Development of metallothionein-based assays for the measurement of zinc status in humans

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Development of metallothionein-based assays for the measurement of zinc status in humans
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xi, 92 leaves : ill. ; 29 cm.
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Sullivan, Vicki K., 1961-
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Food Science and Human Nutrition thesis, Ph. D   ( lcsh )
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Thesis (Ph. D.)--University of Florida, 1997.
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Includes bibliographical references (leaves 81-91).
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by Vicki K. Sullivan.
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Typescript.
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Vita.

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DEVELOPMENT OF METALLOTHIONEIN-BASED ASSAYS FOR ASSESSMENT
OF ZINC STATUS IN HUMANS















By


VICKI K. SULLIVAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1997
























Copyright 1997

by

Vicki K. Sullivan

























To my parents and my husband.














ACKNOWLEDGMENTS

I thank my mentor Dr. Robert Cousins for allowing me to learn and accomplish

exactly what I had hoped to when I arrived here four years ago, for exposing me to a

laboratory that offers a vast array of techniques in basic and applied science, and for

providing an unlimited amount of his time. I also thank my committee members, Drs. Gail

Kauwell, Susan S. Percival, Rachel Shireman and Kuo-Jang Kao for their excellent

expertise and guidance throughout this experience. I also thank Dr. Cousins for

surrounding me with a group of talented and competent co-workers. I thank the following

people for their support: Barbara Davis, Christina Khoo, Lorraine Langerhan-Foster,

Steve Davis, and Karol Smith for sharing this experience with me; Melissa Tuason for her

help with the human study; Drs. Ray Blanchard, Nora Halloquist and Robert MacMahon

for their encouragement and expertise; Warren Clark for putting up with sharing a lab

bench with me, and Virginia Mauldin for always going the extra mile.

I thank both of my parents for their unconditional love, and for teaching me by

example the value of hard work, persistence, and honesty. I also thank them for always

listening. I thank my husband Shawn for his love and devotion, for agreeing to ride this

roller-coaster (graduate school) with me for so many years, and for staying on the ride

through all the ups and downs. I also thank my furry friends Spot, Chewy and Pumpkin,

for making me laugh when no one else could, and for reminding me that the simple things
in life are truly the most important.














TABLE OF CONTENTS

ACKNOW LEDGEM ENTS...................................................................................... iv

LIST OF FIGURES....................................................................................................... vii

ABBREVIATIONS........................................................................................................ xi

ABSTRACT................................................................. ................................................... x

CHAPTERS

1 INTRODUCTION ................................................................................................. 1



2 REVIEW OF THE LITERATURE ........................................................................5
Zinc................ ................................ ...... 5
Distribution and Functions.............................................................................. 5
Absorption and Transport............................................................................... 6
Sources, Bioavailability and Requirements...................................................... 7
Deficiency .......................................................... ....... ................................. 8
Toxicity ...................................................................................................... 9
Assessment of Zinc Status .............................................................. ................ 10
M etallothionein .................................................................................................. 11
M etallothionein for the M easurement of Zinc Status..................................... 13
Reverse Transcriptase Polymerase Chain Reaction ............................................. 13

3 M ATERIALS AND M ETHODS................................................................... 17
Refinement of Human M etallothionein ELISA ................................................... 17
Competitive ELISA ...................................................................................... 17
ELISA Plates ............................................................................................ 17
Source of M etallothionein ............................................................................ 17
Primary Antibody ..................................................................................... 19
Erythrocyte Lysate Preparation..................................................................... 23
Protein Quantitation ................................................................. .. .......... 24
Plasma Zinc and Copper............................................................................... 24
Sandwich ELISA ................................................................................ .......... 24
Development of Competitive Reverse Transcriptase Polymerase Chain
Reaction Assay............................................................................................... .... 26
Cell Culture..................................... ..................................................... ......... 25








Preliminary Hum an Study............................................................................. 26
Isolation of M onocytes.............................................................................. 26
Extraction and Purification of RN A .............................................................. 27
Reverse Transcriptase Polymerase Chain Reaction Assays............................ 28
Competitive RT-PCR ................................................................................... 29
Longitudinal Human Study ................................................ .. ........................ 35
Experim ental Design................................................... ... ........................... 36
Analytical Procedures ...................................................... ........................ 36
Statistical Analysis ........................................................ ..................................... 37

