pigs fed 150,
1000 and 2000 mg Zn/kg (with or without phytase) for 14 d
post-weaning ..................................................................................................... 163

xiv

CHAPTER ONE
Literature Review
INTRODUCTION

The capacity of pigs to adapt to new diets and properly digest dry feed
during the immediate post-weaning period influences their subsequent
performance (1). At weaning, pigs are transferred to a nursery facility,
commingled, vaccinated and exposed to a new social and physical environment.
Collectively, these changes challenge the health status of the animals. Post-
weaning stress symptoms include low feed intake, diarrhea, atrophy of small
intestinal villi and ultimately reduced growth rate, which can translate into
significant financial losses for the swine industry (2).

Nutritional stress arises when piglets feeding patterns are changed, from
consuming sow’s milk (water based and highly digestible) to a dry diet of different
temperature, smell, taste, texture, composition and digestibility (3). Voluntary
feed intake during the first days after weaning is low, thus insufficient to cover the
maintenance energy requirement (4). Low feed intake correlates with alterations
in the gastrointestinal tract, and the combination of these factors may cause
lower nutrient digestive and absorptive capacity (5). Poor performance often
observed in the newly weaned pig might result from an immunological response
to soy protein antigens found in starter diets and may lead to a compromised

intestinal morphology and subsequent diarrhea (6,7).

Digestive capacity and diet composition

The digestive system of the piglet at weaning is adapted to secrete
enzymes for milk digestion. Lactase activity is high, while the activity of Iipases
and proteases is sufficient to digest milk fat and protein (8). As early as one wk
post-weaning (~ 5 wk of age), pigs display lower exocrine pancreatic enzyme
activity compared to pre-weaned or 6 wk old pigs (9). Similar observations were
obtained by Jensen et al. (1997), who speculated that decreases in enzyme
activity might be due to depleted enzymatic stores caused by higher rates of
secretion than synthesis, suggesting that pigs need to adapt by increasing
enzyme secretion immediately post-weaning when they are exposed to the new
dietary regimen (10). Thus the selection of dietary ingredients for post-weaning
diets is crucial for proper digestion and growth.

Nursery diets can be formulated based on a feed budget and/or in several
phases that differ in ingredient composition. Generally, the feedstuffs found in
the initial post-weaning diet are highly digestible quality ingredients, such as
lactose, plasma protein and spray-dried whey. The length of time this first diet is
fed depends on the age at weaning. Many byproducts such as spray-dried red
blood cells, egg protein and fishmeal are also used in early diets. Also, refined
soy products, such as soy protein concentrate, with reduced allergenicity are
utilized.

Corn is high in digestible starch, low in fiber and protein, and deficient in
lysine and tryptophan. These deficiencies are corrected by adding other

feedstuffs to the diet, such as soybean meal. Soybean meal is a good protein

source, due to its amino acid composition and balance. Since, it is high in lysine,
tryptophan and threonine, it supplies the amino acids that are limiting in corn.
However, young pigs may display hypersensitivity to some indigestible
oligosaccharides found in soybean meal. In addition, soybean meal contains
immunologically active proteins that can affect gut morphology, by increasing
crypt depth and reducing villous height of the piglets (6).

Minerals are required nutrients supplied in weanling pig diets, and can be
fed in organic or inorganic forms to meet the pigs’ requirements. Copper (Cu) as
copper sulfate (CuSO4) and zinc (Zn) as zinc oxide (ZnO) have been used in
pharmacological concentrations as growth promoting agents. Interestingly, an
additive growth response is not obtained when these minerals are fed together
(11,12). However, when an antibacterial agent (i.e. Carbadox) is fed together
with pharmacological Cu (13) or Zn (14), the growth response obtained is
additive.

The biological responses seen in pigs fed these minerals are influenced
by chemical form and feeding level (15). However, their mechanisms of action
are not clear. Antibacterial properties are attributed to Cu as CuSO4 when it is
fed at concentrations of 200 to 250 mg Culkg of diet, and Its use is effective in
the presence or absence of antibiotics (16). However, higher levels of Cu (> 500
mg/kg) should not be fed to pigs because they are toxic (17,18).

Numerous studies demonstrate that feeding 2000 to 3000 mg ankg diet
as ZnO to traditionally (18 - 24 d) or early-weaned (10 — 14 d) pigs, results in

enhanced growth performance (11,14,19). Studies feeding pharmacological Zn

as acetate, carbonate, sulfate (20), complexed to individual amino acids such as
methionine or lysine (21), amino acid peptides or polysaccharide complex (22)
are not as effective in enhancing growth as ZnO. However, comparing the
effects of feeding Zn at the required concentration (100 mg Zn/kg) vs. feeding
pharmacological concentrations of up to 4000 mg Zn/kg, as ZnO to newly
weaned pigs on tissue accretion and daily fecal Zn excretion has not been
investigated.
Environmental concerns

Currently, ZnO is included in most nursery diets at a dietary concentration
of 2000 to 3000 mg ankg diet as a growth promotant and it may aid in
preventing post-weaning diarrhea (14). However, supplementing
pharmacological Zn to pigs creates environmental concerns due to increased Zn
excretion. One study estimated that pigs fed 2000 or 3000 mg ankg for 21 d
excrete 14.1 or 21.5 times more fecal Zn, respectively, than pigs fed 150 mglkg
of Zn (23), which is close to the NRC (24) Zn requirement (100 mg Zn/kg).
Furthermore, in a nursery balance study during which pigs were fed
pharmacological Zn for 10 d prior to fecal collection, pigs were in negative
balance, excreting more Zn than they consumed (22). This is likely because the
balance study was conducted following 10 days of feeding pharmacological Zn.

High Zn accumulation in the soil has been implicated in reduced plant
growth (25). Feces and urine composition are related to the diet fed to the
animal; phosphorus (P), nitrogen (N), Cu and Zn are of greatest environmental

concern. Phosphorus concentration in the waste material is often used to

regulate the amount of swine and poultry manure that may be applied as fertilizer
(26).
Phytic acid

Phytic acid is an organic form of P, chemically known as myo-inositol
hexakis dihydrogen phosphate (IP6). Common animal feedstuffs contain 60-80%
of the total P in the phytic acid molecule (27). This molecule is the primary
source of inositol and a storage form of P in plant seeds (28). It contains a six-
carbon ring with six phosphate groups (OPO3H2), making phytic acid a strong
chelating agent that binds cations with different affinities. Phosphate group
number 5 has been shown to preferentially bind Zn in vitro (29).
Phytase

In the past two decades microbial and plant acid phosphatases have been
isolated, purified, characterized and commercialized as feed additives to increase
nutrient availability. The International Union of Pure and Applied Chemistry and
the International Union of Biochemistry recognize two types of phytase (myo-
inositol hexakisphosphate phosphohydrolase) phosphatases (30). The 3-
phytase (EC. 3.1.3.8) attacks phytic acid at the 3-position, and the 6-phytase
(EC. 3.1.3.26) removes the phosphate group at the 6-position of the phytate
molecule. The removal of these groups represents P release, thus any cation
attached to this group is released as well. In theory, once the first phosphate is
released, the resulting IP5 molecule can be re-attacked by the enzyme, releasing
another phosphate group, yielding an IP4 molecule. Hydrolysis of the additional

phosphate groups may continue until an IP molecule is obtained (31).

Commercially available phytases such as Natuphos® (BASF, Mt. Olive,
NJ) and Ronozyme® P (Roche Vitamins, Parsippany, NJ) are examples of the 3
and 6 phytases, respectively. Additional available phytases are PhyzymelM XP
(Diversa Corp., San Diego, CA), Zymetrics QuantumTM (Diversa Corp., San
Diego, CA), Eco-PhosTM (Phytax, Portland, ME), Allzyme PhytaseTM (Alltech,
Nicholasville, KY), Biofeed® (Novo Nordisk, Princeton, NJ), and FinaseTM (Alko
Ltd. Biotechnology, Rajamaki, Finland).

Possible phytase sources for pigs

There are four possible sources of phytases for pigs: 1) endogenous
intestinal phytase produced in digestive secretions, 2) endogenous phytase in
feedstuffs, 3) phytase from resident bacteria, or 4) supplemented phytase
produced by exogenous microorganisms (26).

Endogenous intestinal phytase. The activity of the intestinal phytase in
pigs receiving a corn-soybean meal diet is negligible. As much as 60-74% of the
phytic acid hydrolysis occurs in the stomach and duodenum of pigs fed
supplemental fungal phytase compared to a 10% hydrolysis in pigs fed
unsupplemented diets (32). In vitro studies using rat, human, chicken and calf
intestinal mucosa to determine endogenous phytase activity, conclude that pH
plays a key role in phytase activity. In the rat, optimal activity is achieved at a pH
range from 7.0 to 7.5. In human homogenates, maximal activity is obtained at
pH 7.4, with no detectable activity at pH 9. In chicken and calf intestinal mucosa,

optimal pH is at 8.3 and 8.6, respectively (33).

Endogenous phytase in feedstuffs. Phytase is naturally found in
several dietary feedstuffs such as wheat, rice and barley, all of which could be
used for swine diets. However, it has been reported that the phytic acid P
released from animal diets formulated with wheat is below the amounts released
with phytase supplemented diets (28). Therefore, in order to use wheat as a
phytase source, diets must be formulated with at least 40% wheat to hydrolyze
all the phytic acid P, which is not economically feasible (26,28). In 1992,
Cromwell estimated that 29 to 49% of the P in wheat by-products is bioavailable,
while the P bioavailability from fish meal, blood meal, whey and alfalfa meal is
estimated to be 100% (34). Phytase can also be obtained from soybeans (35).
However, because phytase is an enzyme subject to degradation at high
temperatures, its activity is not sustained during the high temperatures used in
the production of soybean meal (36).

Supplemental and resident bacterial phytase. High temperature
resistance is desirable for phytase use in animal feed, because of feed pelleting
and processing (31). In 1996, Yi and Kornegay suggested that in 17 kg pigs the
main site of dietary fungal phytase activity (51% of maximum activity) was in the
stomach at pH 4, with reduced activity (31% of maximum activity) in the upper
part (2 m section past the pylorus) of the small intestine at pH 6.8 (37). However,
the exact location within the gastrointestinal tract and the pH at which the P is
released from phytic acid in pigs fed corn-soybean meal diets are unknown.
Considering temperature and pH as required characteristics, several microbial

phytases have been developed from the bacteria Bacillus 3p, (38), and

Escherichia coll (E. 00/!) (39), the fungus Aspergillus niger (40), plants, or by
genetic modifications (36).

Bacterial phytase yields are low, their optimum pH for maximum activity is
neutral to alkaline, and since most bacteria produce intracellular phytase that is
unable to hydrolyze phytic acid efficiently, their use as an additive is not practical
(28,30). The fungal phytase produces extracellular enzyme and is widely used
commercially (Natuphos®, Allzyme“, Ronozyme® P) in swine diets as a feed
additive to improve phytate-P bioavailability and to reduce fecal P excretion of
pigs (41,42). Fungal phytase has maximal activity at both pH 2.5 and pH 5.5,
which resembles the pH in the stomach of pigs (3.4 - 4.8), suggesting that the pH
of stomach digesta is favorable for high phytase activity, whereas the pH in the
small intestine (6.4 —7.2) is favorable for pancreatic enzymes which hydrolyze
proteins from the chyme, potentially destroying any leftover phytase (37).

Using genetic engineering techniques, the E. coli phytase gene (appA)
was isolated and incorporated into a yeast expression system (Pichia paston's),
producing a thennostable phytase that works at a pH range of 2.5 - 3.5, which
also resembles the physiological pH of the proventriculus of chickens and
stomach of pigs. Another experimental phytase known as a “consensus”
phytase, is heat-stable, and derived from the sequences of 13 known fungal
phytases. This enzyme’s optimal activity occurs at 71°C, and its catalytic activity
and maximal pH are comparable to those of Aspergillus fumigates at 37°C (43).
Growth performance, bone strength and plasma inorganic P were improved by

supplementation (1250 U/kg) to weanling pigs, however more research is needed

to compare its effects on bone mineralization and mineral tissue concentrations
with other available phytases (44).
Phytase supplementation

Phytic acid is a strong chelating agent, attracting multivalent cations to
form phytic acid-cation complexes. It is considered an important anti-nutritional
factor for the availability of minerals (45). At neutral and alkaline pH, phytic acid
forms insoluble salts with cations like calcium (Ca), Cu, iron (Fe) or Zn (46).
O’Dell and Savage (47) were the first to suggest that phytic acid reduces the
availability of dietary Zn in the chick. When rats are fed diets containing 3000 mg
Zn/kg and phytic acid, less Zn is absorbed and fecal Zn is increased (48). A
plausible mechanism to explain how phytic acid affects Zn absorption was
proposed by Oberleas and co-workers (49). Zinc, phytic acid and Ca in a molar
ratio of 1:1 :2 respectively, form a precipitate in a pH range of 5 to 9, which
contains 97% of the originally added Zn (49), suggesting that Zn combines with
the phosphate groups of the phytic acid and Ca to form a Zn-Ca-phytic acid
insoluble complex. Both the formation of mineral-phytic acid complexes and the
negligible amount of endogenous phytase production in pigs increases the
tendency to excrete phytic acid bound minerals in the feces, resulting in
decreased mineral absorption and increased environmental concerns.

Commercial phytase supplementation to pigs fed corn-soybean meal diets
is a good alternative for enhancing mineral absorption. Phytase addition to
animal feeds is currently widely implemented in the Netherlands and the United

States (50). Phytase hydrolyses the ester bond phosphate groups, and this

hydrolysis is needed for releasing inorganic P and the trace minerals that may be
attached to the phytic acid molecule (51). Phytase supplementation studies in
pigs demonstrate improved performance, increased plasma Zn concentration
(52,53), greater Zn retention (54), and improved bone mineralization (55), while
adding phytase to human diets also results in increased Fe absorption (56).
Thus, phytase supplementation is a promising solution to improve Zn availability
and possibly limit environmental pollution (57).

Zinc essentiality and biochemical properties

In 1869, Raulin first showed that Zn was required for the growth of
Aspergillus niger, and it was later reported that Zn was essential for the growth of
rats (58). In animals, signs of Zn deficiency include growth failure, hair loss,
hyperkeratinization of the skin, and testicular atrophy (59). The discovery that Zn
prevents and cures parakeratosis in pigs (60) and the demonstration of Zn
deficiency and stunted growth in chicks (47), initiated an interest in studying the
nutritional physiology of Zn in livestock. In humans, it was discovered that Zn
deficiency caused growth retardation in male Middle Eastern adolescents
consuming primarily unleveaned bread. Subsequent growth was obtained upon
Zn supplementation (58).

Zinc chemical characteristics include no variable valance (low risk of free
radical production), rapid exchange of ligands, and binding mostly to sulfur or
nitrogen in biological systems (61). These properties enable Zn to play three
major biological roles: catalytic, structural and regulatory (62). Zinc is required by

more than 300 metalloenzymes, and can be directly involved in the catalysis of

10

an enzyme. The removal of the catalytic Zn results in an apoenzyme that retains
its tertiary structure but has decreased activity (61). Zinc is usually bound by
three protein ligands and a water molecule, that work in coordination during
catalysis (63). The function of Zn is to polarize the substrate and activate the
water molecule that later acts as a nucleophile. The Zn acts as a template that
brings together the substrate and the nucleophile (64). This lowers the transition
energy thereby accelerating the conversion of substrates to products.
Structurally, Zn is part of the structure of insulin, DNA and RNA polymerases,
proteins involved in DNA replication, transcription factors and Zn finger proteins
(65). Finally, in its regulatory role, Zn serves as cofactor for enzymes, enhances
protein stability and regulates transcription of several genes. Zinc is essential for
proper immune function, including natural killer cell activity (66), and can
potentially modulate secondary messenger metabolism, thus affecting cell
signaling and transduction (67).
Benefits of zinc supplementation

Children that consume diets that are primarily plant based, with low or no
animal protein, benefit from Zn supplements (68). Zinc supplementation reduces
persistent diarrhea (69) and is used along with oral rehydration therapy to treat
acute watery diarrhea in developing countries (70-72). Supplementing Zn is an
economically feasible alternative for growth enhancement and diarrhea
treatment.

European researchers hypothesize that the mode of action of ZnO in

weanling pigs is by controlling diarrhea caused by E. coli (73). However, studies

11

demonstrate no change in number of E. coli or enterococci per gram of feces of
pigs fed pharmacological Zn diets (74,75). Furthermore, studies in Caco-2 cells
exposed to enterotoxigenic E. coli demonstrate that ZnO protects these cells by
inhibiting bacterial adhesion and internalization into cells, thus modulating
subsequent cytokine expression that might occur in response to pathogenic
exposure, and not by a direct antibacterial effect (76).

Numerous studies of pigs fed pharmacological Zn have been reported in
an effort to explain the mechanism of action of dietary Zn. In 1995, McCully et al.
reported that feeding 3000 mg Zn/kg diet to nursery pigs for 21 d improved fecal
consistency versus animals fed 150 mg Zn/kg (20). These results are in
accordance with an NOR-42 study in which pigs fed 3000 mg Zn/kg and/or 250
mg Culkg had firmer stools compared with animals fed 100-150 mg Zn/kg diet or
10-15 mg Culkg diet (12). The reason for fecal consistency improvement is
unknown. However, immunohistochemical analysis of intestinal tissue of nursery
pigs fed 3000 mg ankg diet for 28 cl post-weaning demonstrated deeper
duodenal crypts and greater total thickness from the villus tip to serosa layer,
thus improving gut morphology and possibly increasing the absorption capacity
of the duodenum (77).

Microscopic examinations of the duodenal and jejunal intestinal segments
from nursery pigs challenged with transmissible gastroenteritis virus (T GE) fed
3000 mg Zn/kg diet for 14 d, suggested an improvement in gut morphology when
compared to TGE—challenged pigs fed 250 mg Zn/kg diet (78). Perhaps the

beneficial effects of ZnO supplementation are the result of a combination of

12

mechanisms including gut morphology improvement, and possible cytokine and
gene expression changes that act in concert to improve growth and fecal
consistency.
Metallothionein

Expression of proteins has been studied to understand Zn metabolism.
Among these is metallothionein (MT), a low molecular weight (< 7 kDa) protein
with a Cys-X-Cys amino acid sequence (X = any non-cyclic, non-Cys or
heterocyclic amino acid), a primary protein structure of 61 to 68 amino acids (79)
and a high metal content of 7 9 atoms of metal / mol of protein (80). This protein
was first isolated from the equine renal cortex in the late 50’s by Margoshes and
Vallee (81), and is found throughout the animal kingdom (82). Binding affinity for
metals is as follows: mercury (Hg) z lead (Pb) > Cu+1 z silver (Ag) > cadmium
(Cd) > Zn (83).

There are two major MT genes: MT—l and MT-ll. They differ slightly in
their sequence, and their respective proteins are separable by high performance
liquid chromatography (84,85). Two additional genes exist, MT-lll and MT-IV,
with heterogeneity being a post-translational acetylation and/or variations due to
metal composition (86). In mammals, MT-l and MT-II are found in all organs,
while MT-lll is mainly expressed in the brain and MT-IV is most abundant in
squamous epithelial tissue (87). In the pig, a total of ten MT isoforrns have been
identified, seven of them for MT-l, two for MT-ll, and one for MT-lll (88).

Despite many decades of research, the precise physiological function of

MT has not been identified. Proposed functions are metal transport and storage,

13

protection against reactive oxygen species (89), detoxification of heavy metals
(90,91), and metal transfer to ligands or proteins with higher affinity for the
particular metal (92). Although this protein is primarily found in the cytoplasm,
nuclear expression has also been observed. Nuclear localization could be
related to DNA protection against oxidative damage, or regulation of Zn supply to
enzymes and transcription factors (93). Therefore, MT does not have a single
biological function but its role is multifaceted (94).

Liver, kidneys and intestinal mucosa play a key role in Zn homeostasis
(95), often with MT, a protein often considered as an intracellular marker of
excess Zn (96). Our laboratory fed 3000 mg Zn/kg for 7, 14, or 28 d post-
weaning and found elevated renal and hepatic Zn, and increased duodenal, renal
and hepatic MT protein concentrations (19). Tran et al. (96) fed 10, 100, 400 or
1000 mg ankg to rats for 7 d and reported that Zn concentrations increased from
the stomach to the colon, with significantly higher concentrations in rats fed 400
and 1000 mg ankg than in rats fed 10 or 100 mg ankg.- In addition, MT
concentrations were higher in the duodenum of rats fed 400 and 1000 mg Zn/kg
than MT concentrations in the jejunum, ileum, caecum, and colon.
Metallothionein was higher in all tissues of these rats than rats fed 10 or 100 mg
ankg. These studies suggest that an adaptive mechanism, which includes MT,
exists to protect from excess Zn accumulation.
Regulation of gene expression by dietary zinc

Nutrition influences gene expression. In the last ten to fifteen years,

nutrients have been recognized as direct and independent regulators of gene

14

expression, having the ability to influence transcription, mRNA processing,
mRNA stability, translation and post-translational modifications (97). Thus,
understanding the interaction between Zn and gene expression may yield
important information (98).

Transcriptional regulation of MT. In 1981, Durnam and Palmiter
identified the MT-l gene in the mouse (99). By combining DNA sequence with
results of deletion mapping studies, short sequences were found to mediate
metal response of the MT gene. These sequences are known as metal response
elements (MREs) and are. present in multiple copies in the 5’ regulatory region
upstream of the transcription initiation start site. Once Zn is inside the cell, it
binds to metal transcription factor-1 (MTF-1), which acts as a Zn sensor
(100,101). The MREs sews as binding sites for transcription factors such as
MTF-1, increasing transcription rate of MT upon metal activation (102).

Mechanisms for MT gene regulation by Zn have been studied. Different
concentrations (5 - 180 mg ankg) of dietary Zn regulate MT in mouse kidney,
liver, and intestine (103). The regulation by Zn is due to increased transcription,
with maximal levels of MT mRNA abundance observed shortly before changes in
protein synthesis are seen (104,105). Richards and Cousins (106) were able to
block MT mRNA induced by high dietary Zn using the transcription inhibitor
actinomycin D, confirming transcriptional regulation. Additional studies in rats fed
a Zn depleted diet for 4 d, then gavaged with either no Zn or 125 mg Zn/kg BW

after an overnight fast showed that MT mRNA abundance was threefold higher in

15

rats fed Zn, and this increase preceded the accumulation of Zn in the liver by a
few hours, indicating a rapid transcriptional induction by Zn (104).
Metallothionein synthesis in mucosal cells is triggered by both fasting

and high luminal Zn concentrations (95). Feeding a Zn-deficient diet to rats for 1
d decreased MT concentrations from normal to nearly undetectable
concentrations in both liver and intestine. However, repletion with a diet
containing 150 mg Zn for one additional day caused a 10-fold increase in MT
synthesis (104). When rats were fed 1000 to 2000 mg Zn/kg diet for up to 8 wks,
increases in hepatic MT concentration were reported, but these dropped to trace
amounts after feeding the same rats a Zn deficient diet for 3 d (107). Similarly,
traditionally and early weaned pigs fed 3000 mg Zn/kg diet for 14 d showed
increased MT protein concentrations in the liver compared to pigs fed 150 mg
ankg (19). These studies suggest that Zn induces MT synthesis de novo, and
that MT is involved in trafficking or processing newly acquired cellular Zn.

Hepatic MT can be induced by an inflammatory response. Glucocorticoids
produced as a consequence of an inflammatory response induced MT-l and MT-
" through sequences known as glucocorticoid response elements (86).
Metallothionein synthesis during inflammation may also be regulated by acute
phase reactant proteins, catecholamines, glucagon, interleukin-6, interleukin-1,
tumor necrosis factor a, and y—interferon (108,109). Additional nucleotide
sequences in the MT promoter known as antioxidant response elements induce
MT synthesis upon activation due to oxidative stress (110). None of these

biochemical mechanisms have been studied in the pig.

16

Transcriptional regulation of additional genes. Gene expression
studies in the intestine by Blanchard and Cousins (111) in male Sprague-Dawley
rats fed Zn deficient diets show upregulation of pre-prouroguanylin (pre-PUG)
and cholecystokinin (CCK). Pre-PUG is the precursor of uroguanylin, an
intestinal hormone that controls electrolyte balance in the small intestine, and
CCK has a neuroendocrine function of satiety control and endocrine roles of gall
bladder contraction and gastric emptying (112). The regulation of pre-PUG
mRNA and uroguanilyn by dietary Zn might have a role in diarrheal disease,
while CCK’s role in satiety control may explain the well-documented anorexia
seen in a Zn deficient state (113). Interestingly, uroguanylin and CCK proteins
show overexpression during Zn deficiency, strengthening the hypothesis that the
involvement of these genes is part of the mechanism that causes diarrhea and
anorexia in Zn deficient animals (114).

Most Zn studies to assess gene expression have focused on deficiency.
To observe effects of Zn deficiency on intestinal mucosa gene expression, rats
were individually fed a Zn deficient or adequate diet for 14 d. Results showed
that expression of genes associated with redox state of the cell, and
carbohydrate and lipid metabolism were affected (115). Also, DDRT—PCR
studies show that the differential expression of thymic genes of rats fed Zn
deficient diets precedes phenotypic effects on thymic function (116).

Zinc homeostasis
Zinc absorption responds to alterations in dietary Zn. Cousins (95)

suggested that high Zn intakes resulted in a decrease in absorption efficiency,

17

however the mechanism involved in Zn transport in tissues within the body has
not been elucidated. To achieve Zn homeostasis, regulation occurs through
intestinal uptake, fecal excretion, renal resorption and excretion, and intracellular
storage (117). On a cellular level, under variable Zn concentrations, an
adjustment in Zn uptake and secretion, intracellular protein binding, re-
distribution and sequestration within vesicles or organelles is required for
homeostatic control (1 18).

When Zn is internalized, it is thought to be available in four intracellular
pools. First, it can bind tightly to metalloproteins, serving a structural role (119).
Second, MT binds Zn with low affinity and may become part of a labile Zn pool
considered a reservoir for cytosolic Zn (86). Third, it may be compartmentalized
in intracellular organelles to supply Zn to dependent proteins, or be stored.
Compartmentalization may occur in organelles such as Golgi apparatus, synaptic
vesicles and secretory granules (120). Fourth, Zn may be free, which is
considered to be in the nanomolar range (121).

Zinc is a charged, hydrophilic ion that cannot move across membranes by
passive diffusion, requiring the presence of transmembrane proteins to aid the
passage of “free” Zn across cell membranes (100). Transporter-mediated
movement, which is a time, concentration, pH, and temperature-dependent
process, is likely responsible for “free“ Zn movement across cellular membranes
(1 18).

Zinc transporters. In eukaryotes, there are two known families of Zn

transporters. The SLC39 transporters belong to the ZIP family and are involved

18

in Zn uptake from outside the cell into the cytoplasm and from intracellular
compartments into the cytoplasm. The second family, SLC30 transporters,
belong to the cation diffusion facilitator (CDF) family and promote Zn efflux by
pumping Zn from the cytoplasm out of the cell and from the cytoplasm into
intracellular organelles and vesicles (122,123).

The CDF family can be divided into three different sub-groups based on
their sequence similarities. The CDF-l subgroup is found only in prokaryotes,
whereas CDF-II and III are found in both eukaryotes and prokaryotes (122). In
mammals, a total of 9 mammalian CDF members have been characterized
including two recently identified members (124). These carriers are known as Zn
transporters (ZnT). All contain 6 transmembrane domains and have both N- and
long C- termini on the cytoplasmic side with the exception of ZnT-5, which has a
large N- terminal extension and an estimated six to nine additional
transmembrane domains (122,124). The ZnT’s contain a cytoplasmic histidine-
rich loop between transmembrane domains IV and V, where it is thought that Zn
may be bound, and transported across the membranes. The greatest degree of
conservation between these transporters is found within transmembrane
domains I, II and V (122,125).

In the rat, the mRNA abundance of ZnT-1 and ZnT-4 is distributed
ubiquitously, with varied mRNA abundance among tissues, while ZnT-2 mRNA
abundance is greatest in the small intestine and kidney (126). Duodenum and
jejunum ZnT-1 mRNA expression is greater in the villus cells than in crypt cells

and the protein islocalized in the basolateral surface in the upper portion of the

19

villus cells (100). The mRNA of ZnT-3 has only been observed in brain and testis
(127), while ZnT—5 is ubiquitously expressed with highest expression in the
pancreas and is associated with Zn enriched vesicles involved in insulin
secretion (128). The ZnT—6 facilitates translocation of cytoplasmic Zn into the
trans Golgi network and vesicular compartment (129). The ZnT-7 is expressed
primarily in the Golgi apparatus membrane of the small intestine, liver and lungs,
and functions in transporting Zn from the cytoplasm into the Golgi apparatus.
Because of ZnT-7’s high abundance in the small intestine, it may play a role in
Zn absorption (125). According to Palmiter and Huang (124), ZnT-8 and ZnT-9
were identified as transporters via database analysis, and no experimental data
are available.