4 RESULTS
Refinement of Human M etallothionein ELISA .................................................... 41
ELISA Plates............................................................................................... 41
Antibodies .......................................................... .......................................... 41
Erythrocyte Lysate Preparation..................................................... .... ...... 42
Source of M etallothionein .......................................................................... 42
Preliminary Experiments for Competitive RT-PCR Assay and Sandwich
ELISA ............................................................................................................... 50
THP-1 Cells ..................... ............................................... ............ ................ 50
Flow Cytometry ...................................................................................... .. 50
Com petitive RT-PCR ........................................... .................................... .... 50
Sandwich ELISA ........................................................ .................................. 54
Longitudinal Hum an Study .......................................... ............................................. 54
Competitive RT-PCR ...................... .................................... ......................... 54
Sandwich ELISA .................. ...................................................... .................. 55
Plasma Zinc .................................................................................................. 55
Plasma Copper............................................................................................. 56

5 D ISCU SSION AND CON CLU SION S ...................................................................... 62

APPEND IX A .............................................................. ...................... .................... 73


APPEND IX B .................................................. ... ................... ................. 80

REFEREN CE................................................................................................................ 81

BIOGRAPHICAL SKETCH .......................................... ............................................... 92










vi















LIST OF FIGURES


Figure page

3-1. Diagram of competitive reverse transcriptase polymerase chain reaction (RT-
PC R ) assay .............................................................................................................. 31

3-2. Overview of metallothionein (MT) cDNA competitor template construction.......... 32

3-3. The metallothionein (MT) cDNA and competitor cDNA were resolved on an
8% polyacrylamide gel stained with ethidium bromide.............................................. 33

3-4. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay
to qualify metallothionein (M T) mRNA................................................................... 34

3-5. Overview of protocol for longitudinal human study .............................................. 38

3-6. Competitive reverse transcriptase chain reaction (RT-PCR) assay to quantify
m etallothionein (M T) m RN A ................................................................................... 39

3-7. Percent integrated intensity for cDNA bands after competitive RT-PCR................ 40

4-1. Titration Curve for Anti-Human Metallothionein Chicken Antibodies.................... 43

4-2. Sandwich ELISA to Test for Human Metallothionein Specificity........................... 44

4-3. Sandwich ELISA for Comparison of Monoclonal Antibodies................................. 45

4-4. Direct ELISA for the Comparison of MT From Different Species.......................... 46

4-5. Effect ofProtease Inhibitors on Erythrocyte Lysate MT Stability Over Time.......... 47

4-6. Zinc-stimulated increase in metallothionein mRNA levels in THP-1 cells using
semi-quantitative reverse transcriptase polymerase reaction (RT-PCR)..................... 48

4-7. Flow cytometry profile of human monocytes.......................................................... 49

4-8. Human Monocyte MT cDNA after Competitive RT-PCR...................................... 51

4-9. Sandwich ELISA for Erythrocyte Metallothionein................................................. 52












4-10. Standard Curve for Sandwich ELISA .................................................................. 53

4-11. Competitive RT-PCR: Monocyte Metallothionein cDNA.................................... 57

4-12. Polyacrylamide gel electrophoresis showing intra-assay variation for
com petitive RT-PCR assay....................................................................................... 58

4-13. Erythrocyte human metallothionein (hMT) as analyzed by sandwich ELISA......... 59

4-14. Plasm a Z inc ......................................................................................................... 60

4-15. C opper Z inc ........................................................................................................ 6 1

5-1. Comparison ofMonocyte MT mRNA and Erythrocyte MT protein Response
to Zinc Supplem entation .......................................................................................... 70

















ABBREVIATIONS


bp basepair
BSA bovine serum albumin
cDNA complementary deoxyribonucleic acid
ddH20 glass distilled deionized water
DEPC diethylpyrocarbonate
DMEM Dulbecco's modification of Eagle's medium
DNA deoxyribonucleic acid
dNTP deoxynucleoside triphosphate
ELISA enzyme-linked immunosorbent assay
FAA flame atomic absorption
FACS fluorescence activated cell sorter
FBS fetal bovine serum
FITC fluoroscein isothyocyanate
FPLC fast protein liquid chromatography
IACUC Institutional Animal Care and Use Committee
ICBR Interdisciplinary Center for Biotechnology Research
IgG immunoglobulin G
hMT human metallothionein
mRNA messenger ribonucleic acid
MT metallothionein
NIH National Institute of Health
PAGE polyacrylamide gel electrophoresis
PBS phosphate-buffered saline
PCR polymerase chain reaction
RIAs radioimmunoassays
RT-PCR reverse transcriptase polymerase chain reaction














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


DEVELOPMENT OF METALLOTHIONEIN BASED ASSAYS FOR THE
MEASUREMENT OF ZINC STATUS IN HUMANS

By

Vicki K. Sullivan

May, 1997

Chairman: Robert J. Cousins
Major Department: Food Science and Human Nutrition


While zinc is an essential mineral with numerous physiological roles, a reliable

index for zinc status in humans has not been developed. Metallothionein (MT), a cysteine-

rich protein of low molecular weight, is induced by zinc. Because the MT gene is

transcriptionally regulated by zinc, MT has great potential for serving as an index of

dietary zinc status. The specific aims of this project were as follows: 1) to develop a new

enzyme-linked immunoassay (ELISA) for the measurement of human MT (hMT) in

erythrocytes; 2) to develop a method for measuring hMT mRNA levels in monocytes; and