A mutation in the ZnT-4 gene causes insufficient Zn transport into milk
from the lactating mammary glands of lethal milk mice (130). As a consequence,
the pups of any genotype that are raised by lethal milk mothers die of Zn
deficiency before weaning (131). However, maternal Zn supplementation
improves pup survival, indicating that there are additional transport mechanisms
within the gland to facilitate Zn export into milk (132).

Palmiter and Findley (133) first discovered ZnT-1 in mutagenized baby
hamster kidney cells (BHK) transfected with a Zn-responsive reporter gene. The
BHK cell line was grown in high Zn culture media and was extensively
mutagenized to screen for genes affected by Zn concentrations. One mutant
variant of these cells showed elevated basal expression of the reporter gene, and

resistance to Zn toxicity. A Zn export function was suggested for this gene.

20

Expression of ZnT-1 as a fluorescent fusion protein showed localization on the
plasma membrane of the BHK cells, supporting the theory of the transporter
having an exporter function. In addition, a study using transient forebrain
ischemic cells in gerbils demonstrated ZnT-1 up-regulation during ischemia, a
condition that causes Zn influx into neurons (134). Thus, ZnT-1 transcriptional
regulation by Zn may contribute to cellular Zn balance by promoting Zn efflux.

Transcription of the ZnT-1 gene is under the control of the MRE-binding
transcription factor MTF-1. The ZnT-1 promoter contains two MRE consensus
sequences and MTF-1 binds to both sites, resulting in transcriptional activation
(135). Expression of MT occurs in a similar fashion; therefore Zn homeostasis
may be achieved through the induction of both these genes.

In the rat, two ZnT-1 transcripts (~2.4 and 7.0 kb) have been identified
(133). Liver and intestinal membrane protein preparations indicate the presence
of ~38 and ~42 kDa proteins, respectively (100). However, the estimated size of
ZnT—1 based on its amino acid sequence is ~55 kDa. Protein size differences
with various tissues suggest that ZnT-1 may be post-translationally modified
(100,132,136). Potential modifications include phosphorylation or glycosylation,
which may modulate the stability and/or transport activity of ZnT-1 (125).
Another possibility for the multiple ZnT-1 sizes may be due to its suggested
multimeric structure (133).

The ZnT-1 transporter is oriented toward the basolateral surface of
enterocytes, consistent with the function of Zn efflux towards the portal circulation

(100). Dietary Zn intakes of 180 mg Zn/kg show a significant increase in rat ZnT-

21

1 mRNA in the intestine but not in kidney or liver. Furthermore, 2 hr after an oral
gavage dose of Zn (32.5 mglkg BVV) to rats, MT and ZnT-1 mRNA increase in
liver and intestine; however, no increases in ZnT-1 protein are obtained. At 6 hr
post-dose, both MT and ZnT-1 mRNA and ZnT-1 protein increase significantly
(100). Therefore, following a high oral Zn dose, up-regulation of ZnT-1 and MT
mRNAs occurs rapidly, although protein induction of ZnT-1 is slightly slower than
MT, suggesting that ZnT-1 and MT translational mechanisms differ.

Palmiter (137) suggests that these proteins work together to prevent Zn
toxicity in cells, however their inductive mechanisms are indeed different. High
intracellular Zn induces apoMT, which sequesters Zn and makes cells more
tolerable to increased Zn concentrations. In contrast, ZnT-1 facilitates Zn efflux,
thus lowering total cellular Zn content (133). However, both mechanisms work

together to handle Zn disposal when excess Zn enters the cell.

22

LITERATURE CITED

1. Marion, J., Rome, V., Savary, G., Thomas, F., Le Dividich, J. & Le
Huerou Luron, l. (2003) Weaning and feed intake alter pancreatic enzyme
activities and corresponding mRNA levels in 7-d-old piglets. J. Nutr. 133: 362-
368.

2. Pollmann, D. S. (1993) Effects of nursery feeding programs on
subsequent grower-finisher pig performance. In: Proceedings of the Fourteenth
Western Nutrition Conference (J. Martin), pp. 243-254. Edmonton.

3. Zijlstra, R. T., Whang, K. Y., Easter, R. A. & Odle, J. (1996) Effect of
feeding a milk replacer to early-weaned pigs on growth, body composition, and

small intestinal morphology, compared with suckled litterrnates. J. Anim. Sci. 74:
2948-2959.

4. Bark, L. J., Crenshaw, T. D. & Leibbrandt, V. D. (1986) The effect of
meal intervals and weaning on feed intake of early weaned pigs. J. Anim. Sci. 62:
1233-1239.

5. Pluske, J. R., Williams, I. H. & Aherne, F. X. (1996) Maintenance of
villous height and crypt depth in piglets by providing continuous nutrition after
weaning. Anim. Sci. 62: 131-144.

6. Li, D. F., Nelssen, J. L., Reddy, P. G., Blecha, F., Klemm, R. &
Goodband, R. D. (1991) lnterrelationship between hypersensitivity to soybean
proteins and growth performance in early-weaned pigs. J. Anim. Sci. 69: 4062-
4069.

7. Bailey, M., Miller, B. G., Telemo, E., Stokes, C. R. & Bourne, F. J.
(1993) Specific immunological unresponsiveness following active primary

responses to proteins in the weaning diet of piglets. Int. Arch. Allergy Immunol.
101: 266-271.

8. Maxwell, C. V. & Carter, S. D. (2001). Feeding the weaned pig. In:
Swine Nutrition (A.J. Lewis, & L.L. Southern) 2nd Ed, pp. 691-706. CRC Press,
Boca Raton, FL.

23

9. Lindemann, M. D., Cornelius, S. 6., el Kandelgy, S. M., Moser, R. L. &
Pettigrew, J. E. (1986) Effect of age, weaning and diet on digestive enzyme
levels in the piglet. J. Anim. Sci. 62: 1298-1307.

10. Jensen, M. S., Jensen, S. K. & Jakobsen, K. (1997) Development of
digestive enzymes in pigs with emphasis on lipolytic activity in the stomach and
pancreas. J. Anim. Sci. 75: 437-445.

11. Smith, J. W., Tokach, M. D., Goodband, R. D., Nelssen, J. L. &
Richert, B. T. (1997) Effects of the interrelationship between zinc oxide and
copper sulfate on growth performance of early-weaned pigs. J. Anim. Sci. 75:
1861-1866.

12. Hill, G. M., Cromwell, G. L., Crenshaw, T. D., Dove, C. R., Ewan, R.
C., Knabe, D. A., Lewis, A. J., Libal, G. W., Mahan, D. C., Shurson, G. 0.,
Southern, L. L. & Venum, T. L. (2000) Growth promotion effects and plasma
changes from feeding high dietary concentrations of zinc and copper to weanling
pigs (regional study). J. Anim. Sci. 78: 1010-1016.

13. Roof, M. D. & Mahan, D. C. (1982) Effect of carbadox and various
dietary copper levels for weanling swine. J. Anim. Sci. 55: 1109-1117.

14. Hill, G. M., Mahan, D. 0, Carter, 8. D., Cromwell, G. L., Ewan, R. C.,
Harrold, R. L., Lewis, A. J., Miller, P. S., Shurson, G. C. & Veum, T. L. (2001)
Effects of pharmacological concentrations of zinc oxide with or without the
inclusion of an antibacterial agent on nursery pig performance. J. Anim. Sci. 79:
934-941.

15. Cromwell, G. L., Stahly, T. S. & Monegue, H. J. (1989) Effects of
source and level of copper on performance and liver copper stores in weanling
pigs. J. Anim. Sci. 67: 2996-3002.

16. Stahly, T. S., Cromwell, G. L. & Monegue, H. J. (1980) Effects of the
dietary inclusion of copper and (or) antibiotics on the performance of weanling
pigs. J. Anim. Sci. 51: 1347-1351.

24

17. Suttle, N. F. & Mills, C. F. (1966) Studies of the toxicity of copper to
pigs. 1. Effects of oral supplements of zinc and iron salts on the development of
copper toxicosis. Br. J. Nutr. 20: 135-148.

18. Suttle, N. F. & Mills, C. F. (1966) Studies of the toxicity of copper to
pigs. 2. Effect of protein source and other dietary components on the response to
high and moderate intakes of copper. Br. J. Nutr. 20: 149-161.

19. Carlson, M. S., Hill, G. M. & Link, J. E. (1999) Early and traditionally
weaned nursery pigs benefit form phase-feeding pharmacological concentrations
of zinc oxide: effect on metallothionein and mineral concentrations. J. Anim. Sci.
77: 1199-1207.

20. McCully, G. A., Hill, G. M., Link, J. E., Weavers, R. L., Carlson, M. S. &
Rozeboom, D. W. (1995) Evaluation of zinc sources for the newly weaned pig. J.
Anim. Sci. 73: 72 (abs).

21. Schell, T. C. & Kornegay, E. T. (1996) Zinc concentration in tissues
and performance of weanling pigs fed pharmacological levels of zinc from ZnO,
Zn-methionine, Zn-lysine, or ZnSO4. J. Anim. Sci. 74: 1584-1593.

22. Case, C. L. & Carlson, M. S. (2002) Effect of feeding organic and
inorganic sources of additional zinc on growth performance and zinc balance in
nursery pigs. J. Anim. Sci. 80: 1917-1924.

23. Meyer, T. A., Lindemann, M. D., Cromwell, G. L., Monegue, H. J. &
Inocencio, N. (2002) Effects of pharmacological levels of zinc as zinc oxide on
fecal zinc and mineral excretion in weanling pigs. Prof. Anim. Sci. 18: 162-168.

24. NRC (1998) Nutrient Requirements of Swine, 10th Ed. Washington,
DC.

25. Chaney, R. L. (1993). Zinc phytotoxicity. In: Zinc in Soils and Plants
(A.D. Robson), pp. 135-150. Kluer Academic Publishers, Dordrecht, Netherlands.

25

26. Kornegay, E. T. (1996). Nutritional, environmental and economic
considerations for using phytase in pig and poultry diets. In: Nutrient
Management of Food Animals to Enhance and Protect the Environment (E.T.
Kornegay), pp. 277—302. CRC Press, Inc., Boca Raton, FL.

27. Harland, B. F. & Morris, E. R. (1995) Phytate: A good or a bad food
component. Nutr. Res. 15: 733-754.

28. Wodzinki, R. J. & Ullah, A. H. (1996) Phytase. Adv. Appl. Microbiol.
42: 263-302.

29. Champagne, E. T. & Fisher, M. S. (1990) Binding differences of Zn (II)
and Cu (ll) ions with phytate. J. Inorg. Biochem. 38: 217-223.

30. Dvorakova, J. (1998) Phytase: sources, preparation and exploitation.
Folia Microbiol. 43: 323-338.

31. Vohra, A. & Satyanarayana, T. (2003) Phytases, microbial sources,
production, purification, and potential biotechnological applications. Crit. Rev.
Biotechnol. 23: 29-60.

32. Jongbloed, A. W., Mroz, Z. & Kemme, P. A. (1992) The effect of
supplementary Aspergillus niger phytase in diets for pigs on concentration and
apparent digestibility of dry matter, total phosphorus, and phytic acid in different
sections of the alimentary tract. J. Anim. Sci. 70: 1159-1168.

33. Bitar, K. & Reinhold, J. G. (1971) Phytase and alkaline phosphatase
activities in intestinal mucosae of rat, chicken, calf and man. Biochim. Biophys.
Acta 268: 442-452.

34. Cromwell, G. L. (1992) The biological activity of phosphorus in
feedstuffs for pigs. Pig News Info. 13: 75N-78N.

35. Li, J., Hegeman, C. E., Hanlon, R. W., Lacy, G. H., Denbow, M. D. &
Grabau, E. A. (1997) Secretion of active recombinant phytase from soybean cell-
suspension cultures. Plant Physiol. 114: 1103-1111.

26

36. Lei, X. & Stahl, C. H. (2001) Biotechnological development of effective
phytases for mineral nutrition and environmental protection. Appl. Microbiol.
Biotechnol. 57: 474-481.

37. Yi, Z. & Kornegay, E. T. (1996) Sites of phytase activity in the
gastrointestinal tract of young pigs. Anim. Feed Sci. Technol. 61: 361-368.

38. Kim, Y., Kim, H. K., Bae, K. 8., Yu, J. H. & Oh, T. K. (1998)
Purification and properties of a therrnostable phytase from Bacillus sp. DS11.
Enzyme Microb. Technol. 22: 2-7.

39. Greiner, R., Kontietzny, U. & Jany, K. D. (1993) Purification and
characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys.
303: 107-113.

40. van Hartingsveldt, W., van Zeijl, C. M., Harteveld, G. M., Gouka, R. J.,
Suykerbuyk, M. E., Luiten, R. G., van Paridon, P. A., Selten, G. C., Veenstra, A.
E. & van Gorcom, R. F. (1993) Cloning, characterization and overexpression of
the phytase-encoding gene (phyA) of Aspergillus niger. Gene 127: 87-94.

41. Matsui, T., Nakagawa, Y., Tamura, A., Watanabe, C., Fujita, K.,
Nakajima, T. & Yano, H. (2000) Efficacy of yeast phytase in improving
phosphorus bioavailability in corn-soybean meal-based diet for growing pigs. J.
Anim. Sci. 78: 94-99.

42. Mullaney, E. J., Daly, C. B. & Ullah, A. H. (2000) Advances in phytase
research. Adv. Appl. Microbiol. 47: 157-199.

43. Lehmann, M., Kostrewa, D., Wyss, M., Brugger, R., D'Arcy, A.,
Pasamontes, L. & van Loon, A. P. (2000) From DNA sequence to improved
functionality: using protein sequence comparisons to rapidly design a
therrnostable consensus phytase. Protein Eng. 13: 49-57.

44. Gentile, J. M., Roneker, K. R., Crowe, S. E., Pond, W. G. & Lei, X. G.
(2003) Effectiveness of an experimental consensus phytase in improving dietary
phytate-phosphorus utilization by weanling pigs. J. Anim. Sci. 81: 2751-2757.

27

45. Ravindran, V., Bryden, W. L. & Kornegay, E. T. (1995) Phytates:
occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol.
Rev. 6: 125-143.

46. Vohra, P., Gray, G. A. & Kratzer, F. H. (1965) Phytic acid-metal
complexes. Proc. Soc. Exp. Biol. Med. 120: 447-449.

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

48. Davies, N. T. & Nightingale, R. (1975) The effects of phytate on
intestinal absorption and secretion of zinc, and whole-body retention of zinc,
copper, iron and manganese in rats. Br. J. Nutr. 34: 243-258.

49. Oberleas, D., Muhrer, M. E. & O'Dell, B. L. (1966) Dietary metal-
complexing agents and zinc availability in rats. J. Nutr. 90: 56-62.

50. Holm, P. B., Kristiansen, K. N. & Pedersen, H. B. (2002) Transgenic
Approaches in Commonly Consumed Cereals to Improve Iron and Zinc Content
and Bioavailability. J. Nutr. 132: 5143-516.

51. Pallauf, J. & Rimbach, G. (1997) Nutritional significance of phytic acid
and phytase. Arch. Anim. Nutr. 50: 301-319.

52. Lei, X., Ku, P. K., Miller, E. W., Ullrey, D. E. & Yokoyama, M. (1993)
Supplemental microbial phytase improves bioavailability of dietary zinc to
weanling pigs. J. Nutr. 123: 1117-1123.

53. Martinez, M. M., Hill, G. M., Link, J. E., Raney, N. E., Tempelman, R.
J. & Ernst, C. W. (2004) Pharmacological zinc and phytase supplementation
enhance metallothionein mRNA abundance and protein concentration in newly
weaned pigs. J. Nutr. 134: 538-544.

54. Adeola, 0., Lawrence, 8., Sutton, A. & Cline, T. (1995) Phytase-
induced changes in mineral utilization in zinc-supplemented diets for pigs. J.
Anim. Sci. 73: 3384-3391.

28

55. Cromwell, G. L., Coffey, M. T., Monegue, H. J. & Randolph, J. H.
(1995) Efficacy of low-activity, microbial phytase in improving the bioavailability of
phosphorus in corn-soybean meal diets for pigs. J. Anim. Sci. 73: 449-456.

56. Sandberg, A. S., Hulthen, L. R. 8. Turk, M. (1996) Dietary Aspergillus
niger phytase increases iron absorption in humans. J. Nutr. 126: 476-480.

57. Jondreville, C., Revy, P. S. & Dourrnad, J. Y. (2003) Dietary means to
better control the environmental impact of copper and zinc by pigs from weaning
to slaughter. Livest. Prod. Sci. 84: 147-156.

58. Prassad, A. (1993). Historical aspects of zinc. Biochemistry of Zinc (E.
Frieden), pp. 1-15. Plemum Press, New York.

59. Prassad, A. (2003) Zinc and Immunity: Molecular Mechanisms of Zinc
Action on T Helper Cells. J. Trace. Elem. Exp. Med. 16: 139-163.

60. Tucker, H. F. & Salmon, W. D. (1955) Parakeratosis or zinc deficiency
disease in the pig. Proc. Soc. Exp. Biol. Med. 88: 613-616.

61. Mocchegiani, E., Muzzioli, M. & Giacconi, R. (2000) Zinc,
metallothioneins, immune responses, survival and ageing. Biogerontology 1:
133-143.

62. Vallee, B. L. & Falchuk, K. N. (1993) The biochemical basis of zinc
physiology. Phys. Res. 73: 79-118.

63. Vallee, B. L. & Auld, D. S. (1990) Active-site zinc ligands and activated
H20 of zinc enzymes. Proc. Natl. Acad. Sci. U S A 87: 220-224.

64. Mathews, B. W. (1988) Structural basis of the action of thermolysis
and related zinc peptidases. Acc. Chem. Res. 21: 333-340.

29

65. Coleman, J. E. (1992) Zinc proteins, enzymes, storage proteins,
transcription factors and replication proteins. Ann. Rev. Biochem. 61: 897-946.

66. Wellinghausen, N., Kirchner, H. & Rink, L. (1997) The immunobiology
of zinc. Immunol. Today 18: 519-521.

67. Beyersmann, D. & Haase, H. (2001) Functions of zinc in signaling,
proliferation and differentiation of mammalian cells. Biometals 14: 331-341.

68. Ferguson, E. L., Gibson, R. S., Opare Obisaw, C., Ounpuu, S.,
Thompson, L. U. & Lehrfeld, J. (1993) The zinc nutriture of preschool children
living in two African countries. J. Nutr. 123: 1487-1496.

69. Sazawal, S., Black, R. E., Bhan, M. K., Jalla, S., Sinha, A. & Bhandari,
N. (1997) Efficacy of zinc supplementation in reducing the incidence and
prevalence of acute diarrhea-a community-based, double-blind, controlled trial.
Am. J. Clin. Nutr. 66: 413-418.

70. Roy, S. K., Tomkins, A. M., Akramuzzaman, S. M., Behrens, R. H.,
Haider, R., Mahalanabis, D. & Fuchs, G. (1997) Randomized controlled trial of
zinc supplementation in malnourished Bangladeshi children with acute diarrhoea.
Arch. Dis. Child. 77: 196-200.

71. Dutta, P., Mitra, U., Datta, A., Niyogi, S. K., Dutta, 8., Manna, B.,
Basak, M., Mahapatra, T. S. & Bhattacharya, S. K. (2000) Impact of zinc
supplementation in malnourished children with acute watery diarrhoea. J. Trop.
Pediatr. 46: 259-263.

72. AI-Sonboli, N., Gurgel, R. Q., Shenkin, A., Hart, C. A. & Cuevas, L. E.
(2003) Zinc supplementation in Brazilian children with acute diarrhoea. Ann.
Trop. Paediatr. 23: 3-8.

73. Holm, A. (1988) Escherichia coli associated diarrhea in weaner pigs:
zinc oxide added to the feed as a preventative measure. Dan. Veterinaertidsskr
72: 1118-1126.

30

74. Jensen Waern, M., Melin, L., Lindberg, R., Johannisson, A.,
Petersson, L. & Wallgren, P. (1998) Dietary zinc oxide in weaned pigs-effects on
performance, tissue concentrations, morphology, neutrophil functions and faecal
microflora. Res. Vet. Sci. 64: 225-231.

75. Katouli, M., Melin, L., Jensen-Waem, M., Wallgren, P. & Mollby, R.
(1999) The effect of zinc oxide supplementation on the stability of the intestinal
flora with special reference to composition of coliforrns in weaned pigs. J. Appl.
Microbiol. 87: 564-573.

76. Roselli, M., Finamore, A., Garaguso, I., Britti, M. S. & Mengheri, E.
(2003) Zinc oxide protects cultured enterocytes from the damage induced by
Escherichia coli. J. Nutr. 133: 4077-4082.

77. Carlson, M. 8., Hoover, S. L., Hill, G. M., Link, J. E. & Turk, J. R.
(1998) Effect of pharmacological zinc on intestinal metallothionein concentration
and morphology in the nursery pig. J. Anim. Sci. 76: 57 (abs).

78. Stanger, B. R., Hill, G. M., Link, J. E., Turk, J. R., Carlson, M. S. &
Rozeboom, D. W. (1998) Effects of high Zn on TGE-challenged, early weaned
pigs. J. Anim. Sci. 76: 57 (abs).

79. Kagi, J. H. R. & Schaffer, A. (1988) Biochemistry of metallothionein.
Biochemistry 27: 8509-8515.

80. Kojima, Y., Berger, 0., Vallee, B. L. & Kagi, J. H. (1976) Amino-acid
sequence of equine renal metallothionein-1 B. Proc. Natl. Acad. Sci. U S A 73:
3413-3417.

81. Margoshes, M. & Vallee, B. L. (1957) A cadmium protein from equine
kidney cortex. J. Am. Chem. Soc. 79: 4813-4814.

82. Hamer, D. H. (1986) Metallothionein. Annu. Rev. Biochem. 55: 913-
951.

31

83. Nielson, K. B., Atkin, C. L. &Winge, D. R. (1985) Distinct metal-
binding configurations in metallothionein. J. Biol. Chem. 260: 5342-5350.

84. Suzuki, K. T. & Maitani, T. (1981) Metal dependent properties of
metallothionein. Biochem. J. 199: 289-295.

85. Miles, A. T., Hawksworth, G. M., Beattie, J. H. & Rodilla, V. (2000)
Induction, regulation, degradation and biological significance of mammalian
metallothioneins. Crit. Rev. Biochem. Mol. Biol. 35: 35-70.

86. Coyle, P., Philcox, J. 0, Carey, L. C. & Rofe, A. M. (2002)
Metallothionein: the multipurpose protein. Cell. Mol. Life Sci. 59: 627-647.

87. Romero lsart, N. & Vasak, M. (2002) Advances in the structure and
chemistry of metallothioneins. J. Inorg. Biochem. 88: 388-396.

88. Huang, M. C., Pan, P. K., Zheng, Z. F., Chen, N. C., Peng, J. Y. &
Huang, P. C. (1998) Multiple isoforms of metallothionein are expressed in the
porcine liver. Gene 211: 49-55.

89. Sato, M. & Bremner, I. (1993) Oxygen free radicals and
metallothionein. Free Radic. Biol. Med. 14: 325-337.

90. Kelly, E. J., Quaife, C. J., Froelick, G. J. & Palmiter, R. D. (1996)
Metallothionein l and II protect against zinc deficiency and zinc toxicity in mice. J.
Nutr. 126: 1782-1790.

91. Klaassen, C. D. & Liu, J. (1998) Metallothionein transgenic and knock-
out mouse models in the study of cadmium toxicity. J. Toxicol. Sci. 23: 97-102.

92. Schmidt, C. & Beyersmann, D. (1999) Transient peaks in zinc and
metallothionein levels during differentiation of 3T3L1 cells. Arch. Biochem.
Biophys. 364: 91-98.

32

93. Cherian, M. G. & Apostolova, M. D. (2000) Nuclear localization of
metallothionein during cell proliferation and differentiation. Cell. Mol. Biol. 46:
347-356.

94. Vallee, B. L. (1991). Introduction to Metallothionein. In: Methods in
Enzymology. Metallobiochemistry. Part B. Metallothionein and Related
Molecules (J.F. Riordan, & B.L. Vallee), pp. 3-8. Academic Press, Inc., Boston,
MA.

95. Cousins, R. J. (1985) Absorption, transport and hepatic metabolism of 1
copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol.
Rev. 65: 238-309.

96. Tran, C. 0., Butler, R. N., Howarth, G. S., Philcox, J. C., Rofe, A. M. &
Coyle, P. (1999) Regional distribution and localization of zinc and metallothionein
in the intestine of rats fed diets differing in zinc content. Scand. J. Gastroenterol.
34: 689-695.

97. Clarke, S. D. (1999) Nutrient regulation of gene and protein
expression. Curr. Opin. Clin. Nutr. Metab. Care 2: 287-289.

98. Hesketh, J. E., Vasconcelos, M. H. & Berrnano, G. (1998) Regulatory
signals in messenger RNA: determinants of nutrient-gene interaction and
metabolic compartmentation. Br. J. Nutr. 80: 307-321.

99. Durnam, D. M. & Palmiter, R. D. (1981) Transcriptional regulation of
the mouse metallothionein-l gene by heavy metals. J. Biol. Chem. 256: 5712-
5716.

100. Mo Mahon, R. J. & Cousins, R. J. (1998) Regulation of the zinc
transporter ZnT-1 by dietary zinc. Proc. Natl. Acad. Sci. USA 95: 4841-4846.

101. Andrews, G. K. (2000) Regulation of metallothionein gene expression
by oxidative stress and metal ions. Biochem. Pharmacol. 59: 95-104.

33

102. Stuart, G. W., Searle, P. F., Cen, H. Y., Brinster, R. L. & Palmiter, R.
D. (1984) A 12-base—pair DNA motif that is repeated several times in
metallothionein gene promoters confers metal regulation to a heterologous gene.
Proc. Natl. Acad. Sci. USA 81: 7318.

103. Dunn, M. A., Blalock, T. L. & Cousins, R. J. (1987) Metallothionein.
Proc. Soc. Exp. Biol. Med. 185: 107-119.

104. Mc Corrnick, C. C., Menard, P. M. & Cousins, R. J. (1981) Induction
of hepatic metallothionein by feeding zinc to rats of depleted zinc status. Am. J.
Physiol. 240: E414-E421.

105. Blalock, T. L., Dunn, M. A. & Cousins, R. J. (1988) Metallothionein
gene expression in rats: tissue-specific regulation by dietary copper and zinc. J.
Nutr. 118: 222-228.

106. Richards, M. P. & Cousins, R. J. (1975) Mammalian zinc
homeostasis: requirement for RNA and metallothionein synthesis. Biochem.
Biophys. Res. Commun. 64: 1215-1223.

107. Chen, R. W., Vasey, E. J. & Whanger, P. D. (1977) Accumulation
and depletion of zinc in rat liver and kidney metallothionein. J. Nutr. 107: 805-
813.

108. Schroeder, J. J. & Cousins, R. J. (1990) Interleukin 6 regulates
metallothionein gene expression and zinc metabolism in hepatocyte monolayer
cultures. Proc. Natl. Acad. Sci. USA 87: 3137-3141.

109. Coyle, P., Philcox, J. C. & Rofe, A. M. (1995) Metallothionein
induction in cultured rat hepatocytes by arthritic rat serum, activated
macrophages, interleukin-6, interleukin-11 and leukaemia inhibitory factor.
lnflamm. Res. 44: 475-481.

110. Lee, D. K., Carrasco, J., Hidalgo, J. & Andrews, G. K. (1999)
Identification of a signal transducer and activator of transcription (STAT) binding
site in the mouse metallothionein-l promoter involved in interleukin-6-induced
gene expression. Biochem. J. 337: 59-65.

34

111. Blanchard, R. K. & Cousins, R. J. (1996) Differential display of
intestinal mRNAs regulated by dietary zinc. Proc. Natl. Acad. Sci. USA 93: 6863-
6868.

112. Greenberg, R. N., Hill, M., Crytzer, J., Krause, W. J., Eber, S. L.,
Hamra, F. K. & Forte, L. R. (1997) Comparison of effects of uroguanylin,
guanylin, and Escherichia coli heat-stable enterotoxin STa in mouse intestine
and kidney: evidence that uroguanylin is an intestinal natriuretic hormone. J.
Investig. Med. 45: 276-282.

113. Chesters, J. K. & Quarterrnan, J. (1970) Effects of zinc deficiency on
food intake and feeding patterns of rats. Br. J. Nutr. 24: 1061-1069.

114. Cui, L., Blanchard, R. K., Coy, L. M. & Cousins, R. J. (2000)
Prouroguanylin overproduction and localization in the intestine of zinc-deficient
rats. J. Nutr. 130: 2726-2732.

115. Blanchard, R. K., Moore, J. 8., Green, C. L. & Cousins, R. J. (2001)
Modulation of intestinal gene expression by dietary zinc status: effectiveness of
cDNA arrays for expression profiling of a single nutrient deficiency. Proc. Natl.
Acad. Sci. U S A 98: 13507-13513.

116. Moore, J. B., Blanchard, R. K. & Cousins, R. J. (2003) Dietary zinc
modulates gene expression in murine thymus: results from a comprehensive
differential display screening. Proc. Natl. Acad. Sci. U S A 100: 3883-3888.

117. Krebs, N. F. (2000) Overview of zinc absorption and excretion in the
human gastrointestinal tract. J. Nutr. 130: 13748-13778.

118. Reyes, J. G. (1996) Zinc transport in mammalian cells. Am. J.
Physiol. 270: 0401-0410.

119. Vallee, B. L. & Auld, D. S. (1990) Zinc coordination, function and
structure of zinc enzymes and other proteins. Biochemistry 29: 5647-5659.

35

120. Zalewski, P. D., Millard, S. H., Forbes, I. J., Kapaniris, 0., Slavotinek,
A., Betts, W. H., Ward, A. 0., Lincoln, S. F. & Mahadevan, l. (1994) Video image
analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent
probe for zinc. J. Histochem. Cytochem. 42: 877-884.

121. Williams, R. J. P. (1989). An introduction to the biochemistry of zinc.
In: Zinc in Human Biology (C.F. Mills), pp. 15-31. Springer-Verlag, New York.

122. Gaither, L. A. & Eide, D. J. (2001) Eukaryotic zinc transporters and
their regulation. Biometals 14: 251-270.

123. Kambe, T., Yamaguchi-lwai, Y., Sasaki, R. & Nagao, M. (2004)
Overview of mammalian zinc transporters. Cell Mol. Life. Sci. 61: 49-68.

124. Palmiter, R. D. & Huang, L. (2004) Efflux and compartmentalization
of zinc by members of the SLC30 family of solute carriers. Pflugers Arch. 447:
744-751.

125. Kirschke, C. P. & Huang, L. (2003) ZnT7, a novel mammalian zinc
transporter, accumulates zinc in the Golgi apparatus. J. Biol. Chem. 278: 4096-
4102.

126. Liuzzi, J. P., Blanchard, R. K. & Cousins, R. J. (2001) Differential
regulation of zinc transporter 1, 2 and 4 mRNA expression by dietary Zn in rats.
J. Nutr. 131: 46-52.

127. Palmiter, R. D., Cole, T. B., Quaife, C. J. & Findley, S. D. (1996) ZnT-
3 a putative transporter of zinc into synaptic vesicles. Proc. Natl. Acad. Sci. USA
93: 14934-14939.

128. Kambe, T., Narita, H., Yamaguchi-lwa, i. Y., Hirose, J., Amano, T.,
Sugiura, N., Sasaki, R., Mori, K., lwanaga, T. & Nagao, M. (2002) Cloning and
characterization of a novel mammalian zinc transporter, zinc transporter 5,
abundantly expressed in pancreatic B cells. J. Biol. Chem. 277: 19049-19055.

36

129. Huang, L., Kirschke, C. P. & Gitschier, R. J. (2002) Functional
characterization of a novel mammalian zinc transporter, ZnT-6. J. Biol. Chem.
277: 26389-26395.

130. Huang, L. & Gitschier, R. J. (1997) A novel gene involved in zinc
transport is deficient in the lethal milk mouse. Nat. Genet 17: 292-297.

131. Piletz, J. E. & Ganschow, R. E. (1978) Zinc deficiency in murine milk
underlies expression of the lethal milk (lm) mutation. Science 199: 181-183.

132. Kelleher, S. L. & Lbnnerdal, B. (2003) Zn transporter levels and
localization change throughout lactation in rat mammary gland and are regulated
by zinc in mammary cells. J. Nutr. 133: 3378-3385.

133. Palmiter, R. D. & Findley, S. D. (1995) Cloning and functional
characterization of a mammalian zinc transporter that confers resistance to zinc.
The EMBO J. 14: 639-649.

134. Tsuda, M., lmaizumi, K., Katayama, T., Kitagawa, K., Wanaka, A.,
Tohyama, M. & Takagi, T. (1997) Expression of zinc transporter gene, ZnT-1, is
induced after transient forebrain ischemia in the gerbil. J. Neurosci. 17: 6678-
6684.

135. Langmade, S. J., Ravindra, R., Daniels, P. J. & Andrews, G. K.
(2000) The transcription factor MTF-1 mediates metal regulation of the mouse
ZnT1 gene. J. Biol. Chem. 275: 34803-34809.

136. Kelleher, S. L. & Lennerdal, B. (2002) Zinc transporters in the rat
mammary gland respond to marginal zinc and vitamin A intakes during lactation.
J. Nutr. 132: 3280-3285.

137. Palmiter, R. D. (2004) Protection against zinc toxicity by
metallothionein and zinc transporter 1. Proc. Natl. Acad. Sci. U S A 101: 4918-
4923.

37

CHAPTER TWO

Martinez, Michelle M., Hill, Gretchen M., Link, Jane E., Raney, Nancy E.,
Tempelman, Robert J., Ernst, Catherine W. (2004) Pharmacological Zinc
and Phytase Supplementation Enhance Metallothionein mRNA Abundance
and Protein Concentration in Newly Weaned Pigs. Mm,
134: 538-544.

38

Pharmacological Zinc and Phytase Supplementation Enhance
Metallothionein mRNA Abundance and Protein Concentration in Newly
Weaned Pigs"2
Michelle M. Martinez, Gretchen M. Hill,3 Jane E. Link, Nancy E. Raney, Robert J.

Tempelman and Catherine W. Ernst

Department of Animal Science, Michigan State University, East Lansing, MI

Running title: ZINC & PHYTASE IMPACT ON METALLOTHIONEIN IN PIGS

1 Presented in part at the 11th meeting of Trace Elements in Man and Animals
(T EMA), in Berkeley, California, June 2-6, 2002 [Martinez, M.M., Hill, G.M., Link,
J.E., Ernst, C.W., and Raney, NE. (2002) Impact of Pharmacological Zinc and
Phytase on Liver Metallothionein Concentration and mRNA Abundance in the
Young Pig. (Abstract 59)], and at the American Society of Animal Science
Midwestern section meeting, March 17-19, 2003 [Martinez, M.M., Hill, G.M., Link,
J.E., Raney, NE, and Ernst, CW. (2003) Pharmacological Zn and phytase
enhance renal and intestinal mucosa cell metallothionein protein and relative
mRNA abundance in the nursery pig. J. Anim. Sci. 81 :56 (Abstract 226).]

2 Supported in part by an Animal Health Formula Fund Grant from the Michigan
Agricultural Experiment Station, East Lansing, Michigan.

3 To whom correspondence should be addressed. E-mail: hillgre@msu.edu.
Michigan State University, Department of Animal Science, 2209 Anthony Hall,
East Lansing, MI 48824. Fax (517) 432-0190. Tel. (517) 355-9676.

39

4 Abbreviations used: APC, American Protein Corporation; CI, confidence
interval; MT, metallothionein; Zn, zinc; ZnO, zinc oxide; Z150, 150 mg ankg;
Zn150P, 150 mg Zn/kg with phytase; 201.000. 1,000 mg Zn/kg; Zn1,oooP, 1,000 mg
Zn/kg with phytase; Zn2,ooo, 2,000 mg Zn/kg; anoooP, 2,000 mg Zn/kg with

phytase.

40

ABSTRACT

The swine industry feeds pharmacological zinc (Zn) to newly weaned pigs
to improve health. Since most swine diets are plant-based with high phytic acid
contents, we hypothesized that adding phytase to diets could reduce the amount
of Zn needed to obtain beneficial responses. Metallothionein’s (MT) role in Zn
homeostasis could be important in this positive response. Thus, the goal of this
study was to investigate the effect of dietary Zn and phytase on relative MT
mRNA abundance and protein concentration in newly weaned pigs. Diets
containing adequate (150 mg ankg) or pharmacological concentrations of Zn
(1 ,000 or 2,000 mg Zn/kg), as zinc oxide, with or without phytase (0, 500 FT Ulkg,
Natuphos® BASF) were fed in a 3 x 2 factorial design. Plasma and tissue
minerals were measured in pigs killed after 14 d of dietary intervention. Hepatic
and renal relative MT mRNA abundance and protein were greater (P < 0.05) in
pigs fed 1,000 mg Zn/kg with phytase, or 2,000 mg Zn/kg with or without phytase
versus the remaining treatments. Intestinal mucosa MT mRNA abundance and
protein were greatest (P < 0.05) in pigs fed 2,000 mg Zn/kg with phytase. Pigs
fed 1,000 mg Zn/kg plus phytase or 2,000 mg Zn/kg with or without phytase had
higher plasma, hepatic and renal Zn than those fed the adequate Zn diets or
1,000 mg Zn/kg. We conclude that feeding 1,000 mg ankg with phytase
enhances MT mRNA abundance and protein and Zn absorption just as 2,000 mg
Zn/kg with and without phytase.

KEY WORDS: - metallothionein - pharmacological zinc - phytase - pig

41

INTRODUCTION

In recent years, Zn supplementation has been used in developing
countries to treat diarrheal disease in young children (1,2). In addition, the US.
swine industry adds 2,000 — 3,000 mg ankg as zinc oxide (ZnO) to the diets of
newly weaned pigs for the first 14 d post-weaning to promote growth (3,4) and
fecal consistency (5,6), both of which can be impaired by weaning stress.
Feeding pharmacological Zn (3,000 mglkg) for 14 d to newly weaned pigs
improves gut morphology by increasing villous height and reducing crypt depth in
the duodenum and jejunum, thus potentially increasing the absorptive capacity of
the small intestine and consequently improving growth (7).

Since the 1960’s there has been increasing evidence that phytic acid, an
organic molecule found in cereal grains, possesses anti-nutritional properties
(8,9). Phytic acid forms insoluble salts with bivalent cations such as calcium
(Ca) and Zn, thus affecting their bioavailability at neutral and alkaline pH, leading
to increased mineral excretion and reduced mineral absorption (10). Since pigs
consume grain diets and lack the ability to produce sufficient phytase
endogenously, supplementation of this enzyme in feed has been shown to
improve mineral bioavailability and decrease nutrient excretion (11). However,
the effect of phytase when pharmacological Zn is fed has not been evaluated.

Metallothionein is a low molecular weight (~7 kDa), cysteine-rich protein
present in many living organisms (12). This protein has been considered an
intracellular marker of excess Zn inside cells, based on the increased induction of

MT when dietary Zn intakes are well above normal (13). Despite many decades

42

of research, MT’s precise physiological function has not been elucidated.
However, some suggested functions attributed to this protein are detoxification of
nonessential heavy metals (cadmium and mercury), homeostasis of Zn and
copper (Cu), metal-transfer, free-radical scavenger, and metal-storage (14,15).

Transcriptional regulation of MT by dietary Zn has been demonstrated in
rats and cultured cells treated with Actinomycin D (16). Moreover, evidence
suggests that transcriptional regulation of the MT gene occurs by activation of
metal response elements (MREs) located in the promoter region of the MT gene,
in response to the binding of intracellular Zn to metal transcription factor-1 (MTF-
1) (17). The MTF-1 acts as an intracellular Zn sensor able to coordinate several
genes involved in Zn homeostasis (18).

The purpose of this experiment was to determine the effects of
supplementing pharmacological Zn with or without phytase on liver, kidney and
intestinal mucosa MT mRNA abundance and protein concentration. In previous
research when swine diets contained pharmacological Zn, an interaction
between Zn, Cu and iron (Fe) was observed (5,19). Thus, the dietary effect of
pharmacological Zn and phytase on hepatic, renal and plasma mineral (Cu, Fe,

Zn and P) concentration of the pigs was also investigated.

43

MATERIALS AND METHODS

Animals and diets. Ninety-six crossbred [(Landrace x Yorkshire) x
Duroc] gilts and barrows (5.5 kg, 18 d of age) were housed in pens (2.23 m2), in
a biosecure, environmentally controlled room (ZS-30°C) at the Swine Teaching
and Research Center at Michigan State University (East Lansing, MI). At
weaning, pigs (4 l pen) were randomly allotted to one of six different treatments
based on weight and litter for a 14-d study. Diets met or exceeded the
recommendations of the National Research Council (20). The Zn source used in
this experiment was ZnO which contained 72% Zn (Prince Agri Products, Quincy,
IL). The phytase was added at 500 FTU/kg (Natuphos®, BASF Corp., Mount
Olive, NJ). One phytase unit (FTU) is the amount of enzyme that releases 1umol
inorganic phosphorus from sodium phytate per minute at pH 5.5 and 37°C.
Treatments in a 3 x 2 factorial arrangement were fed for 14 d (Table 1). The
dietary treatments were: 1) adequate Zn diet containing 150 mg Zn/kg (Zn150), 2)
Zn150 plus 500 FTng (Zn15oP), 3) pharmacological Zn diet containing 1,000 mg
ankg (Zntooo), 4) 201,000 plus 500 FTU/kg (Zn1,oooP), 5) pharmacological Zn diet
containing 2,000 mg Zn/kg (202,000). or 6) 202.000 plus 500 FTUlkg (anoooP).
Pigs were provided feed and water ad libitum. This project was approved by the
Michigan State University All University Committee on Animal Use and Care
(12l99-1 59-00).

Sample collection and measurements. The pigs and feed were
weighed weekly and daily gain, daily feed intake and feed efficiency were

calculated. On d-14, one pig from each pen (4 pigs l treatment) was randomly

44

selected and bled from the anterior vena cava with heparinized vacutainer tubes
(BD Vacutainerm, Becton Dickson, Franklin Lakes, NJ) for determination of
plasma minerals (Cu, Fe, Zn and P). These pigs were then killed by a lethal
injection (0.05 mng BW) of sodium pentobarbital (392 glL). Kidney and liver
samples were excised for mineral (Cu, Fe, Zn and P) and MT protein analysis,
while separate tissue samples were collected and flash frozen in liquid nitrogen
for RNA isolation. The small intestine was ligated at the ligament of Treitz to
remove the first 20 cm segment of intestine. The intestinal sample was cut
longitudinally starting at the pylorus to expose mucosa cells and washed three
times in 20 mL of ice-cold phosphate buffered saline (PBS). The intestinal
mucosa was scraped and cells were prepared following the procedure of Carlson
et al. (4). Half of the cells were weighed and collected in 15 mL polypropylene
tubes (Corning lnc., Corning, NY) followed by the addition of 250 mmol/L sucrose
buffer (a volume of 4 times the weight) for the detection of MT. The remaining
cells were flash frozen in liquid nitrogen for RNA isolation.

Mineral analysis. All glassware used during the analysis was soaked for
12 h in 500 mmol/L nitric acid solution, followed by two rinses with deionized-
distilled water. To determine mineral concentrations, the tissue and feed were
microwave digested (21). The concentrations of Cu, Fe and Zn were determined;
by flame atomic absorption spectometry (Unicam 989 Atomic Absorption
Spectometer, SOLAAR Series, Cambridge, UK) while tissue P concentration was
detected by a colorimetric method (22) utilizing a Beckman DU7400

spectrophotometer (Beckman Coulter lnc., Fullerton, CA). Bovine liver was used

45

as standard reference (15771, US. Department of Commerce, National Institute
of Standards and Technology, Gaithersburg, MD) and was analyzed with the
tissue samples for instrument standardization and quality control.

Plasma samples were deproteinized with 800 mmol/L trichloroacetic acid
(TCA) solution prior to the Cu, P, and Zn analyses. Briefly, plasma samples were
added 1:4 (v/v), vortexed and centrifuged at 2,000 x g for 15 min and analyzed
for Cu and Zn by flame atomic absorption spectometry, and P by the colorimetric
method (22). The number of plasma samples available for the P analysis varied
among the different dietary treatments (Zn150, n=3; Zn150P, n=2; Zn1,ooo, n=4;
Zn1,oooP, n=2; 202.000. n=2; anoooP, n=4) due to inadequate sample volume for
each animal. Plasma Fe was determined by flame atomic absorption
spectrometry (4).

Metallothionein assay. Metallothionein protein concentration was
determined by a modification of the silver saturation assay (23). A red blood
cell hemolysate was prepared from fresh porcine blood (24). Reagents were
prepared daily. Tissue (0.2 - 0.59) was homogenized (Janke & Kunkel Ultramax
T25, Tekmar Company, Cincinnati, OH) in 250 mmol/L sucrose buffer (1 :4) and
centrifuged at 18,000 x g for 20 min. For renal and intestinal samples from pigs
fed 2mm and Zn150P, two additional repetitions of the hemolysate step were
performed.

RNA isolation. Total RNA was isolated from kidney, liver and intestinal
mucosa cells using TRlzol® Reagent (GIBCO BRL, Life Technologies,

Gaithersburg, MD) according to the manufacturers protocol. RNA concentrations

46

were determined by absorption at 260 nm (A250) using a Beckman DU650
spectophotometer (Beckman Coulter, Inc, Fullerton, CA). RNA quality and
integrity were determined by calculating the Ame/280 ratio and by agarose gel
electrophoresis, respectively.

Dot blot and northern blot analysis. Duplicate dot blots were prepared
as previously described (25) by spotting 1, 3 and 5 pg of denatured kidney, liver
or intestinal mucosa RNA (n=6) onto nylon membranes (Hybond-XL, Amersham
Biosciences Corp., Piscataway, NJ). Total RNA samples were also subjected to
northern blot analysis as previously described (25) to determine the number and
size of MT transcripts. The dot and northern blots were hybridized with a mouse
MT-1 cDNA probe (provided by J. Carrasco and J. Hidalgo of the Universidad
Auténoma de Barcelona, Barcelona, Spain) using modifications of standard
procedures (26,27). Blots were also probed with a porcine glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) cDNA probe for normalization (28). Probes
were synthesized using 3zP-dCTP (Perkin Elmer, Life Sciences Inc., Boston MA)
and the Multiprime DNA Labeling System (RPN 16012, Amersham Biosciences
Corp., Piscataway, NJ). Hybridizations were done at 65°C for 16-22 h.
Membranes were rinsed as previously described (25) and exposed to Kodak
BioMax film (Eastman Kodak Co, Rochester, NY) in the presence of intensifying
screens at -80°C for 1-4 d. Quantification of MT and GAPDH relative mRNA
abundance was achieved by densitometry scan of autoradiographs using a

Fluor-S Multilmager (Bio-Rad Laboratories, Hercules, CA).

47

Statistical analysis. The data was analyzed using regular analysis of
variance procedures based on the MIXED procedure of SAS (29). Factors of
interest always included zinc, phytase and their interaction. The experimental unit
in the growth performance analysis was the pen, while for plasma and tissue
minerals, and MT protein concentration, the individual pigs served as
experimental units. For MT mRNA abundance, blot by treatment (zinc-phytase)
interaction was additionally modeled along with pig as random effects to define
the experimental units for this analysis, with Satterthwaite’s approximation used
to determine the error degrees of freedom for test. Furthermore, GAPDH mRNA
abundance was modeled as a covariate for a regression-based normalization of
MT mRNA abundance in accordance with recent recommendations by Poehlman
(30).

With the exception of Fe values, a logarithmic transformation was required
to make the data more normally distributed. Thus, back-transformed means and
their respective 95% confidence intervals (lower and upper limits) are provided
as point and interval estimates, respectively. Scheffé’s test was used to
determine the presence of linearity of response to treatments.

The Fe concentrations and remaining log-transformed values were used
for a residual correlation analysis between plasma and tissue mineral
concentrations, MT relative mRNA abundance and protein concentrations. Each
pair of traits is jointly modeled using a series of bivariate mixed effects models
that each include the specified between-trait residual correlation to be estimated.

SAS PROC MIXED is used to do this in a very similar way to what was also

48

recently considered by Thiebaut et al. (31). The residual correlation is an
adjusted or partial correlation. That is, after the mixed model effects (eg. zinc,
phytase, zinc x phytase, blot, blot x zinc x phytase, pig) are accounted or
adjusted for, the correlation for whatever is left over (hence the term residual
correlation) between responses of interest is estimated. Residual correlations

were considered to be different from 0 when P < 0.05.

49

RESULTS

Plasma and tissue mineral concentrations. Feeding pharmacological
Zn (1 ,000 or 2,000 mg ankg) with or without phytase to the nursery pigs did not
affect plasma Cu (Table 2). However, a significant interaction of phytase and
dietary Zn on renal Cu was obtained (P < 0.05). Different dietary treatments did
not significantly affect hepatic Cu or Fe, or plasma or renal Fe concentrations
(data not shown). Plasma P concentration was higher (P < 0.05) in the pigs fed
the phytase-supplemented diets, except in the pigs fed anoooP. In this study,
the pigs’ renal P concentration was not affected by the dietary treatments.
However, both dietary Zn (P < 0.05) and supplemental phytase (P < 0.05)
increased hepatic P (Table 2).

A statistically significant effect of zinc (P < 0.05) and phytase (P < 0.05)
was observed for plasma Zn concentrations (Figure 1A). In the liver (Figure
18), there was a statistically significant interaction of zinc and phytase
supplementation (P < 0.05). However, renal Zn responded similarly to plasma Zn
(Figure 1C) where a significant effect of dietary Zn (P < 0.05), as well as by
phytase supplementation (P < 0.05) was obtained. The Scheffe’s test confirmed
that regardless of phytase supplementation, increasing the concentration of
dietary Zn caused a linear increase in plasma, kidney and liver Zn (P < 0.0001).

Metallothionein protein and mRNA abundance. Northern blot
analysis was performed to determine the number and size of MT transcripts in
kidney, liver and intestinal mucosa. The analysis revealed the presence of a

single MT transcript (~ 0.5 kb) in all three tissues, although no transcript was

50

detected in the intestinal mucosa of pigs fed Zn150 (data not shown). The blots
were stripped and re-probed with GAPDH and a single transcript (~ 1.2 kb) was
detected in all of the samples (data not shown).

Dot blot analysis was performed to determine relative MT mRNA
abundance in liver (Figure 2), kidney and intestinal mucosa of individual pigs.
The pigs fed Zn1,oooP, 202,000 or anoooP had greater MT mRNA abundance when
compared to pigs fed adequate Zn (Zn150, Zn150P) or 1,000 mg ankg (Zn1,ooo).

To correct for loading differences, all dot blots were striped and re-probed with
porcine GAPDH (data not shown).

Relative metallothionein mRNA abundance (Figure 3) in the liver tissue of
pigs fed Zn1,oooP was not statistically different from that of pigs fed 202.000 or
anoooP. A significant interaction (P < 0.01) of phytase and zinc supplementation
on liver relative MT mRNA abundance was obtained with values being four to
five-fold greater in pigs fed Zn1,oooP, 202.000 or anoooP than for those fed Zn150,
Zn15oP or anoo. No significant interaction was observed for hepatic MT protein
concentration (Figure 3). However, MT protein concentration increased with
dietary Zn (P < 0.05) and with supplemental phytase (P < 0.05).

In the kidney, a significant zinc and phytase interaction on MT mRNA
abundance (Figure 4) was observed (P < 0.04). In addition, there was a
significant effect of Zn on MT protein (Figure 4) concentration in the kidney (P <
0.0003), but no phytase effect was obtained.

The response of MT in the intestinal mucosa (Figure 5) was different than

that observed in the other tissues. The relative MT mRNA abundance and the

51

protein concentration obtained from intestinal mucosa cells of pigs fed Zn15o,
Zn150P or 201.000 was below the detectable limits of the assays (0.29 arbitrary
units and 186 pmol Ag/g, respectively). However, there was a significant Zn and
phytase interaction on relative MT mRNA abundance and MT protein
concentration (Figure 5). In both cases, the greatest response was in pigs that
received anoooP when compared with the pigs that were fed Zn1,oooP or 202.000
(P < 0.05).

Residual correlations. The results of the mineral residual correlation
(Table 3) demonstrated that in the liver and kidney, Cu and Fe were significantly
correlated. Moreover, in the kidney, Zn was correlated with kidney Cu and Fe (P
< 0.01 and P < 0.02, respectively). However, residual correlations between
plasma mineral concentrations and tissue minerals, relative MT mRNA
abundance and protein were not significant (data not shown).

The MT residual correlation (Table 3) confirmed that liver MT mRNA
abundance and liver MT protein were significantly correlated. Furthermore, liver
Zn was correlated with liver relative MT mRNA abundance and protein
concentration (P < 0.03 and P < 0.01, respectively). A similar relationship was
obtained for the correlation analysis performed in the renal tissue. Kidney MT
mRNA and protein were correlated (P < 0.01), while kidney Zn was correlated
with both relative MT mRNA abundance and protein (P < 0.01 and P < 0.01,
respectively). The intestinal mucosa MT mRNA and MT protein displayed a

trend to significantly correlate (r = 0.36, P = 0.08, data not shown).

52

Growth performance. Significant differences in daily gain were observed
on the first week of the study. Pigs fed supplemental phytase gained faster
(203.49 i 33.20 vs. 238.76 :t 34.55, P < 0.03). For the remainder of the

experiment, there were no differences in gains, feed intake or feed efficiency

(data not shown).

53

DISCUSSION

Weaning is a major stress that involves a change of diet and environment
for pigs resulting in decreased performance and increased incidence of diarrhea.
In addition, the corn and soybeanmeal included in swine diets are high in phytic
acid, which causes adverse effects on mineral bioavailability and may contribute
to depressed growth (8). Nutritional approaches have been investigated to
promote nutrient availability and growth in newly weaned pigs. Supplementing
phytase to weaned pig diets results in improved Zn utilization (32), while
pharmacological Zn enhances growth performance and reduces the incidence of
diarrhea in nursery pigs through an undetermined mechanism (33). However,
feeding a combination of supplemental phytase and pharmacological Zn to pigs
has not been reported.

Proposed mechanisms for enhanced growth with pharmacological Zn
include 1) metallothionein, a protein that plays a key role in Zn homeostasis by
sequestering Zn in the intestine, and 2) improved gut morphology (7). In
agreement with studies of rats and mice that demonstrate dietary Zn regulation of
the MT gene (16,34), in this study we confirm that pharmacological Zn and
phytase supplementation increase the abundance of MT mRNA in the liver,
kidney and intestinal mucosa cells of nursery pigs. By performing northern blot
analysis of liver, kidney and intestinal mucosa RNA, we revealed the presence of
an approximately 0.5 kb MT transcript, which is in accordance with a rat

transcript size previously published for MT (35).

The liver, where the highest concentrations of Zn and MT were found, was
the most responsive tissue, similar to the work of Carlson and collaborators (4).
Pigs fed 1,000 mg ankg with no phytase had liver MT mRNA abundance and
protein concentration similar to pigs fed adequate Zn, regardless of phytase
inclusion. However, when phytase is added to 1,000 mg Zn/kg, MT mRNA and
protein concentration increase and are similar to pigs fed 2,000 mg Zn/kg with or
without phytase. Interestingly, this dietary treatment effect was also obtained
with Zn concentrations in the liver. This suggests that supplementing zinc and
phytase increases the amount of Zn absorbed, characterized by an overall
greater MT mRNA abundance, MT protein and Zn concentration.

In renal tissue, a different response pattern for MT mRNA abundance and
protein was seen than that obtained in the liver. A significant interaction of zinc
and phytase on MT mRNA abundance was obtained. Metallothionein protein
concentration increased with dietary Zn, whereas phytase did not have a
synergistic effect. Nevertheless, MT mRNA abundance, protein and Zn
concentrations were positively correlated (P < 0.01), similar to the work of Blalock
et al. (36) where rats fed 180 mg Zn/kg had kidney MT mRNA abundance linearly
correlated with total MT protein. Blalock and collaborators also observed a
tissue difference on the induction of both metallothionein mRNA and protein.

The amount of intestinal mucosa MT differed from that observed in the
liver and kidney, emphasizing tissue specificity. The amount induced in pigs fed
Zn150, Zn150P, or 201.000 was below the detection limits of the MT mRNA

abundance and protein assays, hence no data is reported. Metallothionein

55

mRNA abundance and protein concentrations from pigs fed anoooP were higher
than in pigs fed Zn1,oooP or 202.000. This suggests that in the upper intestine, MT
is induced to a greater extent when exposed to high dietary Zn, 10 to 20 times
the requirement, and this effect is more marked by supplementing phytase along
with pharmacological Zn (2,000 mg Zn/kg), an effect not seen with other tissues.
In addition, the early work of Richards and Cousins (16) showed that dietary Zn
regulates MT, which plays a key role in Zn absorption by controlling the amount
of Zn entering the body and transferred into portal circulation.

Using the Zn chelator Zinquin and immunohistochemical analysis, Tran et
al. (13) reported that rats fed increasing concentrations of Zn (0, 400 mg ankg)
displayed an increased proportion of the Zn attached to the luminal surface of the
gut, but when Zn was fed at a pharmacological concentration (1 ,000 mg Zn/kg)
more Zn was internalized and sequestered by MT synthesized de novo. Perhaps
the unique MT increase observed in the intestinal mucosa in pigs fed anmoP
prevents additional Zn from crossing into circulation, thus preventing higher Zn
concentrations in the liver. Moreover, it is possible that the hepatic Zn pool of
pigs fed Zn15o, Zn150P or anoo, can be processed efficiently by several
mechanisms, one of which might be MT. However, when pigs are fed Zn1,oooP,
anpoo or anoooP the MT concentrations plateau, indicating that an alternate
pathway might be needed to metabolize this Zn overload.

In the plasma, Zn concentrations increased linearly (P < 0.05) with dietary
Zn. However, no statistically significant residual correlations between plasma

and tissue mineral concentrations, MT protein or MT mRNA abundance were

56

obtained. Similar results were observed in young broilers by Sandoval et al. (37).
Thus, the use of plasma Zn as an absorption or mineral status indicator in pigs is
of little value.

Feeding the combination of Zn and phytase to pigs increases renal Cu
concentration (P < 0.05) similar to observations by Carlson et al. (4) when
nursery pigs were fed 3,000 mg ankg (ZnO) for up to 28 d. This documented
effect of Zn on Cu metabolism was previously observed in nursery pigs whose
mothers were fed pharmacological Zn (5,000 mg Zn/kg) for two parities (19).
Hepatic Cu concentrations were depressed in the young pig, which has also
been observed in humans and rats. The lack of response in hepatic Cu in this
study may be due to the limited time of the dietary intervention.

Even though it has been suggested that the Ca to P ratio can be adjusted
to obtain a significant phytase effect (38), our experimental diets were formulated
to meet the P requirement as established by the NRC for swine (20). Pigs fed
the highest Zn diets as well as those fed phytase had increased (P < 0.05) P
concentrations in the liver, in accordance to similar results obtained by our lab,
where a numerical increase in hepatic P concentration was obtained in pigs fed
2,000 mg ankg for 14 d (39). This unique pharmacological Zn effect on P
metabolism deserves further investigation. In addition, plasma P was also higher
(P < 0.05) when pigs were fed supplemental phytase. These results demonstrate
an apparent increase in P availability primarily caused by phytase
supplementation, which is in accordance with previous research demonstrating

that phytase supplementation increases overall P retention in pigs (11).

57

In conclusion, these findings support the hypothesis that pigs fed
pharmacological Zn have increased organ Zn characterized by increased MT
mRNA abundance and MT protein concentrations. Moreover, supplementing
phytase further enhanced these effects when a minimum of 1,000 mg Zn/kg was
fed. Due to MT’s role in sequestering Zn when excess Zn is fed, one might
hypothesize that adequate dietary Zn concentrations (150 mg Zn/kg) did not
provide sufficient Zn to significantly induce MT, even when phytase was
supplemented. However, adding phytase to 1,000 mg Zn/kg diet did provide the
additional Zn necessary for higher MT mRNA abundance and protein compared
to 1,000 mg ankg alone. These data suggest that current pharmacological
doses of Zn fed to pigs (2,000 mg ankg) could be reduced to 1,000 mg Zn/kg by
adding phytase. This dietary modification could result in the enhanced growth
benefits of pharmacological Zn, as well as reduced nutrient excretion, especially

Zn, by newly weaned pigs.

58

ACKNOWLEDGEMENT
The authors wish to express appreciation to J. Carrasco and J. Hidalgo of
the Universidad Autonoma de Barcelona, Barcelona, Spain for providing the
mouse MT-1 cDNA probe, and BASF Corporation, Prince Agri Products and APC
Inc for supplying ingredients used in this study. The technical assistance of

Dana Dvoracek-Driksna and Kim Hargrave is gratefully acknowledged.

59

LITERATURE CITED

1. Sazawal, S., Black, R. E., Bhan, M. K., Jalla, S., Sinha, A., & Bhandari,
N. (1997) Efficacy of zinc supplementation in reducing the incidence and
prevalence of acute diarrhea - a community-based, double-blind, controlled trial.
Am. J. Clin. Nutr. 66:413-418.

2. Dutta, P., Mitra, U., Datta, A., Niyogi, S. K., Dutta, S., Manna, B.,
Basak, M., Mahapatra, T. S., & Bhattacharya, S. K. (2000) Impact of zinc
supplementation in malnourished children with acute watery diarrhoea. J. Trop.
Pediatr. 46: 259-263.

3. Stanger, B. R., Hill, G. M., Link, J. E., Turk, J. R., Carlson, M. S., &
Rozeboom, D. W. (1998) Effects of high Zn on TGE-challenged, early weaned
pigs. J. Anim. Sci. 76: 57.

4. Carlson, M. S., Hill, G. M., & Link, J. E. (1999) Early and traditionally
weaned nursery pigs benefit form phase-feeding pharmacological concentrations
of zinc oxide: effect on metallothionein and mineral concentrations. J. Anim. Sci.
77: 1199-1207.

5. Hill, G. M., Cromwell, G. L., Crenshaw, T. D., Dove, C. R., Ewan, R. C.,
Knabe, D. A., Lewis, A. J., Libal, G. W., Mahan, D. C., Shurson, G. 0., Southern,
L. L., & Venum, T. L. (2000) Growth promotion effects and plasma changes from
feeding high dietary concentrations of zinc and copper to weanling pigs (regional
study). J. Anim. Sci. 78: 1010-1016.

6. McCully, G. A., Hill, G. M., Link, J. E., Weavers, R. L., Carlson, M. S., &
Rozeboom, D. W. (1995) Evaluation of zinc sources for the newly weaned pig. J.
Anim. Sci. 73: 72.

7. Carlson, M. 8., Hoover, S. L., Hill, G. M., Link, J. E., & Turk, J. R.
(1998) Effect of pharmacological zinc on intestinal metallothionein concentration
and morphology in the nursery pig. J. Anim. Sci. 76: 57.

8. O'Dell, B. L., & Savage, J. E. (1960) Effects of phytic acid on zinc
availability. Proc. Soc. Exp. Biol. Med. 103: 304-305.

60

9. Ravindran, V., Bryden, W. L., & Kornegay, E. T. (1995) Phytates:
occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol.
Rev. 6: 125-143.

10. Oberleas, D., Muhrer, M. E., & O'Dell, B. L. (1966) Dietary metal-
complexing agents and zinc availability in rats. J. Nutr. 90: 56-62.

11. Adeola, 0., Lawrence, B. V., Sutton, A. L., & Cline, T. R. (1995)
Phytase-induced changes in mineral utilization in zinc-supplemented diets for
pigs. J. Anim. Sci. 73: 3384-3391.

12. Dunn, M. A., Blalock, T. L., & Cousins, R. J. (1987) Metallothionein.
Proc. Soc. Exp. Biol. Med. 185: 107-119.

13. Tran, C. D., Butler, R. N., Howarth, G. S., Philcox, J. C., Rofe, A. M., &
Coyle, P. (1999) Regional distribution and localization of zinc and metallothionein
in the intestine of rats fed diets differing in zinc content. Scand. J. Gastroenterol.
34: 689-695.

14. Bremner, l., & Beattie, J. H. (1990) Metallothionein and the trace
minerals. Annu. Rev. Nutr. 10: 63-83.

15. Davis, S. R., & Cousins, R. J. (2000) Metallothionein expression in
animals: a physiological perspective on function. J. Nutr. 130: 1085-1088.

16. Richards, M. P., & Cousins, R. J. (1975) Mammalian zinc homeostasis
requirement for RNA and metallothionein synthesis. Biochem. Biophys. Res.
Comm. 64: 1215-1223.

17. Radtke, F., Heuchel, R., Georgiev, 0., Hergersberg, M., Gariglio, M.,
Dembic, Z., & Schaffner, W. (1993) Cloned transcription factor MTF-1 activates
the mouse metallothionein I promoter. EMBO J. 12: 1355-1362.

18. Andrews, G. K. (2001) Cellular zinc sensors: MTF-1 regulation of gene
expression. BioMetals 14: 223-237.

61

19. Hill, G. M., Ku, P. K., Miller, E. R., Ullrey, D. E., Losty, T. A., & O'Dell,
B. L. (1983) A copper deficiency in neonatal pigs induced by a high zinc maternal
diet. J. Nutr. 113: 867-872.

20. NRC (1998) Nutrient Requirements of Swine, 10th Ed. National
Academy Press, Washington, DC.

21. Shaw, D. T., Rozeboom, D. W., Hill, G. M., Booren, A. M., & Link, J. E.
(2002) Impact of vitamin and mineral supplement withdrawal and wheat middling
inclusion on finishing pig growth performance, fecal mineral concentration,
carcass characteristics, and the nutrient content and oxidative stability of pork. J.
Anim. Sci. 80: 2920-2930.

22. Gomori, G. (1942) A modification of the colorimetric phosphorus
determination for use with the photoelectric colorimeter. J. Clin. Lab. Med. 27:
955-960.

23. Scheuhammer, A. M., & Cherian, M. G. (1986) Quantification of
metallothionein by silver saturation. In J.F. Riordan, & B.L. Vallee, Eds. Methods
in Enzymology. Metallobiochemistry. Part B. Metallothionein and related
molecules. Academic Press, Inc, San Diego, Ca.

24. Onosaka, S., & Cherian, M. G. (1982) Comparison of metallothionein
determination by polarographic and cadmium-saturation methods. Toxicol. Appl.
Pharmacol. 63: 270-274.

25. Ernst, C. W., McFarland, D. C., 8 White, M. E. (1996) Expression of
insulin-like growth factor II (IGF-ll), IGF binding protein-2 and myogenin during
differentiation of myogenic satellite cells derived from the turkey. Differentiation
61: 25-33.

26. Brown, T., & Mackey, K. (1997) Analysis of RNA by Northern and Slot
Blot Hybridization. In F.M. Ausubel, ed. Current Protocols in Molecular Biology.
John Wiley & Sons, New York.

62

27. Brown, T. (2000) Dot and Slot Blotting of DNA. In F .M. Ausubel, ed.
Current Protocols in Molecular Biology. John Wiley & Sons, New York.

28. Yao, J., Coussens. P. M., Saama, P., Suchyta, S., & Ernst, C. W.
(2002) Generation of expressed sequence tags from a normalized porcine
skeletal muscle cDNA library. Animal Biotechnol. 13: 211-222.

29. SAS Systems (1999-2000) The SAS System for Windows 98, 8.1 ed.
SAS Institute, Inc., Cary, NC.

30. Poehlman, E. T. (2003) Reduced metabolic rate after caloric
restriction-can we agree on how to normalize the data? J. Clin. Endocrinol.
Metab. 88: 14-15.

31. Thiebaut, R., Jacqmin-Gadda, H., Chene, G., Leport, C., &
Commenges, D. (2002) Bivariate linear mixed models using SAS proc MIXED.
Comput. Methods Programs Biomed. 69: 249-256.

32. Lei, X., Ku, P. K., Miller, E. W., Ullrey, D. E., & Yokoyama, M. (1993)
Supplemental microbial phytase improves bioavailability of dietary zinc to
weanling pigs. J. Nutr. 123: 1117-1123.

33. Poulsen, D. (1989) Zinc oxide for weaned pigs. 40th Annu. Mtg. Eur.
Assoc. Anim. Prod., Dublin, Ireland.

34. Durnam, D. M., & Palmiter, R. D. (1981) Transcriptional regulation of
the mouse metallothionein-l gene by heavy metals. J. Biol. Chem. 256: 5712-
5716.

35. Huber, K. L., & Cousins, R. J. (1988) Maternal zinc deprivation and
interleukin-1 influence metallothionein gene expression and zinc metabolism of
rats. J. Nutr. 118: 1570-1576.

36. Blalock, T. L., Dunn, M. A., & Cousins, R. J. (1988) Metallothionein
gene expression in rats: tissue-specific regulation by dietary copper and zinc. J.
Nutr. 118: 222-228.

63

37. Sandoval, M., Henry, P. R., Luo, X. G., Littell, R. C., Miles, R. D., &
Ammerman, C. B. (1998) Performance and tissue zinc and metallothionein
accumulation in chicks fed a high dietary level of zinc. Poult. Sci. 77: 1354-1363.

38. Wodzinki, R. J., & Ullah, A. H. (1996) Phytase. Adv. Appl. Microbiol.
42: 263-302.

39. Martinez, M. M., Hill, G. M., Link, J. E., Green, J. G., & Driksna, D. D.
(2002) Effects of the addition of phytase and pharmacological concentrations of
zinc oxide on phosphorus excretion in the nursery pig. J. Anim. Sci. 80: 73.

64

Composition of experimental diets for nursery pigs”

TABLE 1

 

 

 

0-7 G 7-14 d
Ingredients gtkg diet (as fed) glkg diet (as fed)
Corn 390.90 531 .40
Soybean meal (44% CP) 202.00 201.80
Spray-dried whey 200.00 200.00
Plasma protein3 60.00 —
Lactose 100.00 —-
Blood cells3 — 20.00
DL-Methionine 1 .10 0.60
L-Lysine - HCL 1.50 1.50
Soybean oil 10.00 10.00
Monocalcium phosphate4 14.10 15.20
Ground limestone 5.50 5.20
Salt 3.50 3.30
Trace mineral pre-mix5 5.00 5.00
Vitamin pre-mix6 6.00 6.00

 

‘The basal diet was formulated to meet or exceed recommended concentrations (20). Analysis of the diets indicated that
dietary Zn was within approximately 10% of the targeted dietary concentration.

2 ZnO (1.25, 2.50 g l kg of final diet) and Natuphos‘” phytase (0.84 g / kg of final diet) were substituted for corn to provide
1,000 and 2,000 mg of Zn and the necessary phytase were appropriate.

3APC, Inc (AP 920 - sprayed dried plasma, AP 3016 - granulated sprayed dried animal blood cells).

‘Monocalcium phosphate = 21% elemental P.

51’ race Mineral pre-mix, (mineral / kg of final diet): CuSO. - 5H20, 10 mg; FeSO. - H20. 100 mg; C;H.(NH2)2 - 2Hl, 0.15
mg; MnSO. - H20, 10 mg; Na23e03, 0.3 mg; ZnO, 100 mg.

Vitamin pre-mix, (vitamin / kg of final diet): retinyl acetate, 1.65 mg; cholecalciferol, 0.165 mg; DL-a-tocopherol, 0.0198
mg; menadione sodium bisulfite, 4.41 mg; niacin, 26.0 mg; riboflavin, 4.41 mg; Ca-D-panthotenate, 17.62 mg; thiamine

mononitrate, 1.10 mg; pyridoxine hydrochloride, 0.991 mg; cyanocobalamin, 33 mg.

65

.2.2 62.0

.522 .900 n.93 mozaaamzv 6.5 one On 13¢ 2:58 Lea 2223 c568 Eat «29.393 25905 Co .oE: F 3822 35 9:35 .6 5:95 05 m_ Sty E5 omegfi 9.0 N

.86 v n. .Letfi 35833 5888 a Song; 39 a s 23.2 .vuc #3235 853:8 $3 9: .0 3:5. .63: new 826: £69: popcohmchomn 9m coacomoa mos? .2;F

 

 

 

Rd 88 88 8:883:28 83.833598 89882983 83882983 828383888 82888.88; 3:955 .03..
28 58 88 388398888 888888388 888889388 $8,838,883 888598388 go.o-39o:8.o 3:255 >655.
as 8.0 28 358888588 88838.38? 88.88.0198: 88.889883 88.839383 83838.88; 32083—3835
adding.“

2.8 $8 88 88.7819 83 837203488 88.788.28.88 85.888.34.38 82.723.85.68 88.720883 3:955 .03..
88 2.0 38 888.884.9388 289889.383 82.8.3388 888839848 288.888.8328 838889388 3205.5 >055.
$8 28 38 88.883.83.83 83883393 808838838 88.488858: 83.888858: 85883383 3:051:83:—
mg

8.... x 5 a... 5 88." 88.. 82 888 88.. S. 9.8:. .osN
3.2 o a... 8 9.5K . .888;

 

 

 

.3883 Eco—coins» 505.? .o 5.3 ass .0 25>». _ao.uo_ooa::u=n .6
82.823 can «a... no 33. use Sect... 683... 5 3.2.32... 9:... cannon ho cozfiucoocoo

N m..m<._.

66

TABLE 3
Residual correlation of the copper, iron, zinc, metallothionein
mRNA abundance and metallothionein protein concentrations in
liver and kidney of pigs fed pharmacological levels of zinc

with or without supplemental phytase

 

Response variable Estimate P value
Lira:
Cu vs. Fe 0.41 0.04
MT mRNA vs. MT protein 0.40 0.04
MT mRNA vs. Zn 0.41 0.03
MT protein vs. Zn 0.63 0.01
m
Cu vs. Fe 0.59 0.01
Zn vs. Cu 0.86 0.01
Zn vs. Fe 0.45 0.02
MT mRNA vs. MT protein 0.66 0.01
MT mRNA vs. Zn 0.48 0.01
MT protein vs. Zn 0.77 0.01

 

67

 

Plasma Zn Concentration
(umol Zn I L)

    

 

 

 

 

 

 

2...“; | 23,“: I 2...,“ WI 2.12.00; [2an

Figure 1A. Concentration of zinc in plasma of pigs fed zinc (150, 1,000
and 2,000 mg Zn/kg) with or without phytase for 14—d post-weaning. Values are
backtransformed means (bar height) with 95% CI (error bars), n=4. Main effects

of zinc and phytase were statistically significant, P < 0.05.

68

 

10.0
9.0
8.0 «
7.0
6.0
5.0 -
4.0 l
3.0 —
2.0
1.0 ,
0.0 -

(mmol Zn I g)

 

Liver Zn Concentration

 

 

an ab

       

33.20-

anp lzm,000 Izmmp I an, IanmP

 

 

 

 

 

 

 

 

Figure 1B. Concentration of zinc in liver of pigs fed zinc (150, 1,000 and
2,000 mg Zn/kg) with or without phytase for 14-d post-weaning. Values are
backtransformed means (bar height) with 95% Cl (error bars), n=4. The zinc x
phytase interaction was significant; means without a common letter differ, P <

0.05.

69

1.0

0.9

0.8 ‘17
0.7

0.6 ,
0.5

522mm

an ‘ anP I 201,000 ,Z'HpooP I 202.0007 lznzmop

 

Kidney Zn Concentration
(mmol Zn I g)

 

 

 

 

 

Figure 1C. Concentration of zinc in kidney of pigs fed zinc (150, 1,000
and 2,000 mg Zn/kg) with or without phytase for 14-d post-weaning. Values are
backtransformed means (bar height) with 95% Cl (error bars), n=4. Main effects

of zinc and phytase were statistically significant, P < 0.05.

70

anP _ Zn.“

 

“IQ—b

5519—.

  

zn1_ooo I zn1'oooP

Figure 2. Dot blot analysis of liver MT mRNA of pigs fed zinc (150,
1,000 and 2,000 mg Zn/kg) with or without phytase for 14-d post-weaning.
Each quadrant contains total RNA from 4 pigs fed the designated diet.

RNA samples from each pig were spotted at 1, 3, and 5 pg.

71

a Relative MT mRNA Abundance I MT Protein

 

 

 

 

 

 

3.5 120
h
30 1'
35 ° 100
3
5 25 ._ 5
fig .. t“ 32
<
252.0 .I 35
n: on
E? 60 ;3
5g :30
._ 0
7'3" .40 a
E 1.0 3
h
9 «20
3 0.5 It.“ [2].; Ill—l
. Jul »0

 

 

 

 

 

 

 

 

 

 

 

 

20150 I meP 201000 Izm'MPI 202.000 Iz'lzmooP

Figure 3. Relative MT mRNA abundance and protein concentration in the
liver of pigs fed zinc (150, 1,000 and 2,000 mg Zn/kg) with or without phytase for
14-d post-weaning. Values are backtransformed means (bar height) with 95% CI
(error bars), n=4. A significant zinc x phytase interaction was detected for
relative hepatic MT mRNA abundance; means without a common letter differ, P <
0.01. Main effects of zinc and phytase on MT protein concentration were

statistically significant, P < 0.05.

72

a Relative MT mRNA AbundanoelMT Protein

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

30 5.0
g h I. ‘I4.5
8 15 2'. ' t 4.0
c .
3 ’5
2.. +3.5 3
< 2-0 o
0‘ : ab "“ 30 I
53 up a q
l- 2‘15 a” —_ , 25°;g
:5 .. =
>‘e J. 2.058
3< A _._ v:I
2~1.0 .. 5
g 1.5 5‘
E " 1.0 3
2 0-5l
X ‘0.5

0.0 L0.0

 

zmmp | anm | anooP

Figure 4. Relative MT mRNA abundance and protein concentration in the
kidney of pigs fed zinc (150, 1,000 and 2,000 mg Zn/kg) with or without phytase
for 14-d post-weaning. Values are backtransformed means (bar height) with
95% Cl (error bars), n=4. A significant zinc x phytase interaction was detected
for relative renal MT mRNA abundance; means without a common letter differ, P
< 0.04. The effect of zinc on MT protein concentration was statistically

significant, P < 0.0003.

73

E] REIatRe MT leilAmAbu‘ndancefl MT Protein

 

 

 

 

 

2.0 14
b .V.

g 18I if “12
1-1.0 ._
512 £3
> .—
ggtzfi -3 a!
'5" . 05
131.0 2°
8; «a 3:
8:00. 05
£3 9. a ;'u
:4: SE
3‘0.” _ __ x '3
c r‘ 2
_ M :L I i

00 ‘ 0

 

 

 

 

 

 

 

 

 

 

 

zmmP I anm I anOP

Figure 5. Relative MT mRNA abundance and protein concentration in the
intestinal mucosa of pigs fed zinc (1 ,000 mg Zn/kg with phytase and 2,000 mg
ankg with or without phytase) for 14-d post-weaning. Data for pigs fed 150 mg
Zn/kg with or without phytase and 1,000 mg ankg without phytase were below
the detectable limits of the assays. Values are backtransformed means (bar
height) with 95% Cl (error bars), n=4. A zinc x phytase interaction for relative
intestinal mucosa MT mRNA abundance and MT protein concentration was

detected; means without a common letter differ, P < 0.05.

74

CHAPTER THREE
Identification of ZnT-1 in Porcine Liver of Newly Weaned Pigs Fed

Pharmacological Zinc and Phytase Supplemented Diets1

Michelle M. Martinez', Gretchen M. Hill', Jane E. Link', Catherine W. Emst', Bo L.
LénnerdaIT, Shannon L. Kelleher’, Emily E. Helman' and Matthew E. Doumit"t

'Department of Animal Science, Michigan State University, East Lansing, MI; ’Department of
Nutrition, University of California, Davis, CA; ‘Department of Food Science and Human Nutrition,

Michigan State University, East Lansing, MI.

Running Title: ZnT-1 IN YOUNG PIGS FED HIGH ZINC

1 Supported in part by Six State Consortium on Animal Waste Management of
Environmental Protection Agency, an Animal Health Formula Funds Grant from
the Michigan Agricultural Experiment Station, and The Graduate School -
Competitive Doctoral Enrichment Fellowship, Michigan State University, East
Lansing, MI.

2Abbreviations used: BCA, bicinchonic acid; EST, expressed sequence tag;
FTU, phytase unit; HES, hepes / sucrose / EDTA; lgG, immunoglobulin; MT,
metallothionein; MRE, metal response element; MTF, metal transcription factor;
TBS-T, tris buffered saline with tween-20; Zn, zinc; ZnO, zinc oxide; ZnT, zinc
transporter; 2150, 150 mg Zn/kg; Zn150P, 150 mg Zn/kg with phytase; Zn1,ooo,
1,000 mg Zn/kg; Zn1,oooP, 1,000 mg Zn/kg with phytase; 202,000. 2,000 mg ankg;

202.0005”, 2,000 mg Zn/kg with phytase.

75

ABSTRACT

Pharmacological zinc (Zn) is routinely fed to newly weaned pigs to
improve growth. We recently demonstrated that pharmacological Zn and phytase
supplementation increases metallothionein (MT) mRNA abundance and protein
in the liver, kidney and intestinal mucosa of newly weaned pigs. Since Zn
transporter-1 (ZnT-1) appears to act in concert with MT to effectively manage Zn,
we hypothesized that ZnT-1 protein is involved in hepatic Zn homeostasis of pigs
fed pharmacological Zn diets. Adequate (150 mg Zn/kg) or pharmacological
(1000 or 2000 mg Zn/kg) Zn diets with or without phytase (0 or 500 phytase units
lkg) were fed to pigs for 14 d post-weaning. The ZnT-1 was identified as a ~50
kDa protein found exclusively in pig liver crude membrane fractions. Pigs fed 150
and 1000 mg Zn/kg had comparable ZnT-1 protein expression; pigs fed 2000 mg
ankg showed reduced expression of this protein. Phytase did not affect ZnT-1
protein. Our results suggests that hepatic cells of pigs fed pharmacological Zn
(2000 mg Zn/kg) for 14 d, preferentially increase intracellular MT rather than ZnT-
1 to manage the excess Zn.

KEY WORDS: - liver - pharmacological zinc - phytase - pig 0 zinc transporter-1

76

INTRODUCTION

The mechanism involved in Zn transport within and between tissues has
not been elucidated (1). To achieve Zn homeostasis, regulation occurs through
intestinal uptake, endogenous secretions, fecal excretion, renal resorption and
excretion, and intracellular storage (2). Under variable Zn concentrations, cells
adjust: i) Zn uptake and secretion, ii) intracellular protein binding, iii) re-
distribution and/or iv) sequestration within vesicles or organelles for homeostatic
control (3).

Numerous studies demonstrate that feeding 2000 to 3000 mg ankg as
zinc oxide (ZnO) to traditionally (18 — 21 d) or early-weaned (10 — 14 d) pigs,
results in enhanced growth performance (4,5), improved fecal consistency (6,7)
and gut morphology (8). However, the mechanism of action has not been
elucidated. Recently, studies from our laboratory reported that feeding 1000 mg
Zn/kg plus 500 phytase units (FTU)/kg of phytase or 2000 mg Zn/kg with or
without phytase for 14 d post-weaning increased relative metallothionein (MT)
mRNA abundance and protein concentrations in liver, kidney and intestinal
mucosa. Furthermore, a positive correlation between Zn concentration and MT
in liver and kidney suggests that MT plays a homeostatic role in pigs fed high Zn
diets (9).

Exposing cells to high concentrations of Zn activates many protective
mechanisms (10), such as downregulation of Zn uptake transporters (11),
sequestration of Zn into intracellular compartments (12), induction of binding

proteins such as MT (13) and increased Zn efflux (14). Palmiter (10) suggested

77

that as excess Zn enters the cell, both MT and the Zn efflux transporter, ZnT-1
compete with each other for handling Zn disposal.

Transcription of the MT and ZnT-1 genes is under the control of the metal
transcription factor —1 (MTF-1). Once Zn is inside the cell, it binds to MTF-1,
which acts as a Zn sensor (15). Short sequences known as metal response
elements (MREs) found in the promoter region of genes serve as binding sites
for transcription factors such as MTF-1 increasing transcription rate of MT (16)
and ZnT-1 (17) upon metal activation. The MT gene contains six MREs in the 5’
regulatory region upstream of the transcription initiation site (18), while the ZnT-1
promoter contains two MRE consensus sequences and MTF-1 binds to both
sites, resulting in transcriptional activation (17). These reports led us to
hypothesize that ZnT-1 plays a key role with MT in hepatic Zn homeostasis of
pigs fed pharmacological Zn diets. Thus, the objectives of this experiment were
to identify ZnT-1 in the liver, and to determine the effects of pharmacological Zn

and phytase supplementation on the expression of ZnT-1.

78

MATERIALS AND METHODS

Animals and diets. Animal description, allotment and diet composition
have been previously published (9). The Zn source, ZnO, contained 72% Zn
(Prince Agri Products), and phytase was added at 500 phytase units (FTU)/kg
diet (Natuphos®, BASF Corp., Mount Olive, NJ). One FTU is the amount of
enzyme that releases 1 pmol inorganic phosphorous from sodium phytate per
minute at pH 5.5 and 37°C. The basal diet was formulated to meet or exceed
NRC recommendations (19). The dietary treatments were: 1) a basal diet
containing 150 mg an kg (Zn150), 2) Zn15o plus 500 FTU/kg (Zn150P), 3) basal
plus pharmacological Zn diet containing 1000 mg Zn/kg (Zn1ooo), 4) Zn1ooo plus
500 FT Ulkg (Zn1oooP), 5) basal plus pharmacological Zn diet containing 2000 mg
Zn/kg (anooo) or 6) anooo plus 500 FT U/kg (anoooP). Pigs consumed feed and
water ad libitum. This project was approved by the Michigan State University All
University Committee on Animal Use and Care (12/99-159-00).

Sample collection. On d-14, one pig from each pen (n = 24) was
randomly selected and killed by an injection (0.22 mL/kg body weight) of sodium
pentobarbital (392 g/L). Liver tissue samples were excised and flash frozen in
liquid nitrogen for protein isolation. Additionally, liver tissue was obtained from a
21 d lactating Sprague-Dawley rat that was fed a standard rat chow for use in
optimizing the western blot analysis, and to compare band patterns.

Total membrane protein preparation. Approximately 500 mg of liver
tissue was homogenized in 10 mL of ice cold HES buffer [20 mM HEPES, pH 7 /

250 mM sucrose / 1 mM EDTA / protease inhibitor mixture containing 4-(2-

79

aminoethyl) benzenesulfonyl fluoride, trans-epoxysuccinyl-L-leucyl-amido(4-
guanidino) butane, bestatin, leupeptin, aprotonin and sodium EDTA (Sigma-
Aldrich, St. Louis, MO)] using a tissue homogenizer (Janke & Kunkel Ultramax
T25, Tekmar Company, Staufeni, Berlin, Germany) for 20 s. The homogenate
was centrifuged at 21000 x g for 30 min at 4°C. The supernatant was removed
and the pellet was resuspended in 0.5 mL HES buffer. An aliquot (0.5 mL) of
both fractions was mixed in electrophoresis treatment buffer (125 mM Tris, 4%
SDS, 20% glycerol) and heated at 50°C for 20 min. Protein concentrations were
immediately determined in both soluble and insoluble protein fractions using the
bicinchonic acid (BCA) protein assay kit (Pierce, Rockford, IL). Beta-
mercaptoethanol was added at 5% (v/v) and bromophenol blue was added at 4%
(v/v) to the samples prior to storage at -20°C. Also, 50 and 100 pg of protein
from all samples were loaded on 10% SDS/PAGE gels stained with CoomassieTM
brilliant blue R250 (Amersham Biosciences, Buckinghamshire, UK).

Production of ZnT-1 antibody. A polyclonal rabbit anti-rat ZnT-1
antibody (GTRPQVHSGKE, Life Technologies, Carlsbad, CA) was used for this
study (20). The only porcine sequence corresponding to ZnT-1 is a 424 bp
expressed sequence tag (EST) consensus sequence (T 0155407) in The Institute
for Genomic Research (TlGR) database. In the identified epitope region within
the translated porcine sequence, 3 out of the 11 amino acids are different in the
pig sequence (GTRPQAQSGKD).

Western blot analysis. Equal amounts of membrane and soluble protein

(50 pg) from one randomly selected pig from each treatment, plus the rat sample

80

were resolved in a 10% SDS/PAGE gel and transferred onto an lmmobilon-PSQ
(Millipore, Billerica, MA) membrane for 2 hr at 100 V. Blots (n = 8) were blocked
for 1 hr at room temperature with 100 g/L nonfat milk in Tris-buffered saline
(20mM Tris, 137mM NaCl, 2mM KCI) I 0.1% Tween-20 (TBS-T). Membrane
strips (0.5 cm) were stained with amido black and destained to visualize protein
presence. Membranes were incubated with the rabbit anti-rat ZnT-1 polyclonal
antibody (1 :1000) for 8 hr at 4°C, and then washed three times at room
temperature with blocking solution.

Membranes were incubated at room temperature with goat-anti-rabbit
immunoglobulin (IgG) conjugated to alkaline phosphatase (1:500, Sigma-Aldrich,
St. Louis, M0) for 1 hr, and washed three times with blocking solution, three
times with TBS-T and one additional time with TBS. Rabbit pre-immune serum
(121000) and goat anti-rabbit lgG (1:500) served as negative controls.
lmmunoreactive bands were visualized by treatment with an alkaline
phosphatase conjugate substrate (Bio-Rad, Hercules, CA) for 2 min.

Blots were re-blocked with 50 g/L nonfat milk in TBS-T for 1 hr, then
incubated with a mouse B-actin (1:10000) monoclonal antibody (Abcam Ltd,
Cambridge, UK) for 45 min at room temperature. Membranes were washed
three times with blocking solution, and subsequently incubated with goat-anti-
mouse lgG conjugated to alkaline phosphatase (1:20000, Sigma-Aldrich, St.
Louis, M0) for 45 min, washed three times with blocking solution, three times

with TBS-T and one additional time with TBS. lmmunoreactive bands were

81

visualized by treatment with an alkaline phosphatase conjugate substrate for 8
min.

Statistical analysis. The data were analyzed using analysis of variance
procedures based on the MIXED procedure of SAS (21). The model included the
main effects (zinc and phytase) and their interaction. Blot by treatment (zinc x
phytase) interaction was additionally modeled with pig as random effect to define
the experimental units for this analysis, with Satterthwaite's approximation used
to determine the error degrees of freedom for test (22). Furthermore, B-actin
values were modeled as a covariate for a regression-based normalization of ZnT-
1 protein expression in accordance with recent recommendations by Poehlman
(23). Due to lack of normal distribution, data were log transformed. Thus, back-
transformed means and their respective 95% confidence intervals (lower and
upper limits) are provided as point and interval estimates, respectively. Scheffé’s
test was used to determine the presence of linearity of response to treatments

(24).

82

RESULTS

Identification of ZnT-1 in porcine liver. Utilizing western blot analysis,
ZnT-1 was identified in the pellet fraction and was absent in the supernatant of
liver homogenates (Figure 1). Specificity was confirmed with rabbit preimmune
serum, a total lgG fraction, or the ZnT-1 antibody (Figure 2).

Effect of diet on zinc transporter protein concentrations. A ~ 50 kDa
lmmunoreactive band was identified as ZnT—1 protein in the rat and pig samples
(Figure 3A). An additional band (~ 38 kDa) was identified in all samples, but
was more intense in rat liver (Figure 3A) in agreement with results reported by
McMahon and Cousins (14). Both sizes are in agreement with the ZnT-1
predicted sizes and the sizes previously observed in rat liver using this antibody
(S. Kelleher, unpublished data). Beta-actin blots exhibited a ~ 42 kDa band,
which was present in all liver samples (Figure 3B), and is in agreement with its
published size (25).

A significant Zn effect (P < 0.02) was detected on the expression of ZnT-1
protein (Figure 3C), which was higher in pigs fed Zn1ooo compared with pigs fed
202000. Liver of pigs fed Zn150 had comparable ZnT-1 expression to that of pigs
fed Zn1ooo. Neither a Zn x phytase interaction nor a main phytase effect were

observed for relative ZnT-1 protein abundance.

83

DISCUSSION

In this study, we present evidence of the presence and regulation of ZnT-1
in the liver of pigs fed adequate or pharmacological Zn concentrations without an
effect by supplemental phytase. Using a highly specific affinity-purified rat
antibody (20), we identified for the first time the presence of ZnT-1 in liver
samples of newly weaned pigs, suggesting the existence of common epitopes for
this protein. Despite the fact that the epitope sequences are only 72%
homologous, ZnT-1 was successfully identified in all hepatic membrane fraction
samples. Zinc transporter - 1 protein had been previously identified in intestinal
and hepatic membrane fractions of rats fed 30 and 180 mg ankg (14), as well as
in the serosal membrane of cells in the mammary gland of lactating rats fed 10
and 25 mg Zn/kg (20).

Young pigs adapt and benefit from pharmacological dietary Zn, which
suggests that regulation of Zn in tissues, such as the liver, is effective in
managing excess Zn. Previously, we documented that hepatic Zn concentration
of pigs fed phytase and pharmacological Zn diets increased linearly, and these
values were correlated to the increase in relative MT mRNA abundance and
protein (9). The role of ZnT-1, a member of the ZnT transporters, has not been
investigated in the pig when Zn is fed at 10 to 20 times its requirement. The
function of the transporters in the ZnT family is to reduce cytoplasmic Zn through
efflux or secretion (26). Therefore, we propose that ZnT-1 is part of the
mechanism involved in regulating Zn homeostasis in newly weaned pigs fed

pharmacological Zn diets.

84

Feeding a marginal Zn diet (10 mg Zn/kg) to lactating rats decreased
mammary gland ZnT-1 and MT proteins while plasma, milk or mammary gland
Zn concentrations remained unchanged (20) suggesting regulation of Zn upon
exposure to a low Zn diet. An oral Zn dose (35 mg Zn/kg BW) given to rats has
been shown to increase liver ZnT-1 protein and serum Zn 6 h after dosing
compared with rats receiving saline (14). These results suggest that rats
receiving 2.5 to 3 times adequate dietary Zn (0.17 - 0.22 mmol/kg BW) (27),
have increased hepatic Zn efflux via ZnT-1 transporter. In our study, ZnT-1
protein expression was greater in pigs fed Zn1ooo than in those fed 202000. but
hepatic MT protein and Zn were greater in pigs fed anooo than those fed anoo
These data suggest that Zn efflux through this transporter was reduced. The
decrease in ZnT-1 combined with the increase in hepatic Zn and MT
concentrations suggests an altered hepatic Zn efflux mechanism that has not
been previously reported.

Feeding 500 or 1000 mg ankg diet is not as effective as 2000 or 3000 mg
Zn/kg for promoting growth (5,28) in the newly weaned pig. Perhaps reduced
ZnT-1 expression in pigs fed anooo occurs as a response to an overwhelmed
homeostatic system or to the adequate sequestering methods of MT. This
intracellular Zn increase may affect other signaling pathways (29), which may
also affect the expression of the transporter. Recently, Palmiter (10) suggested
that when cells are suddenly exposed to increased Zn concentrations, both MT
and ZnT-1 work in concert, and as intracellular Zn increases, Zn efflux by pre-

existing ZnT-1 predominates. Perhaps when pigs are exposed to high Zn

85

concentrations for 14 d, cells preferentially increase intracellular MT rather than
ZnT-1 to manage the excess Zn.

Preferential MT gene amplification compared to ZnT-1 was observed in
baby hamster kidney cells (BHK) exposed to high concentrations of Zn (~600
0M). Palmiter proposed that this could be due to: 1) the close linkage of the MT-
1 and MT-2 genes (7 kb apart) in the hamster cells and consequent amplification
of both genes upon high Zn exposure, and/or 2) an energetic advantage to
sequester Zn rather than effluxing it (10). Also, there are more MRE sequences
present in the promoter region of MT (16), compared to ZnT-1 (17), which may
affect the differential regulation of these proteins at the gene level, upon
exposure to Zn. Although we demonstrated that ZnT-1 plays a role in regulating
Zn in the liver of pigs fed pharmacological Zn, there are still unanswered

quesfions.

86

ACKNOWLEDGEMENTS
The authors thank Dr. Jeanne Burton at Michigan State University, for
providing the mouse B-actin antibody; BASF Corporation, Prince Agri Products,
International Ingredients Corporation, and American Protein Corporation for

supplying ingredients used in this study; and Guillermo Ortiz-Colcn, and Chuck

Allison for technical assistance.

87

LITERATURE CITED

1. Cousins, R. J. (1985) Absorption, transport and hepatic metabolism of
copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol.
Rev. 65: 238-309.

2. Krebs, N. F. (2000) Overview of zinc absorption and excretion in the
human gastrointestinal tract. J. Nutr. 130: 13748-13778.

3. Reyes, J. G. (1996) Zinc transport in mammalian cells. Am J Physiol
270: 0401410.

4. Smith, J. W., Tokach, M. D., Goodband, R. D., Nelssen, J. L. & Richert,
B. T. (1997) Effects of the interrelationship between zinc oxide and copper
sulfate on growth performance of early-weaned pigs. J. Anim. Sci. 75: 1861-
1866.

5. Hill, G. M., Mahan, D. 0., Carter, S. D., Cromwell, G. L., Ewan, R. C.,
Harrold, R. L., Lewis, A. J., Miller, P. 8., Shurson, G. C. & Veum, T. L. (2001)
Effects of pharmacological concentrations of zinc oxide with or without the

inclusion of an antibacterial agent on nursery pig performance. J. Anim. Sci. 79:
934-941.

6. Stanger, B. R., Hill, G. M., Link, J. E., Turk, J. R., Carlson, M. S. &
Rozeboom, D. W. ( 1998) Effects of high Zn on TGE-challenged, early weaned
pigs. J. Anim. Sci. 76: 57 (abs).

7. Hill, G. M., Cromwell, G. L., Crenshaw, T. D., Dove, C. R., Ewan, R. C.,
Knabe, D. A., Lewis, A. J., Libal, G. W., Mahan, D. C., Shurson, G. 0., Southern,
L. L. & Venum, T. L. (2000) Growth promotion effects and plasma changes from
feeding high dietary concentrations of zinc and copper to weanling pigs (regional
study). J. Anim. Sci. 78: 1010-1016.

8. Carlson, M. 8., Hoover, S. L., Hill, G. M., Link, J. E. & Turk, J. R. (1998)
Effect of pharmacological zinc on intestinal metallothionein concentration and
morphology in the nursery pig. J. Anim. Sci. 76: 57 (abs).

88

9. Martinez, M. M., Hill, G. M., Link, J. E., Raney, N. E., Tempelman, R. J.
& Ernst, C. W. (2004) Pharmacological zinc and phytase supplementation
enhance metallothionein mRNA abundance and protein concentration in newly
weaned pigs. J. Nutr. 134: 538-544.

10. Palmiter, R. D. (2004) Protection against zinc toxicity by
metallothionein and zinc transporter 1. Proc. Natl. Acad. Sci. U S A 101: 4918-
4923.

11. Gaither, L. A. & Eide, D. J. (2001) Eukaryotic zinc transporters and
their regulation. Biometals 14: 251-270.

12. Cousins, R. J. & McMahon, R. .r. (2000) Integrative aspects of zinc
transporters. J. Nutr. 130: 13848-13878.

13. Hamer, D. H. (1986) Metallothionein. Annu. Rev. Biochem. 55: 913-
951.

14. McMahon, R. J. & Cousins, R. J. (1998) Regulation of the zinc
transporter ZnT-1 by dietary zinc. Proc. Natl. Acad. Sci. USA 95: 4841-4846.

15. Andrews, G. K. (2000) Regulation of metallothionein gene expression
by oxidative stress and metal ions. Biochem. Pharmacol. 59: 95-104.

16. Stuart, G. W., Searle, P. F., Cen, H. Y., Brinster, R. L. & Palmiter, R.
D. (1984) A 12-base-pair DNA motif that is repeated several times in
metallothionein gene promoters confers metal regulation to a heterologous gene.
Proc. Natl. Acad. Sci. USA 81: 7318.

17. Langmade, S. J., Ravindra, R., Daniels, P. J. & Andrews, G. K. (2000)
The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1
gene. J. Biol. Chem. 275: 34803-34809.

18. Thiele, D. J. (1992) Metal-regulated transcription in eukaryotes.
Nucleic Acids Res. 20: 1183-1191.

89

19. NRC (1998) Nutrient Requirements of Swine, 10th Ed. Washington,
DC.

20. Kelleher, S. L. & Lcnnerdal, B. (2002) Zinc transporters in the rat
mammary gland respond to marginal zinc and vitamin A intakes during lactation.
J. Nutr. 132: 3280-3285.

21. SAS Systems (1999-2000) The SAS System for Windows 98, 8.1 Ed.
SAS Institute, Inc., Cary, NC

22. Satterthwaite, F. E. (1946) An approximate distribution of estimates of
variance components. Biom. Bull. 2: 110-114.

23. Poehlman, E. T. (2003) Reduced metabolic rate after caloric
restriction-can we agree on how to normalize the data? J. Clin. Endocrinol.
Metab. 88: 14-15.

24. Scheffé, H. (1956) A method for judging all contrasts in an analysis of
variance. Biometrika 40: 87-104.

25. Huang, P., Steplock, D., Weinman, E. J., Hall, R. A., Ding, Z., Li, J.,
Wang, Y. & Liu-Chen, L.-Y. (2004) {kappa} Opioid receptor interacts with
Na+/H+-exchanger regulatory factor-1IEzrin-Radixin-Moesin-binding
phosphoprotein-50 (NHERF-1/EBP50) to stimulate Na+IH+ exchange
independent of Gi/Go proteins. J. Biol. Chem. 279: 25002-25009.

26. Palmiter, R. D. & Huang, L. (2004) Efflux and compartmentalization of
zinc by members of the SLC30 family of solute carriers. Pflugers Arch. 447: 744-
751.

27. Davis, 8. R., McMahon, R. J. & Cousins, R. J. (1998) Metallothionein
knockout and transgenic mice exhibit altered intestinal processing of zinc with
uniform zinc-dependent zinc transporter-1 expression. J. Nutr. 128: 825-831.

28. Case, C. L. & Carlson, M. S. (2002) Effect of feeding organic and
inorganic sources of additional zinc on growth performance and zinc balance in
nursery pigs. J. Anim. Sci. 80: 1917-1924.

90

29. Watjen, W., Benters, J., Haase, H., Schwede, F., Jastorff, B. &
Beyersmann, D. (2001) Zn2+ and Cd2+ increase the cyclic GMP level in PC12
cells by inhibition of the cyclic nucleotide phosphodiesterase. Toxicology 157:
167-175.

91

26:0 :0 3 c2650 0_ _.-._.cN Co c0308.. £0. 05 :o 950% 0.0 2235: 0.9.5:. 2902, 5.80.22
40:3 :0. .dm ”#:3050030 :9. .zwm 30:00 90 NE ucEmE0qzm 9a .29: .m0cmBE0E Co>nc 028:: 0523:3200
2 00:05:02 .80 0.0m mO<n=wow $2. 5 002000. 0.03 20>: am. can ma E0: 81 omv 0:280: 5083 0:05E0E
Lo 0_n_.__ow cm. new 09a :0 moumc0moEoc .02. c_ 90:03 5:00: 0:05E0E 02:0 05 5 0052.02 new 9:32.039

c260: 5065 03200 05 5 E030 002, 0:80:05 0:... .mcozom: £085 95. c_ .3ch :0 530900 ._. 0.59".

c.mv

«.00

«III! ._ . QKO

.la -- I . 0:.

am zmm on 20¢ 5:5: «9.

92

50:0 :0 >0 :50:0 0_ _.-._.:N :0 55000.. .50. 05 :0 :50:0 0.0

223:6 0.900.: 2905 0500.05. 05050000 0050:0005. 05.050 :0 5:5 005.0003 :05 0:0 0003:00 0050:0005.
05050500555 .N 0 5:5 00:00:05 0.05 0.2m .5520 cost: $00555 :....:N 5:5 00500305 0_QE00 50. .Nm H:0_S:0
coo: .5200 0::EE_0.0 :30. 5:5 00:05.05 0.0E00 50. .E ”:0_S:0 com; .30500005 >005E0 .: 05 :0500: 00.
.052 .00.. ”55:0 coat: .7th 5:5 00505.05 0.0E00 90 .Nn. €05.50 coo: .EE00 0::EE_0.0 :30. 5:5 00:00:05
0.:E00 90 ._n_ .00:0.0E0E Co>dv 00:03: 0:00__>5>>_0a 0. 00:80:05 0:0 000 $00.0:wa «so: 5 002000. 0.05

0.02. :0. 0:0 90 E0: 81 08 05:00.: 50.0.: 0:0.nE0_2 .>005E0 _.-._.:N “0.45:0 :30. :0 50500aw .N 0.09".

adv
we. omrllv 1':

I
1'91!!! 1 .E ..
'III‘ I

l

ENG
o —. —.

lati- .3...

N: E 00. N: E 5.5: one.

93

059.0 .3 :50:0 0. :09. on 0:0 mm... 7th .0 55000.. 50. 05 :0 :50:0 0.0 2235: 0.9.0:. 5.90.5 .0.300.0s.
550:0 0. TEN «0.5:0 :30. 5.5 00503305 8.: 050500050. < .0.0>.0:0 “0.: E0005 .0. 0003 0.0.5 .0E.:0 :000
Eo... 63 cm. 5050.: 0:0.0E0E .0 55080 .033m. .50. :300 :0 .0 .0>: 5 0:0 .0. 0000.5. .0E0E0aa30 “305:5 .0 5:5

EVEN m... ooom .0 83 .85 :N E0520 00. 03.3 .0 00:0:030E0: .0>: 5 508.0 7th :0 8.000.qu .<n 0.39".

00.. 00:1
3...... 3.00

Gnu—ontlv I‘ll II, II I I
1. N60
‘55

u r r
“a“ cog-LN occur—N looSCN LooouCN omFCN heave—N 5;: “Dv—

94

0.5050 5: 550:0 0.0 A09. «01. 550-: 0:0 509. om 0:0 0sz 7th h.0 0:0..000: .00. 0:. :0 550:0

0.0 223.2. 0.0505 50.05 .0_:00.0_2 550:0 0. 00050.2 0:0 0.5.90.2 5 005.0000 00 550-: 00:05.50 5::0. 0:0
7...:N :0.-.50 5::0. :55 00:05:05 5.: 050500050. < 0.05.0:0 5.: 50:00.5 .0. 000: 0.0.5 .0550 :000 50.0 a: om.
505.0 0:05.505 50 050050 .035 .50. 5:00 :0 0.0 .02. 5 0:0 An: 0005:: .050505030 50:55 .0 :55 EVEN 9: ooow

.0 89 .005 :N 5.80.0 005 0a.: .0 00.0:00050: .02. 5 0505.0 550-: 0:0 7th 00 :0.000.0xm. .m« 0.50.".

09. «~11 , -. i. -- ..le . E. . -. .0 .
an: NQIIV . .. . x 9m?
00.. 00al|v \lll - 1.!!! .1};
«.00
.. i 0.50
m. ; 0:.

~01 8:ch SaucN moooEN mosucN SEN QSEN 23.2 09.

95

0.30 -

0.25

0.20

0.15 -

0.10 ,

Liver Relative ZnT-1 Protein Abundance
(Arbitrary Units)

 

0.05 —

 

 

 

 

 

0.00 ~ ‘ - . 5
20150 Zn150P zn1ooo anoP Zl‘lzooo Zflzooop

Figure 30. Relative ZnT-1 protein abundance in liver homogenates of
pigs fed dietary Zn (150, 1000 or 2000 mg Zn/kg) with or without supplemental
phytase. Equal amounts of membrane protein (50 pg) from each animal were
used for western blot analysis. Densitometric analysis of relative hepatic ZnT-1
protein abundance was performed. Values are backtransformed means with

95% Cl, n = 4. A significant zinc effect was detected, P < 0.02.

96

CHAPTER FOUR
Dietary Pharmacological or Excess Zinc and Phytase Effects on Tissue
Mineral Concentrations, Metallothionein, and Apparent Mineral Retention in
the Newly Weaned Pig
M. M. Martinez, J.E. Link and GM. Hill
Department of Animal Science, Michigan State University,

2209 Anthony Hall, East Lansing, MI 48824

Running title: High Zinc & Phytase on Tissue Minerals & MT in Pigs

97

ABSTRACT

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and
dietary phytase improves phosphorus and Zn availability. Metallothionein (MT)
increases in the duodenum, kidney and liver of pigs fed 1,000 mg Zn/kg with
phytase or 2,000 mg Zn/kg with or without phytase when fed for 14 d post-
weaning. The goal of this study was to determine the effects of feeding
pharmacological Zn and phytase on tissue minerals, MT, mineral excretion and
apparent retention. Twenty-four newly-weaned pigs (20 d, 7.2 kg) were
individually fed twice daily, a basal diet supplemented with 0, 1,000 or 4,000 mg
Zn/kg as Zn oxide, without or with phytase (500 FTU/kg) for 14 d, followed by a
basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4,000 mg Zn/kg
without phytase had higher (p = 0.01) plasma, hepatic, renal Zn, renal Cu, and
hepatic, renal, and jejunal MT than pigs fed the basal diet or 1,000 mg Zn/kg.
Duodenal MT was higher (p = 0.0001) in pigs fed 1,000 and 4,000 mg Zn/kg than
in pigs fed the unsupplemented diets. In pigs fed 1,000 and 4,000 mg Zn/kg, Zn
loading occurred during the first 11 d of supplementation; by d 14 excess Zn was
being excreted in the feces.
Index Entries: apparent retention, metallothionein, mineral excretion,

pharmacological zinc, phytase, nursery pig

98

INTRODUCTION

The US. swine industry adds pharmacological (1,500 to 3,000 mg ankg)
zinc (Zn) as zinc oxide (ZnO) to post-weaning diets to enhance growth (1, 2) and
improve fecal consistency (3). The National Research Council estimates that a 5
to 10 kg pig requires 100 mg Zn/kg (4). Supplementing Zn at 30 times (3,000 mg
Zn/kg) its requirement has been shown by our laboratory to increase villous
height and reduce crypt depth, potentially increasing intestinal absorptive
capacity (5). The effects of even higher dietary Zn concentrations in the newly
weaned pig on tissue accretion and Zn excretion have not been investigated.

Phytate phosphorus (P) found in cereal grains is known to complex with
cations reducing their bioavailability (6—8). Because non-ruminants produce
negligible amounts of phytase (9), exogenous supplementation of phytase is an
alternative for enhancing mineral availability (10), and retention (11, 12).

Metallothionein (MT) is a cysteine rich protein that is involved in Zn and
copper (Cu) homeostasis, transfer and storage (13, 14), and is considered an
intracellular Zn marker when Zn is fed in excess (15). Our laboratory has shown
that feeding pharmacological Zn (1 ,000 and 2,000 mg ankg) with 500 phytase
units (FTU)/kg of phytase (Natuphos®, BASF Corp, Mount Olive, NJ) or 2,000
mg Zn/kg without phytase for 14 d post-weaning increases kidney Cu, liver Zn,
and MT mRNA and protein concentrations in the duodenum, kidney and liver
(16). Liver, kidneys and intestinal mucosa play a key role in Zn homeostasis
(17). No studies have focused on the effects of adequate and pharmacological

Zn on MT concentration in jejunum in the young pig. Our laboratory fed 3,000

99

mg Zn/kg for 7, 14, or 28 d post-weaning and found elevated renal and hepatic
Zn and MT protein concentrations (2). Tran et al. (15) fed 10, 100, 400 or 1,000
mg Zn/kg to rats for 7 d and demonstrated that Zn concentrations increased
constantly from the stomach to the colon, with higher concentrations in rats fed
400 and 1,000 mg ankg than in rats fed 10 or 100 mg Zn/kg. In addition, MT
concentrations were higher in the duodenum of rats fed 400 and 1,000 mg Zn/kg
compared with MT concentrations in the jejunum, ileum, caecum, and colon, and
compared with rats fed the remaining treatments. However, Reeves et al. (18)
reported that feeding 350 mg Zn/kg to rats initially increased intestinal mucosa,
liver and kidney Zn and MT, until d 14 of supplementation and declined to
concentrations similar to those of rats fed 50 mg ankg by the end of the 42 d
experiment. These results suggest that an adaptive mechanism exists which
protects tissues when pharmacological Zn concentrations are fed. This adaptive
mechanism may include MT and increased mineral excretion.

The objectives of this study were to determine the effects of feeding
adequate (100 mg ankg), pharmacological (1 ,000 mg ankg) or excess (4,000
mg Zn/kg) Zn without or with phytase (500 FTU/kg) on tissue minerals [calcium
(Ca), P, Cu, iron (Fe) and Zn], MT concentrations, mineral excretion and

apparent retention.

100

MATERIALS AND METHODS
Animals, Diets and Measures

At weaning, 24 [(Landrace x Yorkshire) x Duroc] gilts and barrows (20 d of
age, 7.2 kg) were blocked by weight, sex and litter and individually penned in
stainless steel pens (1.40 m x 0.56 m) equipped with a stainless steel self-feeder
and nipple waterer in an environmentally controlled room (ZS-30°C) for a 21 d
study. This study was approved by the Michigan State University All University
Committee on Animal Use and Care (AUCAUC, 12/99-159-00).

The six dietary treatments were fed in two phases (d 1 to 7 and 8 to 14) in
a complete randomized block design with a 3 x 2 factorial arrangement of
treatments, followed by a common diet from d 15 to 21(T able 1). The basal diet,
fed in a meal form, was formulated to meet or exceed the recommendations of
the National Research Council (4) and contained 100 mg Zn/kg provided as ZnO.
The dietary treatments were: 1) adequate Zn diet with 100 mg Zn/kg (Zn1oo), 2)
Zn1oo plus 500 FTU/kg (Zn1ooP), 3) pharmacological Zn diet with 1,000 mglkg
added Zn (Zn1,ooo), 4) anoo plus 500 FTU/kg (Zn1,oooP), 5) excess Zn diet with
4,000 mglkg added Zn (Zn4,ooo), or 6) Zn4,ooo plus 500 FTU/kg (Zn4,oooP). The
dietary phytase used in this experiment was Natuphos® (BASF Corp, Mount
Olive, NJ). One phytase unit (FTU) is defined as the amount of enzyme that
releases 1 pmol inorganic phosphorus from sodium phytate per minute at pH 5.5

and 37°C. The Zn source was ZnO which contained 72% Zn (Prince Agri

101

Products, Quincy, IL). Pigs were fed to appetite twice daily with water available
ad libitum.

During the study, feed waste, total feces, and urine were collected twice
daily. Fecal samples were stored (-20°C) in whirl pack bags (NASCO, Modesto,
CA). Urine was filtered through glass wool and volume was recorded. An aliquot
was frozen (—20°C) in 50 mL screw cap polypropylene tubes (Corning Inc.,
Corning, NY). Body weight of pigs was measured weekly and ADG, ADFl and
G:F were calculated.

Tissue Mineral and Metallothionein Analysis

On d-21, blood samples were collected in heparinized evacuated tubes
(BD Vacutainer, Becton Dickson, Franklin Lakes, NJ) from the anterior vena
cava. Pigs were then killed by intra-cardiac injection (0.22 mng body weight) of
sodium pentobarbital (392 g/L). Plasma was collected by centrifugation (2,000 x
g, 10 min, 4°C, GS-6KR, Beckman Coulter, Palo Alto, CA) and frozen (-80°C) for
later determination of plasma minerals (Ca, Cu, Fe, and Zn) by atomic absorption
spectroscopy (Unicam 989, Thermo Electron Corp., Franklin, MA) as previously
described (16). Plasma P was determined spectrophotometrically (DU 7400,
Beckman Coulter, Palo Alto, CA) by the method of Gomori (19). Kidney and liver
samples were excised and frozen (-80°C) for later analysis. Intestinal mucosa
cells were collected and processed from the duodenum and jejunum according to
the method described by Carlson et al. (2). Tissues were microwave-digested
(MARS 5, CEM Corp., Matthews, NC) for mineral analysis as described by Shaw

et al. (20). The MT protein concentration was determined by modification of the

102

silver saturation assay of Scheuhammer and Cherian (21) as described by
Martinez et al. (16).
Fecal and Urine Analysis

Feces were oven (lsotemp 650G, Fisher Scientific, St. Louis, MO) dried at
80°C for 24 h, then ground (Thomas-Wiley Mill model ED-5, Thomas Co.,
Philadelphia, PA). They were further processed through a 1 mm screen in a
Cyclotec 1093 Mill (Foss Tecator, HOQanas, Sweden). Fecal mineral
concentrations were determined after microwave digestion as described above
for tissues. Urine samples were thawed and centrifuged at 1,000 x g for 15 min
prior to mineral analysis. They were diluted as needed for determination of the
individual mineral elements.

All glassware used for mineral analyses was washed in 30% nitric acid
and rinsed with double-deionized water. Instrument accuracy for all mineral
analyses was established using bovine liver standard (1577b; NIST,
Gaithersburg, MD).
Statistical analysis

Feed intake and performance of one pig fed the Zn1ooP diet was extremely
low throughout the 21 cl study. Residual and scatter plots (22) characterized it as
an outlier, and its data was excluded from the experiment. Weight gain, feed
intake, feed efficiency and apparent mineral retention were analyzed using the
MIXED procedure of SAS (23) with repeated measures. The mixed model
included the pig as random effect, while the fixed effects included zinc, phytase,

their interaction, day as the repeated measures variable, and the subject of the

103

repeated measures specified as the pig within the dietary treatments. The
following covariance structures for the repeated measurements were compared:
compound symmetry, autoregressive order one, and heterogeneous
autoregressive order one (24). The appropriate covariance structure was
determined utilizing Akaike's information criterion (25). Apparent retention was
calculated as mineral intake minus mineral in feces and urine. For fecal, urinary
and retained Ca, P, Cu, and Fe values, their respective mineral intake was
modeled as a covariate for a regression based-normalization (26). Apparent Ca,
P, Cu, and Fe retention are expressed as least square means i standard error.

For plasma and tissue mineral concentrations, the experimental unit was
the individual pig nested within dietary treatments. Least square mean estimates
were used as treatment means, and Bonferroni adjustments were performed as
multiple comparison test. Residual diagnostic plots revealed that Zn
concentration in the feces, urine, and tissues, as well as, Zn retention and tissue
MT concentration required logarithmic transformation to improve normal
distribution. Back-transformed means and their respective 95% Cl (lower and
upper limits) are given in tables and figures. Differences were considered

significant at p < 0.05.

104

RESULTS AND DISCUSSION
Growth Performance

Pharmacological Zn and supplemental phytase did not affect (p = 0.66)
ADG or ADFl during the 21 d study (data not shown). There was a significant Zn
and phytase interaction (p = 0.04) on feed efficiency during d 15-21 (data not
shown). The pigs may not have responded to pharmacological Zn
concentrations because of their high health status, the small number of pigs per
treatment (n = 4) or because newly weaned pigs were fed and kept in individual
stainless steel pens. Our results are in agreement with Schell and Kornegay
(27), Smith et al., (1), and Hill and Miller (28) where 1,000, 3,000 or 5,000 mg
Zn/kg respectively, did not improve growth parameters and with studies reported
by Augspurger et al. (29) and Stahl et al. (30), where phytase supplementation
did not improve growth.
Plasma, Kidney and Liver Mineral Analysis

Feeding newly weaned pigs Zn4,ooo resulted in higher plasma Zn (p =
0.01), and kidney Zn (p = 0.01) concentrations than when pigs were fed Zn1oo or
201.000; no phytase effect was detected (Table 2). Lei et al. (10) reported that
feeding 1,350 FTU/kg to pigs increased plasma Zn. Others have observed that
feeding 3,000 mg Zn/kg resulted in higher plasma and kidney Zn (2, 31, 32).
There was a significant Zn and phytase interaction (p = 0.01) on hepatic Zn
concentrations (Figure 1), which is in agreement with previous reports from our
laboratory where Zn and phytase interacted when pigs were fed 150, 1,000 or

2,000 mg Zn/kg with or without phytase for 14 d post-weaning (16). This

105

interaction resulted in pigs fed Zn1,oooP having a greater hepatic Zn
concentrations than pigs fed Zn1oo, Zn1ooP or 201.000. Pigs fed Zn4,ooo and
Zn4,oooP had similar hepatic Zn concentrations that were greater than liver Zn of
pigs fed the other diets. As with our previous work ( 16) feeding phytase caused
an increase in hepatic Zn concentrations with 1,000 mg Zn/kg, but not with 2,000
mg ankg (16) or 4,000 mg Zn/kg (present study). This finding may be due to the
4,000 mg Zn/kg diet masking the effect of phytase or reducing its usefulness.
Also, when Zn is fed in excess of requirement with or without phytase, its
concentration remains elevated in plasma and storage organs even when dietary
Zn concentration was decreased 7 d before the pigs were killed indicating that Zn
from the diet (100 mg Zn/kg) is adequate to meet the pigs needs and that the
tissue concentrations are not toxic to these young animals.

Supplementing Zn or phytase did not affect Ca, P, Cu or Fe in plasma, or
Ca, P, or Fe concentrations in tissues (Table 3). Similarly, Adeola et al. (11)
found no change in plasma Ca or Cu concentrations after feeding 1,500 FT U/kg
of phytase to pigs for 21 d post-weaning. Murry et al. (33) found that the addition
of phytase (700 or 1,000 FTU/kg) to adequate P diets for 35 d post-weaning did
not affect serum Ca, P or Cu concentrations. In contrast to our results, Meyer et
al. (34) found that feeding 2,000 or 3,000 mg ankg to 22 old pigs for 21 (I post-
weaning decreased liver Cu. In the present study, pigs fed Zn4,ooo had greater (p
= 0.01) kidney Cu concentration than pigs fed Zn1oo or Zn1,ooo, independent of
phytase supplementation. Similar results were observed by Carlson et al. (2),

after 28 d of supplementing 3,000 mg ankg, and Hill et al. (28) when feeding

106

5,000 mg ankg to gilts and sows for two parities. The increase in renal Cu may
be due to MT sequestering Cu resulting in increased urinary Cu excretion, which
we observed (data not shown).
Tissue Metallothionein Protein Analysis

The MT response in duodenum and jejunum differed. Duodenal MT was
greater (p = 0.0001) in pigs fed anoo and Zn4,ooo compared with pigs fed ano
(Figure 2A). However, in the jejunum pigs fed Zn4,ooo had greater (p = 0.009) MT
concentrations than pigs fed the remaining treatments (Figure 28). Furthermore,
the jejunal MT concentrations were less than those observed in the duodenum,
regardless of phytase addition. Carlson et al. (2) reported that feeding 3,000 mg
Zn/kg for 14 d post-weaning resulted in increased duodenum MT compared to
pigs fed 100 mg ankg. In mice, MT concentrations were greater in the
duodenum when 400 mg Zn/kg was fed compared with MT in remaining
segments of the intestine (35), and with mice fed 10 and 150 mg ankg. In
addition, feeding 400 mg ankg or 1,000 mg Zn/kg to rats for 7 d increased
duodenal MT compared to jejunal, ileal, caecal and colon MT concentrations
(15). Furthermore, MT concentrations were greater in rats fed 400 and 1,000 mg
Zn/kg than those fed 10 and 100 mg Zn/kg. These data suggest that pigs fed
1,000 and 4,000 mg Zn/kg have an increased need to synthesize duodenal MT to
sequester and/or transfer Zn into portal circulation. However, after Zn uptake by
the duodenum, the cells of the jejunum are not stimulated to synthesize as much
MT when fed 100 or 1,000 mg ankg as when 4,000 mg ankg is fed indicating

that the Zn concentration in the jejunum of pigs fed 4,000 mg Zn/kg was greater

107

and stimulated the production of MT. This indicates that the duodenum and
jejunum respond independently to Zn concentrations. The increased MT
sequesters excess Zn, which can be excreted in sloughed mucosal cells (17).

Similar to kidney Zn, MT was greater (p = 0.01) in pigs fed Zn4,ooo than in
pigs fed the other treatments (Figure 20). However in the liver (Figure 2D), pigs
fed 201,000 had greater (p = 0.01) MT protein concentration than pigs fed Zn1oo,
while those fed Zn4,ooo had greater liver MT than pigs fed ano and Zn1,ooo.
Metallothionein concentration was not affected by phytase in any other tissue.
Our renal and hepatic MT results are in agreement with other studies where MT
responds to the amount of Zn fed in the diet (2, 36). We have also shown that
pharmacological Zn (2,000 mg Zn/kg) increased kidney and liver MT mRNA as
well as protein concentration in pigs 14 d post-weaning, and these values were
positively correlated with their respective tissue Zn concentrations (16). The
present study suggests that after removing pharmacological or excess dietary
Zn, endogenous Zn continues to stimulate MT production in the duodenum
and/or jejunum, and that liver MT continues to sequester and manage excess Zn.
Fecal, Urinary and Apparent Zn Retained

Intake, fecal, urinary and retained Zn during phase I (d 1-7), H (d 8-14), lll
(CI 15-21) and overall (21 d) are shown in Table 4. During phase I and II, Zn in
feces, urine and that retained increased as the concentration of Zn in the diet
increased, with no effect of supplemental phytase. In this study, pigs fed 1,000
and 4,000 mg Zn/kg for 14 d, excreted 12.2 (292 mg/d) and 43.5 (1,046 mg/d)

times more fecal Zn respectively, than pigs fed 100 mg Zn/kg (24 mg/d). The

108

greater Zn retention of pigs fed diets that exceed NRC recommendations (201.000
and 204.000) is due to Zn loading of tissues prior to increased fecal and urinary Zn
excretion. This is consistent with results of previous studies (32, 34, 37).

During phase III after pharmacological and excess Zn feeding was
discontinued and all pigs were fed 100 mg Zn/kg, pigs previously fed 1,000 and
4,000 mg ankg diets continued to excrete more urinary and fecal Zn than pigs
fed 100 mg ankg; pigs fed 4,000 mg Zn/kg were in negative balance. Pigs fed
the NRC requirement (Zmoo) excreted a consistent amount of fecal and urinary
Zn throughout the 21 d of the study, maintaining their balance thus indicating that
Zn was not in excess but adequate. Case and Carlson (32) fed pharmacological
Zn (3,000 mg ankg) to 17 d old pigs for 10 d prior to fecal collections. Their pigs
were in negative balance, most likely due to Zn loading occurring for 10 d prior to
collection which was similar to what we observed after 11 d of dietary
intervention (data not shown).

Overall, fecal Zn excretion was greater (p = 0.01) for pigs fed Zn4,ooo, than
pigs fed 201.000 and Zn1oo (Table 4). However, there was a significant Zn by
phytase interaction (p = 0.03) on urinary Zn excretion, where pigs fed 204,000 and
meoP had greater urinary Zn excretion compared with pigs fed Zn1,oooP, but not
201,000. Zinc intake and retention was greater (p = 0.01) for pigs fed Zn4,ooo and
Zn4,oooP compared with pigs fed the remaining diets.

Daily Zn Fecal Excretion and Retention Patterns
We are the first to report day by day fecal Zn excretion (Figure 3) starting

on d 1 after weaning. Fecal Zn excretion for all treatments was similar for d 1 to

109

5. On (I 6, pigs fed Zntooo excreted 3 times more Zn than pigs fed 201.000 and
about 10 times more than pigs fed ano. From d 8—14, the elevated Zn excretion
pattern plateau. In phase III, when dietary Zn was fed at 100 mg Zn/kg, within 3
d all pigs had comparable fecal Zn excretion values averaging 55 mgld, (Figure
3). These data confirm that when Zn is fed at a greater concentration than its
requirement, Zn loads into tissues during the first 11 d of supplementation before
increased Zn excretion occurs. This pattern is absent in pigs fed Zn at their
requirement (100 mg Zn/kg) for 21 d post-weaning. According to our data, if
anoo or Zn4_ooo were fed for only 11 d instead of 14 d, fecal Zn excretion would
be reduced by 50% (168 mg/d and 578 mgld, respectively).
Mineral (Ca, P, Cu, Fe) Apparent Retention

Apparent Ca, P, Cu, and Fe retention are presented for phase I (d 1-7), ll
(d 8-14), Ill (d 15-21) and overall (d 1-21) in Table 5. Supplementing phytase
increased (p = 0.05) Ca retention throughout the entire study, and enhanced P (p
= 0.02) apparent retention, during phase II, III and overall. These results are
consistent with those of Murry et al. (33) who fed weanling pigs phytase (0, 700
or 1,000 FTU/kg) and adequate P diets and reported increased P retention and a
tendency for increased Ca retention. Lei et al. (38) showed that Ca retained as a
percent of intake was increased with supplemental phytase. While retention was
improved, Ca and P concentrations were not increased in kidney, liver and
plasma. Perhaps the retained Ca and P was present in bone tissue, which was
not measured in this study. The retention of Cu and Fe was not affected by

feeding phytase.

110

Feeding Zn4,ooo increased urinary and decreased fecal Cu (data not
shown) resulting in increased Cu retention (p = 0.05). Similarly, Carlson et al.
(37) showed that pigs fed 3,000 mg Zn/kg as ZnO retained more Cu. However,
Meyer et al. (34) showed decreased Cu and Fe retention due to greater fecal Cu
and Fe excretion in pigs fed 3,000 mg Zn/kg for 21 d. Our results of increased
urinary Cu could be associated with the increased renal Cu concentrations
(Table 3) observed in pigs fed Zntooo, confirming an increased urinary Cu output
as a result of feeding high Zn.

Phytase supplementation and pharmacological Zn improved Ca, P, Cu
and Zn retention with no interaction. It is unclear why phytase did not respond
with Zn in an additive manner. In vitro studies by Maenz et al. (39) showed that
at pH 7 and a Zn to phytic acid molar ratio of 0.35:1, phytate-P hydrolysis by
phytase was reduced by 50%. It was proposed by these authors that two phytic
acid molecules binding Zn, may bridge together, causing a conformational
change in the phytic acid molecule that is inaccessible for phytase hydrolysis.
However, in this study Ca and P retention were improved by phytase
supplementation, an indication that some hydrolysis indeed occurred.

Our study results suggest that supplementing phytase (500 FTU/kg) plus
1,000 or 4,000 mg Zn/kg increases liver Zn but does not affect Ca, P, Cu or Fe in
tissues, MT or reduce Zn excretion. Feeding 4,000 mg ankg as ZnO to newly
weaned pigs for 14 d, does not produce toxic effects. Supplementing dietary Zn
at 10 or 40 times the Zn requirement resulted in an adaptive response,

characterized by increased MT concentrations in the intestinal mucosa, liver and

111

kidney, which persist 7 d after pharmacological or excess Zn are withdrawn.
Daily fecal Zn excretion plot confirms that Zn loading occurs by d 11 of feeding
pharmacological or excess Zn, suggesting a shorter supplementation period (11
d) may be effective in reducing Zn excretion by 50% thus reducing environmental
concerns about feeding pharmacological Zn as a growth stimulant in the swine

industry.

112

ACKNOWLEDGEMENTS

This work was supported in part by a Cooperative State Research
Education and Extension Service - Initiative for Future Agriculture and Food
Systems grant from the United States Department of Agriculture, and by The
Graduate School — Competitive Doctoral Enrichment Fellowship, Michigan State
University, East Lansing, MI. The authors express their appreciation to BASF
Corporation, Prince Agri Products, American Protein Corporation, and
International Ingredient Corporation for supplying dietary ingredients used in this

study.

113

LITERATURE CITED

1. Smith, J. W., Tokach, M. D., Goodband, R. D., Nelssen, J. L. and Richert, B.
T., Effects of the interrelationship between zinc oxide and copper sulfate on
growth performance of early-weaned pigs. J. Anim. Sci. 75, 1861-1866 (1997).

2. Carlson, M. S., Hill, G. M. and Link, J. E., Early and traditionally weaned
nursery pigs benefit form phase-feeding pharmacological concentrations of zinc
oxide: effect on metallothionein and mineral concentrations. J. Anim. Sci. 77,
1199-1207 (1999).

3. McCully, G. A., Hill, G. M., Link, J. E., Weavers, R. L., Carlson, M. S. and
Rozeboom, D. W., Evaluation of zinc sources for the newly weaned pig. J. Anim.
Sci. 73 (Supplement 2), 72 (1995).

4. NRC, Nutrient Requirements of Swine, 10th, Washington, DC (1998).

5. Carlson, M. 8., Hoover, S. L., Hill, G. M., Link, J. E. and Turk, J. R., Effect of
pharmacological zinc on intestinal metallothionein concentration and morphology
in the nursery pig. J. Anim. Sci. 76, 57 (1998).

6. O'Dell, B. L. and Savage, J. E., Effect of phytic acid on zinc availability. Proc.
Soc. Exp. Biol. Med. 103, 304-305 (1960).

7. Oberleas, D., Muhrer, M. E. and O'Dell, B. L., Dietary metal-complexing agents
and zinc availability in rats. J. Nutr. 90, 56-62 (1966).

8. Davies, N. T. and Nightingale, R., The effects of phytate on intestinal
absorption and secretion of zinc, and whole-body retention of zinc, copper, iron
and manganese in rats. Br. J. Nutr. 34, 243-258 (1975).

9. Jongbloed, A. W., Mroz, Z. and Kemme, P. A., The effect of supplementary
Aspergillus niger phytase in diets for pigs on concentration and apparent
digestibility of dry matter, total phosphorus, and phytic acid in different sections of
the alimentary tract. J. Anim. Sci. 70, 1159-1 168 (1992).

114

10. Lei, X., Ku, P. K., Miller, E. W., Ullrey, D. E. and Yokoyama, M.,
Supplemental microbial phytase improves bioavailability of dietary zinc to
weanling pigs. J. Nutr. 123, 1117-1123 (1993).

11. Adeola, 0., Lawrence, B., Sutton, A. and Cline, T., Phytase-induced changes
in mineral utilization in zinc-supplemented diets for pigs. J. Anim. Sci. 73, 3384-
3391(1995)

12. Yi, Z., Kornegay, E. T., Ravindran, V., Lindemann, M. D. and Wilson, J. H.,
Effectiveness of Natuphos phytase in improving the bioavailabilities of
phosphorous and other nutrients in soybean meal-based semipurified diets for
young pigs. J. Anim Sci. 74, 1601-1611 (1996).

13. Bremner, l., Metallothionein Ill. Biological Roles and Medical Implications, in
K. T. Suzuki, N. lmura and M. Kimura, Birkhauser, Basel, Switzerland, pp. 111-
124 (1993).

14. Davis, S. R., McMahon, R. J. and Cousins, R. J., Metallothionein knockout
and transgenic mice exhibit altered intestinal processing of zinc with uniform
zinc-dependent zinc transporter-1 expression. J. Nutr. 128, 825-831 (1998).

15. Tran, C. 0., Butler, R. N., Howarth, G. S., Philcox, J. C., Rofe, A. M. and
Coyle, P., Regional distribution and localization of zinc and metallothionein in the
intestine of rats fed diets differing in zinc content. Scand. J. Gastroenterol. 34,
689-695 (1999).

16. Martinez, M. M., Hill, G. M., Link, J. E., Raney, N. E., Tempelman, R. J. and
Ernst, C. W., Pharmacological zinc and phytase supplementation enhance
metallothionein mRNA abundance and protein concentration in newly weaned
pigs. J. Nutr. 134, 538-544 (2004).

17. Cousins, R. J., Absorption, transport and hepatic metabolism of copper and
zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65,
238—309 (1985).

18. Reeves, P. G., Adaptation responses in rats to long-terrn feeding of high-zinc
diets: emphasis on intestinal metallothionein. J. Nutr. Biochem. 6, 48-54 (1995).

115

19. Gomori, G., A modification of the colorimetric phosphorus determination for
use with the photoelectric colorimeter. J. Clin. Lab. Med. 27, 955-960 (1942).

20. Shaw, D. T., Rozeboom, D. W., Hill, G. M., Booren, A. M. and Link, J. E.,
Impact of vitamin and mineral supplement withdrawal and wheat middling
inclusion on finishing pig growth performance, fecal mineral concentration,
carcass characteristics, and the nutrient content and oxidative stability of pork. J.
Anim. Sci. 80, 2920-2930 (2002).

21. Scheuhammer, A. M. and Cherian, M. G., Quantification of metallothionein by
silver saturation, in Methods in Enzymology. Metallobiochemistry. Part B.
Metallothionein and related molecules, 205, J. F. Riordan and B. L. Vallee,
Academic Press, Inc, San Diego, Ca, pp. 78-83 (1986).

22. Ott, R. L., An Introduction to Statistical Methods and Data Analysis, 4th ed.,
Duxbury Press, Belmont, California (1993).

23 SAS Systems (1999-2000) The SAS System for Windows 98, 8.1 ed. SAS
Institute, Inc., Cary, NC.

24. Littell, R. 0., Henry, P. R. and Ammerman, C. B., Statistical analysis of
repeated measures data using SAS procedures. J. Anim Sci. 76, 1216-1231
(1998).

25. Akaike, H., Fitting autoregressive models for prediction. Ann. Inst. Stat. Math.
21, 243—247 (1969).

26. Poehlman, E. T., Reduced metabolic rate after caloric restriction-can we
agree on how to normalize the data? J. Clin. Endocrinol. Metab. 88, 14-15
(2003)

27. Schell, T. C. and Kornegay, E. T., Zinc concentration in tissues and
performance of weanling pigs fed pharmacological levels of zinc from ZnO, Zn-
methionine, Zn-lysine, or ZnSO4. J. Anim. Sci. 74, 1584-1593 (1996).

28. Hill, G. M. and Miller, E. R., Effect of dietary zinc levels on the growth and
development of the gilt. J. Anim Sci. 57, 106-113 (1983).

116

29. Augspurger, N. R., Spencer, J. D., Webel, D. M. and Baker, D. H.,
Pharmacological zinc levels reduce the phosphorus-releasing efficacy of phytase
in young pigs and chickens. J. Anim Sci. 82, 1732-1739 (2004).

30. Stahl, C. H., Roneker, K. R., Pond, W. G. and Lei, X. 6., Effects of combining
three fungal phytases with a bacterial phytase on plasma phosphorus status of
weanling pigs fed a com-soy diet. J. Anim Sci. 82, 1725-1731 (2004).

31. Hill, G. M., Miller, E. R. and Stowe, H. D., Effect of dietary zinc level on health
and productivity of gilts and sows through two parities. J. Anim Sci. 57, 114-122
(1983).

32. Case, C. L. and Carlson, M. 8., Effect of feeding organic and inorganic
sources of additional zinc on growth performance and zinc balance in nursery
pigs. J. Anim. Sci. 80, 1917-1924 (2002).

33. Murry, A. C., Lewis, R. D. and Amos, H. E., The effect of microbial phytase in
a pearl millet-soybean meal diet on apparent digestibility and retention of

nutrients, serum mineral concentration, and bone mineral density of nursery pigs.
J. Anim Sci. 75, 1284-1291 (1997).

34. Meyer, T. A., Lindemann, M. D., Cromwell, G. L., Monegue, H. J. and
Inocencio, N., Effects of pharmacological levels of zinc as zinc oxide on fecal
zinc and mineral excretion in weanling pigs. Prof. Anim. Sci. 18, 162-168 (2002).

35. Tran, C. 0., Butler, R. N., Philcox, J. C., Rofe, A. M., Howarth, G. S. and
Coyle, P., Regional distribution of metallothionein and zinc in the mouse gut.
Comparison with metallothionein-null mice. Biol. Trace. Elem. Res. 1998, 239-
251(1998)

36. Blalock, T. L., Dunn, M. A. and Cousins, R. J., Metallothionein gene
expression in rats: tissue-specific regulation by dietary copper and zinc. J. Nutr.
1 18, 222-228 (1988).

37. Carlson, M. S., Boren, C. A., Wu, C., Huntington, C. E., Bollinger, D. W. and
Veum, T. L., Evaluation of various inclusion rates of organic zinc either as
polysaccharide or proteinate complex on the growth performance, plasma, and
excretion of nursery pigs. J. Anim Sci. 82, 1359-1366 (2004).

117

38. Lei, X. G., Ku, P. K., Miller, E. R. and Yokoyama, M. T., Supplementing corn-
soybean meal diets with microbial phytase linearly improves phytate phosphorus
utilization by weanling pigs. J. Anim Sci. 71, 3359-3367 (1993).

39. Maenz, D. D., Engele-Schaan, C. M., Newkirk, R. W. and Classen, H. L., The
effect of minerals and mineral chelators on the formation of phytase-resistant and

phytase-susceptible forms of phytic acid in solution and in a slurry of canola
meal. Anim. Feed. Sci. Tech. 81, 177-192 (1999).

118

Table 1

Composition of experimental diets for nursery pigs (9 I 100 g diet)1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Ingredients Phase I Phase II Phase III

Corn 39.09 53.14 54.75

Soybean meal (44% GP) 20.20 20.18 23.75

Spray-dried whey 20.00 20.00 10.00

Plasma protein2 6.00

Lactose3 10.00 —-

Blood cellsz 2.00 ,

Menhaden fish meal 5.00

DL-Methionine 0.11 0.06

L-LysineHCL 0.15 0.15 0.15

Choice White Grease‘ 3.00

Soybean oil 1.00 1.00

Monocalcium Phosphate5 1.45 1.52 1.35

Ground limestone 0.55 0.52 0.60

Salt 0.35 0.33 0.30

Trace mineral pre-mix6 0.50 0.50 0.50

Vitamin pre-mix7 0.60 0.60 0.60
Analyzed total zinc concentration

Dietary treahnents (as fed), mglkg diet

ano = (100 mglkg Zn, ZnO) 130.14 129.40 117.73

anoo = (anO + 1,000 mg Zn/kg) 1,281.46 1,114.30

204,000 = (ano + 4,000 mg Zn/kg) 4,654.90 3,913.15

anP = (ano + 500 FTU/kg) 130.30 131.84

Zn1.oooP= (ano + 500 FTU/kg + 1,000 mg Zn/kg) 1,226.48 1,110.05

zn4,000P = (20100 T 500 FTU/k9 1‘ 4.000 mg Zn/kg) 4,841.74 4,044.98

 

1The basal diet was formulated to meet or exceed nutrient needs (4).
Phase I (d 0-7), Phase II (d 8-14), Phase III (d 15-21). Zinc as zinc oxide was added at
the expense of corn.

2American Protein Corp., Inc (AP 920 - sprayed dried plasma, AP 3016 - granulated

sprayed dried animal blood cells)

3lntemational Ingredients Corp.

‘Titre = 36°C min., 4% max. free fatty acids, 1% MIU max.

5Monocalcium phosphate = 21% elemental P

6Trace Mineral pre-mix (g mineral/kg premix): CuSO4- 5H20, 2; FeSO4- H20, 20;

CZH4(NH2)2.2HI, 0.03; MnSO4- H20, 2; NaZSeoa, 0.06; ZnO, 20.

7Vitamin pre-mix (lU/kg premix): vitamin A, 91,8562; vitamin D, 91,865; vitamin E, 11,022;
(g vitamin lkg premix): vitamin K, 2.20; niacin, 4.40; riboflavin, 0.73; panthotenic acid,
2.94; thiamine, 0.18; vitamin B6, 0.17; vitamin B12, 5.50.

119

.8": .QSEN 58x8 2.95am: .8 v": .20 $3 05 .0 £8: .39. new .950; 2.3:. 38.828808. 8... 68:38.. 323 of.n

4me u .8 Eu 5888 a 83 .v: u .8 8:85am: €866 68 88> mm... .2 N

.633 u N a .8 no»: 863 new 2.8 68... $6.58». 5:228: 5 page: 863 9. mum .0 £995 3:... 3886 cm 55, moi 3.5 3:23..

86 85 6.0 8.8 - m. 5. new We. - m8... «.5 8.8 - .3: IN 38 - 33 4.8 2.8 - SN. new 6.8 - 3: 3m Eu... ”2:5.
as... 8.0 8.0 $3 - mm. 5 m: a? - 8.8 2.. $3 - 5.8 88 a? - 2. s «3 63 - 8.9 So 83 - 8.9 56 3.9... 2.35

 

 

1:25
.395 Sn. 5 8a.: 8.... 2: So... 8.... 2: «in... can
2...; a 8... a 9.3K 88......

 

 

:n «:65?» .o 5.3 o£~ «388 ..o .3. 830553 .8258; be 3083
N e...-....

120

.m:.m> 38.88 6... n6 03o. 52...... 3. 2mm 363.3. 6.... .838. 33:338.: cm 8 can Sum 3894. ..

8a.. .n.8...N 38...... v": .mcmm... 83 38:33... 38.35 8....n

..Nm. 3 .8 .6... 58.5... .... 8.3 .v.-. u .8 85830.. >83... 38 6.8.. mm... .2. ...

.80.. .33 83.583 3.6.23... ... 33:8. 6.6.... 9. m.m .6 £3.32. 3...... 68.96 ..m 2...... mm... 3.... 3:83.

 

 

 

 

 

 

8... 8... ...... ....m. .8. 3.... 3... ...... 4.8. ...8. 3.9.. .3...
8... ...... ...... 2... .8 ...8 4.8 3.... ..8 3.8 3.9.. .355.
8... .3... 8... 8... .... ... 3.. 3.. .... N. 335.388...
48':

2... ...... ...... 8.3 ..8 3.? N8 ...... o...” 3.8 33... .2...
8... E... ...... 83 3.8 ...o. 3.... ...... ...... 3.. 33... 8.6.x
N... 8... 8... 8... 3.. ... v. m. m. N. 335.358...
88m

...... 8... .4... 8.... 8... 8.3 8... .3... .3... 8... 33.... .2...
3... 8... .... «...... 8... mm... 8... 8... R... 8... 332.889
3... 8... ...... N8... 8.... .8... 8.... 83... 8.... .8... 33.358...
.mauocmmocn—

8... 9.... 8... 8... 8.... ...... ...... 8.... me... 3.... 33.... .9...
8... ...... 8... 8... 8... 8... 8... 8... 8... 8... 33.... .23...
...... ...... ...... .8... 8.... 8.... 3.... ...... ...... «.... 33.358...
Eng

......N ....n. ..N ....mm 38.4 8.... 8. 38... 8.... 8. 9.3... JEN
2...... .. 8... .. 9.5... 85......

 

Q“;

acct-.8533 on. 35.... :O .n. .50 .32. can .35.... .mEmmfl— ..o 385...

3388......» .365...» .o ......s 0:.N mmooxo ..o 3930.03.58... 6.8.33 .0 Bootm

n 033.

121

...-o... v a 32.... 3:33.83 .5888

m 52...; Bo. 3 ... 8.322 .8": .....S...N .3396. v": .2322... 8.33:8 3333 a... .o 3...... .03.... 9.3 .96.. 3:30... 3E0.3:3...xoan 93 8.58.... 3323.. 3......3

.33. u .2 .3... .8888 3 9.... ....-. u .2 3.5.532. E83... 3. 3.2... 33... ..< 3
.633 u .3 3 .... 303: 3.33 3:3 3:8 .093 330.523 ...—.223... c. 303...... 3.3.5 9. 3.3 .o 23.0.... 3...... 033.96 cm .....s 33... 3.... 3:03....

 

 

 

3o... ..3... .3... .32.. - 3.... ...3 3.333 - 33.. 3.3 ...333 - 33-. 8. ..33 - 3.3. 8. ..333 - 333. 3.. ...3... - 33-. 33 38.3.3.
3..... ...... ...... N.33......3...3..3 3.333-333.333...333-333.3..3 ..33...-3.......3.3 N..3...3.33......... u........-.......33... 2...:
..3... 33... ...... .3... - 333. 333 .33 - .3. 33 .33 - 3.. .3 .333 - 333. 3.3 ..3. - 33. 8. .33 - 3.. .3 .38..
333 3..... ...... 333.333....2. 333-8.333.. 33.-33.8. ...3..-...3.o..o.. 333-3.3.333 23.-3..... 9.8...
3333343
33... R3 ...... 3.... - 33-. .3- ...3 - «3.. 3 .8 - 3.-. 33 .33.- - 33-. ..3- .33 - 33.. 3 .... - ..-. .3 38.833.
33... 33 3..... 33.3-23. 33.3 33.-333.333 ..3.3-..3....3.3 3.3-83.8. 333-333.33.. 33.-3.3.33 3......
3.... 333 ...3 .3... - 3. 3. ..3 - ..3. 3e 3.. - 3.. .3 .3... - .3. 3.. .33 - .v. .3 .33 - ..3. ..3 .33..
3.... ...... 3e... .33 - 3... .3 .33 - 3... ..3 .33 - 3... 33 ..3 - 33. 33 .3.- - 33. .3 .33 - .... 33 9.8...
3393
.33 ..33 .3... .3.3.3 - 333. .33.. 3.3 - .3. .3. .3... - ..-. 33 3.. - 33. 333 ...... - 33-. 333 .3... - ..-. 8 32.533.
..3 33... ...... 633-3....33 33.-33.3... 333-3.3.333 63.73.3393 333-33....33 333-33...... 2...:
R3 333 .3... ...33.3-33..3..3...3 233333.33. ...-3-33.... 3333-39.33.33 333-33.33. 8.33.33 .83..
33... ...... ...... .3..3...-..3..3.333.3 ..3...-3......33 33.-33.3... ate-3333.33... 2.1.3.333... 83.-33.8. 9.9...
33.33
..33 333 ...... .3.3.3-3.v...333.3 .333..-33..333 33.-3-..... .33...3-..3v...33..3 .333..-333.333 ..:-33.3.. 32.89.
...... 333 .3... ...3.3-.3...8.3 ..3..-3v3.33 3..-33.33... 333-33.3.33... 5.3-33.533. 333-3.3.333 3......
3..... 3.... .33 .3... - 3.3. .3.- ..3. - 3... 3.. .3.‘ - .... .3 .33 - 333. 3.3 .33. - 33. ..3. .3... - 3.. 33 .83..
3..... 83 ...... .333.3-333.3.333.3 233-3335... 33.-.....33 33.3-33.3.383 63.33.3333 3..-33.3.. 9.9...
3
..:...cN ...... :3 83... 8.... 8. 38.3 8.... 8. 9....3 9.. .353
3.3.. .. 83 .. 9.5.... 68......

 

...3..oocu_an ..N ..o maggicoEmiqsu 505...... .o 5.3 053 3398 .o .8.uo_33....a...m .3333 9.58. .o Boatm
v 333...

122

.m:_m> notoao. m... .0 8... 55.3 m. 2mm .899 on... 698... 38:53:: an 0. can 2mm 03802 .
..muc .a8.cN 33.8. W: .33... 2a 33:30... 33...... of...
..N6. 3 .0. .0... 5.530 a new ....-. u .0. 3:05.89. E523 um. 22> am... ..< N
.95.. .00.» 80.59... 3:223... c. 3330.. 203 9. m... .0 £903 3...... 3805 cm 5.3 mm... 09... 3:93.

 

 

 

 

 

 

 

8... .... R... 8.” .3... 8.8 «N... ..:... ....8 8.... 63......2.
8... 8... 8... ...... 8a ...N ....N 98 N... «.3 635.888
...... ...... 8... 8... 8.. .... 3... .... o... .... 63.32.82.
R... ...... 8... 8... mm... 8... ...... 8.~ .3 3... 63.5228
3-...

8... 8... 8... cm... ...8 3.8 8.... 3.8 8.8 8.... 635.5...
8... n... ...... 8... 8.. 8.. ....N 8.. ...... 8.. 635.3960
8... mo... «a... 8... SN 8.~ 8.... 8a 8a .3 63.52.32...
8... 8... 8... ...o 8... t... 2... 8... .N... 8... 63.8228
.3...

av... 8... 8... 8... ......o 8.... ....8 8.8 8.8 a... 635.5...
8... ...... .N... 8... ....m .3 8.. am... .3 8a 632.3338
...... 8... 8... ...... 8.. .... 8.. 8.. .... 8.. 63.32.38...
8... 8... ...... ...... ...N ....N 8.... RN .3 8N 63.53.28
fl...

8... ..m... ...... 8... 2.8 8.8 3.8 8.8 8.8 8.8 63.5.8...
8... ...o ...... 5... R... 88 ....u ....N 8.. ....N 635.888
.... n... ...... 8... 3.. ...... 3... ...... ...... 5.. 63.32385.
8... 8... 8... ...... 8a SN RN 8.. 8.. ....N 63.2228
......

$5.5. ...... ..N Ema 2...... 8.... 8. 2...; 8.... 8. 9...... u... can
2...; a ...... .. 9.5... 88......

 

...q.:oz:82 c...— 05 :0 ... .30 “:23...“
ca 38...... 3:25.39.» 505.3 .0 5...: 2i 33.8 .o _8_uo_ooa=:u..n .8252... 9:33. u... floctm

m 03¢...

123

 

600

500

uh
O
O

300

Liver Zinc Concentration
(ug Zn I g wet tissue)
N
O
O

100

 

 

 

ZI11oo anP zn1'ooo anmP 2'14'000 ZH4'oooP

Figure 1. Concentration of zinc in liver of pigs fed 100, 1,000 or 4,000 mg
Zn/kg as zinc oxide, with or without 500 FTU/kg of phytase for 14 d post-weaning
and a common diet (100 mg Zn/kg) for the subsequent 7 d. Values are
backtransformed means with 95% Cl, n = 4 (except Zn1ooP, n=3). The zinc x
phytase interaction was significant. Means without a common superscript differ

(p = 0.01).

124

30

 

N
O
l

Duodenum Metallothionein Protein Concentration
(ug Ag I g wet tissue)
A .8
0 0|
‘ l
l l

 

 

   

 

 

 

Znioo 201.000

Figure 2A. Concentration of metallothionein in duodenum of pigs fed 100,
1,000 or 4,000 mg Zn/kg as zinc oxide, with or without 500 FT Ulkg of phytase for
14 d post-weaning and a common diet (100 mg Zn/kg) for the subsequent 7 d.
Values are backtransformed means with 95% confidence interval, n = 8, (except

Zn1oo, n=7). Main effect of zinc was significant (p = 0.0001).

125

 

Jejunum Metallothionein Protein Concentration
(mg Ag I kg wet tissue)
.5
l

 

 

 

 

 

20100 201,000 Zn4,ooo

Figure 28. Concentration of metallothionein in jejunum of pigs fed 100,
1,000 or 4,000 mg Zn/kg as zinc oxide, with or without 500 FTU/kg of phytase for
14 d post-weaning and a common diet (100 mg Zn/kg) for the subsequent 7 d.
Values are backtransformed means with 95% confidence interval, n = 8, (except

Zn1oo, n = 7). Main effect of zinc was significant (p = 0.01).

126

 

300

250

200 ,

150

100

50

Kidney Metallothionein Protein Concentration
(ug Ag I g wet tissue)

 

 

 

20100 201,000 204.000

Figure 20. Concentration of metallothionein in kidney of pigs fed 100,
1,000 or 4,000 mg ZnIkg as zinc oxide, with or without 500 FTU/kg of phytase for
14 d post-weaning, and a common diet (100 mg ZnIkg) for the subsequent 7 d.
Values are backtransformed means with 95% confidence interval, n = 8, (except

Zn1oo, n=7). Main effect of zinc was significant (p = 0.01).

127

1,600

 

1,400 -« - - if

1,200 -, ~ -- - ~~

 

.5
O
O
O

800 -.

600 ,

400 ,

200

Liver Metallothionein Protein Concentration
(ug Ag I g wet tissue)

 

 

 

Zl‘l1oo zn1,000 2.14.000

Figure ZD. Concentration of metallothionein in liver of pigs fed 100, 1,000
or 4,000 mg ZnIkg as zinc oxide, with or without 500 FT U/kg of phytase for 14 d
post-weaning and a common diet (100 mg ZnIkg) for the subsequent 7 d. Values
are backtransformed means with 95% confidence interval, n = 8, (except Zn1oo,

n=7). Main effect of zinc was significant (p = 0.01).

128

... n .5339... m... .o. EVEN m6 8.. .m... .8888 m ..cm 9.503.803 u v. .0. $33.... .0 35...“. com

505.2. .0 .....s 62.8 0:... mm 9.3.: oooé .o 8.... do. no. mm... .0 2.2.8 8.8.9.... oc_~ .80. ....mn. .n 2:2“.

 

 

 

.30

..u .... .. ..
1'13 1.. (INN l c O
l..l/ .1. .../...:J H \ ....N m
. . m
z .....t . ...... m
/ s . ..8 m.
\ z . ..8 w
... x . . m.
\. . 8.... n.
/ x \ . \ m
/ \ / \ s... ..
< < ....e. M
(Di

8....

 

 

 

 

. ooo.Vch .I ooo..cN - .. u oorcN ,

129

CHAPTER FIVE
Identification of Differentially Expressed Genes in Hepatic Tissue of Newly
Weaned Pigs Fed Pharmacological Zinc and Phytase Supplemented Diets"2
Michelle M. Martinez, Gretchen M. Hill, Jane E. Link, Nancy E. Raney and
Catherine W. Ernst

Department of Animal Science, Michigan State University, East Lansing, MI

Running title: ZINC & PHYTASE AFFECTS GENE EXPRESSION IN PIGS

1 Presented in part at the American Society of Animal Science Midwestern
section meeting, March 17-19, 2004 [Martinez, M.M., G.M. Hill, J.E. Link, N.E.
Raney and C.W. Ernst. (2004) Pharmacological zinc supplementation in nursery
pigs regulates glyoxalase 1 and peroxiredoxin 4 gene expression. J. Anim. Sci.
82: 19 (Supplement 2)].

2 Supported in part by an Animal Health Formula Funds Grant from the Michigan
Agricultural Experiment Station, by a Six State Consortium on Animal Waste
Management of Environmental Protection Agency, and by The Graduate School
- Competitive Doctoral Enrichment Fellowship, Michigan State University, East
Lansing, MI.

3 Abbreviations used: ACY1, aminoacylase 1; CPBZ, carboxypeptidase U;
DDRT—PCR, differential display RT-PCR; EST, expressed sequence tag; FTU,
phytase unit; GLO1, glyoxalase I; MT, metallothionein; MRE, metal response
element; MTF, metal transcription factor; ORM1, orosomucoid 1; PRDX4,

peroxiredoxin 4; Zn, zinc; ZnO, zinc oxide; Z150, 150 mg ZnIkg; Zn150P, 150 mg

130

ZnIkg with phytase; Zn.,ooo, 1,000 mg ZnIkg; Zn..oooP, 1,000 mg ZnIkg with

phytase; 312,000. 2,000 mg ZnIkg; anoooP, 2,000 mg ZnIkg with phytase.

131

ABSTRACT

Porcine post-weaning stress symptoms include low feed intake,
diarrhea, small intestinal villi atrophy and reduced growth. Pharmacological zinc
(Zn) improves fecal consistency, intestinal morphology, and growth. Furthermore,
adding phytase to high Zn diets increases hepatic Zn concentration and
metallothionein (MT) mRNA abundance and protein. We hypothesized that
pharmacological Zn and phytase supplementation to newly weaned pigs would
result in changes in gene expression. The goal of this study was to investigate
the effects of feeding newly weaned pigs dietary Zn (150, 1000, or 2000 mg
ZnIkg) as Zn oxide (ZnO) with or without phytase (0 or 500 phytase units
(FTU)/kg) for 14 d on hepatic differential gene expression using differential
display reverse transcriptase polymerase chain reaction (DDRT-PCR). Liver
DNAase treated RNA was reverse transcribed using a 3’ poly-T primer ending in
the anchor bases AC, labeled with 33P-dATP and amplified with six 5’arbitrary
primers. A total of 52 amplicons were putatively identified as differentially
expressed, cloned and sequenced. Five transcripts exhibiting increased
expression in pigs fed pharmacological Zn that had sequence similarities to
genes encoding glyoxalase 1 (GLO1), peroxiredoxin 4 (PRDX4), aminoacylase l
(ACY1), orosomucoid 1 (ORM1) and carboxypeptidase U (CPBZ) were selected
for confirmation. Relative hepatic GLO1 (P < 0.0007), PRDX4 (P < 0.009) and
ACY1 (P < 0.01) mRNA abundances were greater in pigs fed 1000 and 2000 mg
Zn/kg than in pigs fed 150 mg Zn/kg. Relative ORM1 and CPBZ mRNA

abundances were not affected by dietary treatments. Results under these

132

experimental conditions suggest that feeding pharmacological Zn (1000 or 2000
mg ZnIkg) affects genes involved in reducing oxidative stress and amino acid
metabolism, which are essential for cell detoxification and proper cell function.
KEY WORDS: o differential display RT-PCR 0 liver a nursery pig .

pharmacological zinc o phytase

133

INTRODUCTION

The ability of pigs to adapt to and digest dry feed during the post-
weaning period influences their subsequent performance (1). Changes in the
pig’s social and physical environment, as well as in texture and nutritional
composition of the diet, preset a stressful setting. These stressors alter many
systems including immunity and are involved in the etiology of common diseases
(2). Post-weaning stress symptoms include low feed intake, diarrhea, atrophy of
small intestinal villi and ultimately reduced growth rate, which can translate into
significant financial losses for the swine industry (3).

Pharmacological Zn has been successful in increasing growth and
improving fecal consistency (4,5). Furthermore, feeding pharmacological Zn
(3000 mg ZnIkg) to newly weaned pigs for 14 d improves gut morphology by
increasing villous height and reducing crypt depths in the duodenum and jejunum
(6).

Differential gene expression is responsible for morphological and
phenotypical differences, since the transcriptome is dynamic (7). Studies on how
micronutrients affect gene expression will help to clarify the role of trace
elements in health and metabolism and their connection to biochemical events
(8).

Mechanistically, Zn is involved in gene expression in numerous ways
including DNA replication, RNA transcription, through the activity of transcription
factors, DNA and RNA polymerases and playing a role in programmed cell death

(9). Metallothionein (MT), a Zn binding protein, exerts a protective effect against

134

stress by acting as an antioxidant, a Zn storage protein and a metal transfer
protein (10). Transcriptional regulation of the MT gene by dietary Zn has been
demonstrated in rats (11).

Recently, we reported that feeding newly weaned pigs phytase (500
FTU/kg) and pharmacological Zn (1000 - 2000 mglkg) increased MT mRNA
abundance and protein concentrations in the liver, kidney and intestinal mucosa
(12). Other genes may be affected by this dietary intervention. Therefore, the
objective of this experiment was to determine the identity of genes that are
differentially expressed in the liver of pigs fed a pharmacological Zn diet with or

without phytase supplementation for 14 d post-weaning.

135

MATERIALS AND METHODS

Animals and diets. Animal description, allotment and diet composition
have been previously described (12). The dietary treatments fed to pigs for 14 d
after weaning were: 1) adequate Zn diet containing 150 mg Zn/kg (Zn15o), 2)
2mm plus 500 FTU/kg (Zn150P), 3) pharmacological Zn diet containing 1000 mg
ZnIkg (anoo), 4) Zn1ooo plus 500 FTU/kg (anooP), 5) pharmacological Zn diet
containing 2000 mg ZnIkg (202000). or 6) 202000 plus 500 FTU/kg (anoooP). Pigs
were provided feed and water ad libitum. This project was approved by the
Michigan State University All University Committee on Animal Use and Care
(12/99-1 59-00).

Sample collection and total RNA isolation. Details about euthanasia,
liver sample collection methods and total RNA isolation were previously
published (12). RNA concentrations were determined with the RNA 6000 Pico
LabChip® kit using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto,
CA). Additionally, RNA quality and integrity were determined by calculating the
Ammo ratio and by agarose gel electrophoresis, respectively.

Differential display reverse transcription polymerase chain reaction.
Using modifications (13) of Liang and Pardee (14), genomic DNA contamination
was minimized by treating 1 pg of total RNA with 1 U of amplification grade
DNAse I, (lnvitrogen, Life Technologies, Carlsbad, CA) according to the
manufacturer’s protocol. Individual DNAase treated RNA samples (200 ng) from
animals fed Zn150, anoo, Zn.oooP and anooo (n = 4 per treatment), were reverse

transcribed by using a common anchor primer (5'-d(T).2AC-3') and RT-mix [1X

136

Buffer, 25 uM dNTP’s, 10 mM D‘l'l' and 40 U of Superscript II (lnvitrogen, Life
Technologies, Carlsbad, CA)]. Samples were incubated at 40°C for 5 min, 50°C
for 50 min, and 70°C for 15 min in a Peltier Thermal Cycler (PTO-200, MJ
Research, Waltham, MA). A final temperature drop to 4°C stopped the reaction.

Oligonucleotide primers for DDRT—PCR were obtained from the US. Pig
Genome Coordination Program (http://www.genome.iastate.edu/ resources]
ddprimer.html), and were randomly selected (Table 1). Using one anchor primer
paired with six arbitrary primers on 16 cDNA samples, a total of 96 PCR
reactions were generated and ~ 2% of all mRNA species present were screened.
PCR reactions were performed using cDNA (400 ng) in a solution containing 0.2
pM 3’- anchor primer, 20 uM of each dNTP, 1.5 mM MgCIz, 1X MgClz - free PCR
buffer, 0.2 uM 5' - arbitrary primer, 2.5 uCi [0-33P] dATP (Perkin Elmer, Life
Sciences Inc., Boston, MA) and 0.5 U Taq DNA Polymerase (Promega, Madison,
WI). PCR cycling parameters were: 95°C for 2 min, 4 cycles at 92°C for 15 s,
50°C for 30 s, and 72°C for 2 min, followed by an additional 25 cycles with
annealing at 60°C for 30 s and extension at 72°C for 2 min.

Denaturing loading dye (formamide, bromophenol blue and xylene cyanol)
was mixed with each DDRT-PCR sample (8 pl), and samples were dried on
medium heat for 5 min using a speed-vacuum (Savant Instruments Inc.,
Farrningdale, NY) followed by a 3 min 95°C denaturing step. The samples were
electrophoresed on 0.4 mm 5.2% polyacrylamide denaturing gels. Following a
5-6 hr run at 60 W on a vertical gelbox (Model 82, lnvitrogen, Life Technologies /

GIBCO BRL Sequence Systems, Carlsbad, CA), gels were transferred onto

137

Whatman chromatography paper (Whatman®, Maidstone, England) and dried at
80°C for 30 min using a slab dryer (SGD 2000, Savant). Gels were exposed to
BiomaxTM film (Eastman Kodak Co., Rochester, NY) overnight.

Excision and re-amplification of DDRT-PCR products. Radiographs
were developed and the selected bands were circumscribed and rehydrated in
100 pl DEPC treated water and heated at 50°C for 30 min. Re-amplification
reactions included 2 pl of gel band eluate and the anchor and arbitrary primers
used in the DDRT-PCR step under the same reaction conditions and cycling
parameters described above, excluding the isotope. To assess quality of the
reactions, 1 and 2 pl of re-amplification products were electrophoresed with a A
Hind lll marker (lnvitrogen, Life Technologies, Carlsbad, CA) in 1% agarose gels
stained with ethidium bromide (Sigma-Aldrich, St. Louis, MO).

Cloning and sequencing of DDRT-PCR products. Products of interest
obtained from the DDRT-PCR gels were cloned into pGEM-T-Easy Vector-
System I (Promega, Madison, WI). Recombinant vectors were transformed into
E. coli DH5a competent cells (lnvitrogen, Life Technologies, Carlsbad, CA).
Plasmid DNA was purified with the QlAprep Spin Miniprep kit (Qiagen, Valencia,
CA) and digested with EcoR l to confirm the presence of an insert. Sequencing
was performed using SP6 or M13 forward primers (Michigan State University
Genomics Technology Support Facility). Putative identification of the amplicons
was determined using the basic local alignment search tool (BLASTn) software,
with the non-redundant and EST databases of GenBank and The Institute for

Genomic Research (T IGR) database.

138

Independent confirmation by relative real-time PCR. Transcript levels
for 5 genes from 23 liver samples were compared. The hepatic RNA sample for
one pig fed Zn15oP was of poor quality, and was excluded from the analysis. The
genes were aminoacylase-1 (ACY1), glyoxalase l (GLO1), peroxiredoxin 4
(PRDX4), orosomucoid 1 (ORM1), carboxipeptidase U (CPBZ) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the
normalizing gene (Table 2). Relative real-time PCR primers were designed
using Primer Express v. 2.0 (Applied Biosystems, Foster City, CA), and the
assays were performed on an ABI Prism 7000 Sequence Detection System
(Applied Biosystems, Michigan State University Center for Animal Functional
Genomics).

First strand cDNA synthesis was performed with an oligo (dT)... primer
using SuperScript |l RNase H (lnvitrogen, Life Technologies, Carlsbad, CA),
following the manufacturers protocol. The cDNA was purified with QuickClean
resin (BD Biosciences Clontech, Palo Alto, CA) followed by precipitation with
sodium acetate and ethanol. Purified cDNAs were suspended in DNase/RNase-
free sterile water, and quantified using a spectrophotometer. The cDNA samples
were diluted to a final concentration of 10 ng/pl, and stored at -20°C until use.
The real time PCR reactions included 50 ng of cDNA, 300 pM primer, and 1X
SYBR Green Master Mix (Applied Biosystems, Foster City, CA).

Relative quantification was determined using duplicate cDNA samples
from each animal. PCR amplification efficiency plots were generated using

serially diluted cDNA (4 dilutions) to confirm the use of GAPDH as the

139

normalizing gene. Results were recorded relative to a common liver cDNA
sample from a pig fed Zn15o after normalizing for GAPDH. Relative gene
expression changes were then computed using the 2'“Ct method (15).

Independent confirmation by northern blot hybridization. When PCR
amplification plots did not reveal equal amplification efficiency for GAPDH with
the target gene, confirmation of DDRT-PCR results was done by northern blot
analysis using the cDNA clones obtained by DDRT-PCR, labeled as described
previously (12) with 188 rRNA for normalization.

Statistical analysis. The data were analyzed using analysis of variance
based on the MIXED procedure of SAS (16). If Zn, phytase (main effects) or
their interaction, were not statistically significant, the datasets were merged. For
the relative real time PCR analyses, the model included pig nested within the
treatment interaction with the plate as random effect. Satterthwaite’s
approximation was used to determine the error df for test (17). A base 2
logarithmic transformation (Log) of the target and normalization gene Cts was
made to account for the exponential amplification of cDNA prior to analyzing Ct
values with the MIXED procedure of SAS. The transformed target Cts and
GAPDH Cts were modeled as covariates and used in a regression-based
normalization of Cts in accordance with recommendations by Poehlman (18).
These values were compared to the results calculated by using the 2'AACt method
(15), and data are presented as fold changes.

For northern blot analyses, the blot by treatment interaction was used in

the model with pig as a random effect. The 18S rRNA values were modeled as

140

covariates for a regression-based normalization of the genes’ relative mRNA

abundance. Differences were considered to be significant when P < 0.05.

141

RESULTS

Identification of expressed sequences displayed by DDRT-PCR. A
total of 56 putatively differentially expressed amplicons were cloned and
sequenced. High quality nucleotide sequence data was obtained for 52 of these
ESTs and submitted to GenBank (accession numbers CF 1 06636 - CF 1 06687).
A FASTA search of the GenBank and TIGR databases revealed that 24 of the
ESTs (46%) had significant similarities to genes with known identities, 7 were
similar to mitochondrial DNA (13%), and the remainder had unknown identities
(40%). Identified genes were similar to genes involved in protein and amino acid
metabolism, oxidative stress response, regulation of transcription, and
membrane transport (Table 3). From the dietary treatment groups on the DDRT-
PCR gels (Table 3), 5 genes were selected for subsequent mRNA abundance
analyses.

Confirmation of differential expression as afiected by dietary
treatment. From the display gels, the five selected genes exhibited increased
relative mRNA abundance in the liver of pigs fed pharmacological Zn diets
(anooo, anooo) without phytase. To confirm differential expression, a
combination of relative real time PCR and northern blot analyses was utilized.

Two oxidative stress response genes involved in the reduction of
peroxides [peroxiredoxin 4 (PRDX4)], and in detoxification of glycating agents
[glyoxalase l (GLO1)] showed differential expression. Northern blot analysis of
PRDX4 revealed a single transcript of ~0.95 kb (Figure 1). Relative abundance

of PRDX4 mRNA (Figure 2) in liver of pigs fed pharmacological Zn diets was 2

142

fold higher in pigs fed anoo, and 4 fold higher in pigs fed 202000 when compared
with pigs fed Zn150 (P < 0.009). PCR amplification efficiency plots revealed that
GAPDH was not a suitable normalizing gene for GLO1, thus northern blot
hybridization was performed. A single transcript (~ 1.9 kb) was obtained for
GLO1 (Figure 1). Relative GLO1 mRNA abundance was greater (P < 0.0007) in
pigs fed pharmacological Zn diets (Zn1ooo and anooo) when compared with pigs
fed Zn150 (Figure 3). Supplemental phytase did not affect the mRNA abundance
of PRDX4 or GLO1.

N-aminoacylase l (ACY1), which is a gene involved in amino acid
metabolism, also displayed differential expression. Northern blot analysis
revealed the presence of a single transcript (~ 1.3 kb, Figure 1), and that relative
ACY1 mRNA abundance increased (P < 0.01) in pigs fed pharmacological Zn
(anoo and anooo). No phytase effect was observed (Figure 4).

ORM1 and CP82 were not confirmed to have differential expression in

pigs fed any of the dietary treatments using real time PCR (data not shown).

143

DISCUSSION

Differential display reverse transcription PCR is a technique that allows
the simultaneous comparison of multiple treatments, the identification of
differentially regulated mRNAs, and generation of ESTs which can be compared
with DNA sequences in the databases (14,19).

The PRDX (EC 1.11.1.-) family is the most recently identified oxidative
stress enzyme to be characterized. It encodes for a ubiquitous cytosolic enzyme,
which reduces hydrogen peroxide via redox-active cysteine residues. In addition
to hydrogen peroxides, PRDXs can also regulate peroxide-mediated signaling
cascades, and their overexpression reduces hydrogen peroxides in response to
tumor necrosis factor a (TNF-a). In this study, a single transcript for PRDX4
was revealed, which agrees with the size reported for human PRDX4 (20).

Peroxiredoxin 4 overexpression in mice suppresses Nf-KB activation
through regulation of I-ch phosphorylation (20). Additionally, it has been
reported that Zn supplementation (1000 mg ZnIkg) to streptozotozin treated mice
inhibits Nf-KB activation, thus providing protection against oxidative stress and
inflammatory responses (21). Similarly, MT has also been implicated in
inhibiting TNF-induced activation of Nf-KB DNA binding, by inhibition of lKB
degradation and subsequent NF-xB suppression (22). Kim et al. (23) suggest
that MT modulates intracellular signaling molecules such as transcription factors
(Nf-KB) by sequestering intracellular Zn, which is required for its DNA binding

activity. Peroxiredoxins are considered redox regulators of signal transduction

144

(20), acting like switches to control intracellular pathways. Perhaps the increase
in intracellular Zn causes an alteration in cell signaling (24), and PRDX4
upregulation occurs as a result. The suppressive effect by PRDX4 on Nf-KB may
be a mechanism involved in pharmacological Zn’s enhancement of health in the
newly weaned pig.

Glyoxalase 1 (EC 4.4.1.5) is a cytosolic ubiquitous Zn metalloenzyme that
catalyzes the glutathione dependent conversion of glycating agents such as
methylglyoxal to S-D—lactoylglutathione via a 1.2-hydrogen transfer (25). This
enzyme works in conjunction with glyoxalase II to further convert S-D-
lactoylglutathione to D-lactate (26). Methylglyoxal can react with arginine and
lysine residues in proteins, and at high concentrations it inhibits glycolytic
enzymes (27), enhances lipid peroxidation, and binds DNA and RNA causing
macromolecular damage (28). Genomic analysis of the GLO1 gene revealed the
presence of functional metal response elements (MREs) located 647 bp
downstream of the transcription initiation site (27). Another gene containing
MREs in its promoter region is the MT gene (29). The MREs control
transcriptional activation of MT upon exposure to Zn, through the action of metal
transcription factor-1 (30). Transcriptional activity of GLO1 in transfected HepG2
cells was elevated twofold upon exposure to 25 and 75 pM ZnClz for 48 hr(27).
We have previously shown that MT mRNA abundance and protein concentration
are increased in liver, kidney and intestinal mucosa of pigs fed pharmacological

Zn (12), similar to the liver expression patterns observed for PRDX4 and GLO1 in

145

this study. The present study showed a single GLO1 transcript, which was in
accordance with the size of human GLO1 (27).

Zinc involvement in oxidative stress is well known. Its deficiency causes
oxidative DNA damage by impairment of antioxidant defense, and compromises
DNA repair mechanisms (31). Pharmacological Zn supplementation (3000 mg
ZnIkg) to early weaned pigs for 21 d post-weaning has been shown to reduce red
blood cell superoxide dismutase (EC 1.15.1.1) activity compared with pigs fed
150 mg ZnIkg (6). However, the mechanism of how Zn exerts its antioxidant
action is not well defined. It has been suggested that MT induction, and the
ability of Zn to occupy iron and copper binding sites on proteins and DNA, may
contribute toward its antioxidant action (32). The present study shows that
pharmacological Zn supplementation is associated with increased mRNA
abundance of PRDX4 and GLO1, genes that participate in the oxidative stress
response through differing mechanisms. Our results for hepatic GLO1
expression are in agreement with observations in GLO1 transfected HepG2 cells
(27). Perhaps Zn transcriptionally regulates GLO1 through MRE activation, in a
similar fashion to the MT gene, therefore offering protection against glycating
agents. To our knowledge, we provide the first evidence that pharmacological Zn
in the diet increases PRDX4 mRNA abundance.

Of the three additional genes selected for confirmation: 1) ACY1, encodes
for a Zn metalloenzyme involved in peptide metabolism, 2) ORM1, encodes for
an acute phase reactant protein, and 3) CPB2, encodes a plasma

carboxypeptidase. N-acyl-L-amino-acid aminohydrolase (EC 3.5.1.14) is a

146

cytosolic enzyme with greatest abundance in pig kidney versus the liver (33,34).
The biological role of ACY1 is to hydrolyze neutral and hydrophobic ci-N-acyl-L-
amino acids generated during protein degradation (35). This Zn dependent
enzyme has been identified in mammals, primarily in kidney and liver (34), and
contains a single Zn atom per subunit; it is proposed to have catalytic roles (36).
However, Heese et al. (37) suggested that Zn plays a structural role, because the
Zn binding site was too far from the catalytic site of the enzyme. In the present
study, the presence of a single ACY1 transcript was revealed, which is in
accordance with the size reported for pig ACY1 by Mitta et al. (33). Furthermore,
we confirmed that pharmacological Zn supplementation increased the relative
abundance of ACY1 mRNA. No differences were observed on the abundance of
this transcript between animals fed anoo and 202000. suggesting that at dietary
concentrations greater than 1000 mg ZnIkg, relative ACY1 abundance reaches a
plateau. To our knowledge, this study provides the first evidence of Zn effects on
the relative ACY1 mRNA abundance.

Orosomucoid 1 and carboxypeptidase U are genes that encode an acute
phase reactant protein known as a—1-acid glycoprotein, and plasma pro-
carboxypeptidase (EC 3.4.17.20), respectively. These plasma proteins are
secreted into the blood after being synthesized in the liver (38,39). Serum
concentrations of ORM1 are high in fetal (40) and neonatal pigs, decreasing
markedly to a constant level by 2 wk of age (41) and declining further by 112 d.
In a similar pattern, liver ORM1 mRNA is relatively abundant in late fetal and

neonatal pigs then declines rapidly after birth (42). Alpha-1-acid glycoprotein

147

plasma concentration rises during inflammation, thus its function is related to
modulation of an inflammatory response primarily caused by interleukin 1,
interleukin 6 and glucocorticoids (43). Sequence analysis of the murine ORM1
gene revealed the presence of four MRE sequences in the 5’ flanking region, and
one in intron 5 (44). Furthermore, relative liver ORM1 mRNA abundance in mice
injected with 0.5 mg Hg/kg (i.p.) was increased in a time dependent manner.
Zinc was also used to investigate ORM1 mRNA response upon heavy metal
exposure, but its effect on ORM1 mRNA was not as marked as Hg (45). The
authors concluded that Hg regulates ORM1 at the transcriptional level. In our
study, we did not observe a significant effect of dietary Zn on liver ORM1 mRNA
abundance. Additional studies on ORM1 protein indicated that 2000 mg ZnIkg
diet or the antibiotic, Tylan® (88 g/kg), fed to newly weaned pigs for 14 d did not
affect ORM1 plasma protein concentrations (46).

Plasma CP82 was reported in 1988 as a carboxypeptidase involved in
blood clotting (47). Several names have been used for this enzyme according to
its general catalytic mechanism, including carboxypeptidase U (U = unstable),
carboxypeptidase R (R = cleaves Arg and Lys residues in C-terrnini), plasma
carboxypeptidase B (B = basic) and most recently Thrombin-Activatable
Fibrinolysis Inhibitor (TAFI) (48). The activity of CPBZ has been shown to be
affected by the availability of Zn, as demonstrated by the loss of activity with a Zn
chelating agent, 1,10-phenantroline (49). The presence of CPBZ has been
demonstrated in the pig, rabbit and mouse, with activity ranging from 20%

(mouse) to 500% (pig) compared to human serum CP82 (50).

148

Our display gels indicate that pharmacological Zn increases the mRNA
abundance of ORM1 and CPBZ in pig liver. However, relative real time PCR
results did not confirm their differential expression. This may be due to the low
number of pigs in our treatments (n = 4) or the increased sensitivity of the real
time PCR assays. Future research should be conducted to evaluate additional
genes affected by the dietary treatments and to confirm the roles of these genes

at the protein level.

149

ACKNOWLEDGEMENTS
The authors express their appreciation to Dr. Max Rothschild, U.S. Swine
Genome Coordinator, for distribution of the DDRT-PCR oligonucleotide primers,
Dr. Robert J. Tempelman for assisting with the statistical analysis, and the staff
of the Michigan State University Center for Animal Functional Genomics for

technical assistance.

150

LITERATURE CITED

1. Marion, J., Rome, V., Savary, G., Thomas, F., Le Dividich, J. & Le
Huerou Luron, l. (2003) Weaning and feed intake alter pancreatic enzyme
activities and corresponding mRNA levels in 7-d-old piglets. J. Nutr. 133: 362-
368.

2. Kelley, K. W. (1980) Stress and immune function: a bibliographic
review. Ann. Rech. Vet. 11: 445-478.

3. Pollmann, D. S. (1993) Effects of nursery feeding programs on
subsequent grower-finisher pig performance. In: Proceedings of the Fourteenth
Western Nutrition Conference (J. Martin), pp. 243-254. Edmonton, Canada.

4. McCully, G. A., Hill, G. M., Link, J. E., Weavers, R. L., Carlson, M. S. 8.
Rozeboom, D. W. (1995) Evaluation of zinc sources for the newly weaned pig. J.
Anim. Sci. 73: 72 (abs).

5. Hill, G. M., Cromwell, G. L., Crenshaw, T. D., Dove, C. R., Ewan, R. C.,
Knabe, D. A., Lewis, A. J., Libal, G. W., Mahan, D. C., Shurson, G. 0., Southern,
L. L. & Veum, T. L. (2000) Growth promotion effects and plasma changes from
feeding high dietary concentrations of zinc and copper to weanling pigs (regional
study). J. Anim. Sci. 78: 1010-1016.

6. Carlson, M. S., Hill, G. M. & Link, J. E. (1999) Early— and traditionally
weaned nursery pigs benefit from phase-feeding pharmacological concentrations
of zinc oxide: effect on metallothionein and mineral concentrations. J. Anim. Sci.
77: 1199-1207.

7. Junghans, P., Kaehne, T., Beyer, M., Metges, C. C. & Schwerin, M.
(2004) Dietary protein—related changes in hepatic transcription correspond to
modifications in hepatic protein expression in growing pigs. J. Nutr. 134: 43-47.

8. Hesketh, J. E., Vasconcelos, M. H. & Berrnano, G. (1998) Regulatory
signals in messenger RNA: determinants of nutrient-gene interaction and
metabolic compartmentation. Br. J. Nutr. 80: 307-321.

151

9. Falchuk, K. H. (1998) The molecular basis for the role of zinc in
developmental biology. Mol. Cell. Biochem. 188: 41-48.

10. Kagi, R. H. J. (1993) Evolution, structure and chemical activity of class
I metallothioneins: overview. In: Metallothioneins lll (K.T. Suzuki, N. lmura, 8. M.
Kimura), pp. 29-56. Birkhauser Verlag, Basel, Switzerland.

11. Richards, M. P. & Cousins, R. J. (1975) Mammalian zinc homeostasis
requirement for RNA and metallothionein synthesis. Biochem. Biophys. Res.
Comm. 64: 1215-1223.

12. Martinez, M. M., Hill, G. M., Link, J. E., Raney, N. E., Tempelman, R.
J. & Ernst, C. W. (2004) Pharmacological zinc and phytase supplementation
enhance metallothionein mRNA abundance and protein concentration in newly
weaned pigs. J. Nutr. 134: 538-544.

13. Wesolowski, S. R., Raney, N. E. 8. Ernst, C. W. (2004) Developmental
changes in the fetal pig transcriptome. Physiol. Genomics 16: 268-274.

14. Liang, P. 8. Pardee, A. B. (1992) Differential display of eukaryotic
messenger RNA by means of the polymerase chain reaction. Science 257: 967-
971.

15. Livak, K. J. 8. Schmittgen, T. D. (2001) Analysis of relative gene
expression data using real-time quantitative PCR and the 2-[Delta][Delta]CT
method. Methods 25: 402-408.

16. SAS Systems (1999-2000) The SAS System for Windows 98, 8.1 ed.
SAS Institute, Inc., Cary, NC.

17. Satterthwaite, F. E. (1946) An approximate distribution of estimates of
variance components. Biom. Bull. 2: 110-114.

18. Poehlman, E. T. (2003) Reduced metabolic rate after caloric
restriction-can we agree on how to normalize the data? J. Clin. Endocrinol.
Metab. 88: 14-15.

152

19. Cousins, R. J., Blanchard, R. K., Moore, J. B., Cui, L., Green, C. L.,
Liuzzi, J. R, Cao, J. 8. Bobo, J. A. (2003) Regulation of zinc metabolism and
genomic outcomes. J. Nutr. 133: 15218-1526.

20. Jin, D. Y., Chae, H. 2., Rhee, S. G. & Jeang, K. T. (1997) Regulatory
role for a novel human thioredoxin peroxidase in Nf-KB activation. J. Biol. Chem.
272: 30952-30961.

21. Ho, E., Quan, N., Tsai, Y. H., Lai, W. 8. Bray, T. M. (2000) Dietary zinc
supplementation inhibits NF-KB activation and protects against chemically
induced diabetes in CD1 mice. Exp. Biol. Med. 226: 103-111.

22. Sakurai, A., Hara, 8., Okano, N., Kondo, Y., Inoue, J.-i. 8. lmura, N.
(1999) Regulatory role of metallothionein in NF-[kappa]B activation. F EBS
Letters 455: 55-58.

23. Kim, C. H., Kim, J. H., Lee, J. & Ahn, Y. S. (2003) Zinc-induced NF-KB
inhibition can be modulated by changes in the intracellular metallothionein level.
Toxicol. Appl. Pharmacol. 190: 189-196.

24. Watjen, W., Benters, J., Haase, H., Schwede, F., Jastorff, B. 8.
Beyersmann, D. (2001) Zn2+ and Cd2+ increase the cyclic GMP level in PC12
cells by inhibition of the cyclic nucleotide phosphodiesterase. Toxicology 157:
167-175.

25. Creighton, D. J. & Hamilton, D. S. (2001) Brief history of glyoxalase l
and what we have learned about metal ion—dependent, enzyme catalyzed
isomerizations. Arch. Biochem. Biophys. 387: 1-10.

26. Creighton, D. J., Migliorini, M., Pourmotabbed, T. 8. Guha, M. K.
(1988) Optimization of efficiency in the glyoxalase pathway. Biochemistry 20:
7376-7384.

27. Ranganathan, S., Ciaccio, P. J., Walsh, E. S. 8 Tew, K. D. (1999)
Genomic sequence of human glyoxalase-l: analysis of promoter activity and its
regulation. Gene 240: 149-155.

153

28. Brambilla, G., Sciaba, L., Faggin, P., Finollo, R., Bassi, A. M., Ferro,
M. 8. Marinari, U. M. (1985) Methylglyoxal-induced DNA-protein cross-links and
cytotoxicity in Chinese hamster ovary cells. Carcinogenesis 6: 683-686.

29. Stuart, G. W., Searle, P. F., Cen, H. Y., Brinster, R. L. & Palmiter, R.
D. (1984) A 12-base-pair DNA motif that is repeated several times in
metallothionein gene promoters confers metal regulation to a heterologous gene.
Proc. Natl. Acad. Sci. USA 81: 7318.

30. Radtke, F., Heuchel, R., Georgiev, O., Hergersberg, M., Gariglio, M.,
Dembic, Z. & Schaffner, W. (1993) Cloned transcription factor MTF-1 activates
the mouse metallothionein I promoter. EMBO J. 12: 1355-1362.

31 . Ho, E. 8. Ames, B. N. (2002) Low intracellular zinc induces oxidative
DNA damage, disrupts p53, NFkappaB and AP1 binding and affects DNA repair
in a rat glioma cell line. Proc. Natl. Acad. Sci. U S A 99: 16770-16775.

32. Har-el, R. & Chevion, M. (1990) Zinc (II) protects against meta-
mediated free radical induced damage: studies on single and double-strand DNA
breakage. Free. Radic. Res. Commun. 12-13: 509-515.

33. Mitta, M., Ohnogi, H., Yamamoto, A., Kato, l., Sakiyama, F. &
Tsunasawa, S. (1992) The primary structure of porcine aminoacylase 1 deduced
from cDNA sequence. J. Biochem. 112: 737-742.

34. Lindner, H., Hopfner, S., Tafler-Naumann, M., Miko, M., Konrad, L. &
Rohm, K. H. (2000) The distribution of aminoacylase l among mammalian
species and localization of the enzyme in porcine kidney. Biochimie 82: 129-137.

35. Giardina, T., Biagini, A., Dalle Ore, F., Ferre, E., Reynier, M. 8.
Puigserver, A. (1997) The hog intestinal mucosa acylase I: Subcellular
localization, isolation, kinetic studies and biological function. Biochimie 79: 265-
273.

36. Loffler, H. G., Rohm, K. H. & Schneider, F. (1986) Studies on metal
ion dependence, kinetics and SH (S-S) groups of acylaminoacid aminohydrolase.

154

In: Zinc Enzymes (In I. Bertini, C. Luchinat, W. Maret, 8. M. Zeppezauer), pp.
281-288. Birkhauser, Boston, MA.

37. Heese, D., Berger, S. 8. Rohm, K. H. (1990) Nuclear magnetic
relaxation studies of the role of the metal ion in Mn‘Z-substituted aminoacylase l.
Eur. J. Biochem. 188: 175-180.

38. Dente, L. (1989) Human alpha-1-acid glycoprotein genes. Prog. Clin.
Biol. Res. 300: 85-98.

39. Tan, A. K. & Eaton, D. L. (1995) Activation and characterization of
procarboxypeptidase B from human plasma. Biochemistry 34: 5811-5816.

40. Lampreave, F. & Pineiro, A. (1984) The major serum protein of fetal
and newborn pigs: biochemical properties and identification as a fetal form of
alpha 1-acid glycoprotein. Int. J. Biochem. 16:47-53.

41. Son, D. S., Shimoda, M. 8 Kokve, E. (1996) Implication of altered
levels of plasma alpha (1)-acid glycoprotein and its derived sialic acid on plasma
protein binding of trimethoprim in pigs in physiological and pathological states.
Vet. Q. 18: 10-13.

42. Stone, R. T. & Maurer, R. A. (1987) Cloning and developmental
regulation of alpha 1 acid glycoprotein in swine. Dev. Genet. 8: 295-304.

43. Mackiewicz, A., Dewey, M. J., Berger, F. G. 8. Baumann, H. (1991)

Acute phase mediated change in glycosylation of rat a1-acid glycoprotein in
transgenic mice. Glycobiology 1: 265-269.

44. Cooper, R., Eckley, D. M. & Papaconstantinou, J. (1987) Nucleotide
sequence of the mouse alpha 1-acid glycoprotein gene 1. Biochemistry 26: 5244-
5250.

45. Yiangou, M., Ge, X., Carter, K. & Papaconstantinou, J. (1991)
Induction of several actute-phase protein genes by heavy metals: a new class of
metal-responsive genes. Biochemistry 30: 3798-3806.

155

46. Green, J. G., Hill, G. M., Link, J. E., Martinez, M. M., Dvoracek-
Driksna, D. M., Raney, N. E. & Ernst, C. W. (2003) Comparison of tylosin and
pharmacological zinc on acute phase reactant proteins and minerals. J. Anim.
Sci. 81: 112 (abs).

47. Hendriks, D., Scharpé, S., van Sande, M. & Lommaert, M. P. (1989)
Characterization of a carboxypeptidase in human serum distinct from
carboxypeptidase N. J. Clin. Chem. Clin. Biochem. 27: 277-285.

48. Bouma, B. N., Marx, P. F., Mosnier, L. O. 8. Meijers, J. C. M. (2001)
Thrombin-Activatable Fibrinolysis Inhibitor (TAFI, Plasma Procarboxypeptidase
B, Procarboxypeptidase R, Procarboxypeptidase U). Thromb. Res. 101: 329-354.

49. Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W. 8. Drayna, D. (1991)
Isolation, molecular cloning, and partial characterization of a novel
carboxypeptidase B from human plasma. J. Biol. Chem. 266: 21833-21838.

50. Schatteman, K. A., Goossens, F. J., Scharpe, S. S. 8. Hendriks, D.
(1999) Activation of plasma carboxypeptidase U in different mammalian species
points to a conserved pathway of inhibition of fibrinolysis. Thromb. Haemost. 82:
1718-1721.

156

TABLE 1
Anchor and arbitrary primers used in this experiment1

 

 

No. Primer sequence
Anchor # 9 d(T)12 AC 5'-l I I I I I I I I I ITAC-3'
Arbitrary # 4 5'-GCTAGCAGAC-3'
Arbitrary # 7 5'-TGGATTGGTC-3'
Arbitrary # 9 5'-TAAGCCTAGC-3'
Arbitrary # 13 5'-GTTGCACCAT-3'
Arbitrary # 16 5'-TCGGTCATAG-3'
Arbitrary # 20 5'-TCGATACAGG-3'

 

1 Source: http://www.genome.iastate.edu/resources/ddprimer.html

157

Soomémm: oN .> $290 LoEtn. mafia cocgmou 2o; QoEtn:

oflm>mm Ian—<0
BEECH. Iom<0

.m-._r_. ._.<0 000 00._. <00 <00-.m
.m-._.0 ._.<0 0<0 ._.<0 000 <<< 00.7%

 

.m-._.0 ._.0< 9.0 E 0._.< 00... 0t. 0<0 <0<..m omeo>om 38mm
.mFO ._.<0 50 00._. 0.5 00... 00< 0._.<..m 282cc“. 38mm
.m-<0< 0<0 000 000 <00-.m oweo>om EEO

.m-< 00... <<0 000 <00 ._.0< 0t-.m 2920.... Sam—O

.mfiOh 0<0 E <<< ._.0< 0h< <00 ...00 ......0-.m omeo>om «moo
.m-._. ...00 h<< <0< 0H< 0._.0 H<0 00.7% 295.0“. «ma—U
cocoa—coo 3.55 22.025 0:00

 

pecan—Ease mundane Lo.— to»: 225.5 mon— oEz .3. no a»:

N mr.m<._.

158

.80000000 Any oESmQEoo
.832 2 Z .852 5: .8 E85 9: s 8238 Egan. .8853. ”0825".” 50258 5253 .28 2000.200 5 358° 8523... 8528 so 550 m
020:. 208.2090 60E .6. 23E? 0280000 0.000000 60.000?
0009200 .2 08: 5&5 2.55 22985 .35: 50 5 205 oz 2.2:.U
0N ,> 903:9. ._.w<._m 05 05m: 30m .3 >05. 09000: o.m.> x005 0000 £060 mam... moi 0. gmnEoo 063 0:02.022. «
$5009 Esocxc: 52> 3:00 9 nocoumE u :02 .voom :9 >05. mdN .> 903:8 ...wfim 05 05m: 30030.... 0.00960 0. .020an0 903 mcoo__aE<

 

 

     

 

T048 9818 83.805 .228 .m <20 35552.2 2.08:6 B« 58
8an 88:8 «8325 £28 .0 <20 35828:: 0.88:6 $80 2.9
.841 8838 «8325 .298 .m <20 3552822 508:6 A88 :2?
T38 8828 «8325 £98 .0 <20 355525 8896 an: 88
F883. 8838 08385 £28 .0 <20 E55525 {08:6 :80 «mm:
vumAé 945 Be 08385 .228 .m <20 3552822 8896 E: «:8
«88¢ 9398 8.0385 .298 .w <20 E58525 «886 soc EB
:23. 8899 082825 229..» .... 55>: 5290 05588 52:3 .mE__>> 68:6 85 «09
Xian 5839 0888422 \ «.508 .2 053.: seen 552.8 9.285853 9e < 8282.855-” 5 9.08:6 6««v E8
".880 8839 «5858422 \ 82005: .m 3560 m« 5220 58> 052.6 58:6 233 <8
72?”. 8839 E8802 \ mass .0 :5va h: 529.. .8580: 88:6 880 mm?
548 8890» 085.322 < .298 .m 8382 22% 255:. 953:0 88:6 .«80 «08
e884 €889 e. e 881.22 < .298 .m 30268 e 5220 8555 .9089 8923020 308Eo 680 ES
F848 .5838 005005 < 858 .0 803 5552.8 .88:6 28$ 58
F083. 8839 0880-22 < 22%... .... 2862.5 5655.98 8896 as; :9
Earn K850» 0<F<0§ < 5.8 .0 SEE F 285820 8826 2030 2.8
:23. 5860 0<e<0a \ £28 .0 3220. s 285820 808:6 283 53
Fauna». 8839 $22005 «.98 .w C.>o$ _ 82885552 088:6 28: E?
$3.8 «986» «888-22 \ 2203 .: $<0§ m. - 522.. 8.588 55555 «55.0.2 808Eo :30 «5.
e058 8899 38:80 < 22%... .2 3.550 22882 55.. 88:6 28$ «09
21.83 0885» $08800 \ e553 .: 28.05 92% e525 8.08:6 $8. 5....
T248 @8835 «$82.22 < 853 .m 202105220820 :22 852... «88:6 80$ 2.2
$23. 885» 505600 \ ”5:59 .0 $35: 5220. 85:5 05 28:6 :80 68
XXX. 9820+ F. 32800 < 82%... .... €0.60 _ 08.9620 808Eo at; no?
4“??? 8838 08803.5 maze .0 8035 m 888.285.: 9.25:8 as»... 308:6 $80 88
«AWE. 8828 0520.20: \ 22%.. .: 300$ 50 __ 35528 <20 508:6 89 «mo«
Puma... 8298 099.55: < 8.2%... .... a": x. Lose 555.80 888:6 is «03
T38 88... 5e «,«Beoorzz \ 22%.. .x @018 0 8558550 508:6 880 3.8
F80... 3888 0808on < 2.208 .: 5:00 ... .28... 5558...; 280 «88:6 28$ 69
.048 85:80 «82800 3523 .2 :52: v 88.95; 8&2 525 «00.8 0.85. 282 852.2 808Eo cm: «29
T«Aflm 8888 «88800 < 22%.. .: 251$ e 80205; 8&2 5205 «00.8 0.85 same .2922 488:6 am: ES
"00:00:30 Eton—=5 .6025: 0... ..02 00.00003. < 020on

5 E350 a... 6m
«..:oaanuE

159

   
 

Z"1000 zn1oooP Err‘znzooo ZI‘lzoooP
‘ ‘ GLO1

.§.. ~1.9kb
.. . ACY1

... .‘ 1‘ fiihqm
PRDX4

."N‘ “0 tit-~0.95...

1 85
~1.8 kb

 

Figure 1. Northern blot analysis of liver GLO1, ACY1 and PRDX4 mRNA
of pigs fed 150, 1000 or 2000 mg Zn/kg (with or without phytase) for 14 d post-
weaning. Liver total RNA (12 pg) isolated from each pig was analyzed using the
cDNA clones derived from differential display gels as probes. Also shown is 18S

rRNA hybridization to assess equal loading of mRNA.

160

6.0

5.0

4.0

3.0

2.0

Relative PRDX4 mRNA Abundance
(Fold Change)

1.0

 

0.0
Zn15o 201000 202000

Figure 2. Relative PRDX4 mRNA abundance in the liver of pigs fed 150,
1000 or 2000 mg Zn/kg (with or without phytase) for 14 d post-weaning. Relative
real time PCR was performed and fold changes relative to GAPDH and a
common Zn150 reference sample are presented. Values are means i SEM, n=8,
(except Zn15o, n=7). A significant zinc effect was detected for relative PRDX4
mRNA abundance, P < 0.009. No Zn by phytase interaction, or phytase effect

were detected.

161

1.2

1.0

0.6 '

0.4 a

Relative GLO1 mRNA Abundance
(Arbitrary Units)

0.2

 

0.0 ‘
ZI'I150 ZI'I1ooo ZI'Izooo

Figure 3. Relative GLO1 mRNA abundance in the liver of pigs fed 150,
1000 or 2000 mg Zn/kg (with or without phytase) for 14 d post-weaning.
Northern blot analysis was performed and values are mean optical density
readings 1- SEM, n=8 (except Zn150, n=7). A significant zinc effect was detected
for relative hepatic GLO1 mRNA abundance, P < 0.0007. No Zn by phytase

interaction, or phytase effect were detected.

162

0.6 .

0.5

0.4

0.3

0.2 .

Relative ACY-1 mRNA Abundance
(Arbitrary Units)

 

0.0 -*

an ano zn2000

Figure 4. Relative ACY1 mRNA abundance in the liver of pigs fed 150,
1000 or 2000 mg ZnIkg (with or without phytase) for 14 d post-weaning.
Northern blot analysis was performed and values are mean optical density
readings i SEM, n=8 (except Zn150, n=7). A significant zinc effect was detected
for relative hepatic ACY1 mRNA abundance, P < 0.01. No Zn by phytase

interaction, or phytase effect were detected.

163

lES

l’ll'l'll'llllllllllllll'lllll’lll”