3) to test these methods in a human zinc supplementation study. A sandwich ELISA was

developed for the measurement of hMT in erythrocytes. A competitive reverse

transcriptase polymerase chain reaction (RT-PCR) assay was developed for the

measurement of monocyte hMT mRNA. Twenty-five healthy male subjects participated in

an 18 day zinc supplementation study. Erythrocyte hMT, monocyte hMT mRNA, plasma

zinc and plasma copper were evaluated. Zinc supplementation resulted in a significant








increase in erythrocyte hMT by day 8 of supplementation. Erythrocyte hMT returned to

baseline levels by eight days after the end of zinc supplementation. Monocyte hMT

mRNA levels were significantly increased by day 2 of supplementation, remained

significantly elevated through day 18, and returned to baseline levels four days later.

Plasma zinc was significantly increased by day 6 and decreased to control levels by day 18

of supplementation. The results of this study show that 1) hMT mRNA levels change in

monocytes in vivo in response to dietary zinc supplementation, 2) the sandwich ELISA

provides a convenient method for the measurement of erythrocyte hMT, and 3) monocyte

hMT mRNA is more sensitive to the acute elevation of dietary zinc status than erythrocyte

hMT. Further studies are needed to determine if the sandwich ELISA for evaluation of

erythrocyte hMT and the competitive RT-PCR assay of monocyte hMT mRNA are of

value for assessment of zinc deficiency.













CHAPTER 1
INTRODUCTION


While zinc is an essential mineral with numerous physiological roles, a specific,

reliable marker for assaying zinc status in humans has not been developed. Without such a

marker it is difficult to ascertain the amount of dietary zinc required to maintain normal

zinc status. Zinc, which is present in all cells, is a component of more than 60 enzymes

and is found among all enzyme classes (reviewed by Hambidge et al., 1986). In addition,

zinc is thought to play a role in cellular division and control mechanisms through structural

and regulatory functions. These functions influence many physiological factors including

bone development, skin integrity and cellular immunity. Symptoms of severe zinc

deficiency include retardation of growth, depressed immune function, anorexia, dermatitis,

altered reproductive performance, skeletal abnormalities, diarrhea, and alopecia (reviewed

by Walsh et al., 1994). Recently, zinc has been linked to the formation of A 13-amyloid

plaques in the brains of Alzheimer's patients (Bush et al., 1994). Zinc supplementation has

also been positively correlated to the susceptibility to AIDS (Graham et al., 1991).

Considering these findings, a method for definitively monitoring zinc levels would allow us

to identify individuals with suboptimal zinc status. Metallothionein (MT), a ubiquitous

cysteine-rich protein of low molecular weight, is inducible by various metal ions,

particularly zinc, and also binds these metals in specific geometries. Zinc is believed to be

a physiologically important regulator of MT transcription. In terms of nutritional

significance, copper and zinc are the most important metals that are bound to MT (Dunn

et al., 1987). The major proposed functions of MT include detoxification and storage of

heavy metals, scavenging of free radicals, and regulation of cellular copper and zinc

metabolism in response to dietary and physiological changes (reviewed by Hambidge et









al., 1986; Dunn et al., 1987). In general, MT may serve as a highly efficient storage and

retrieval system for zinc (Vallee et al., 1991). A direct relationship exists between dietary

zinc levels and MT concentrations in rat liver, kidney and intestines (Bremner and Davies,

1975; Richards and Cousins, 1976), and bone marrow (Huber and Cousins, 1993b). MT

mRNA levels have also been shown to be proportional to dietary zinc levels in rat, kidney,

liver, intestine, spleen, and ;heart (Blalock et al., 1988; Cousins and Lee-Ambrose, 1992).

Because the MT gene is transcriptionally regulated by zinc, MT has great potential

for serving as an index for assessment of dietary zinc status in humans. An enzyme-linked

immunoassay (ELISA) has been developed in our lab which was designed to measure MT

in human erythrocytes (Grider et al., 1989). The ideal method for assessment of zinc

status in humans must be practical, inexpensive, sensitive, and reproducible. While the

ELISA has shown promise for evaluation of both acute (Grider et al., 1990) and chronic

(Thomas et al., 1992) changes in dietary zinc status, it must be perfected for improved

reliability and tested further before it can be used as a standard measurement for humans.

In addition to developing a simple clinical assay, it is also possible to definitively determine

changes that are occurring in zinc status at the molecular level. Specifically, MT mRNA

levels should reflect changes that occur in MT protein. Therefore, an assay based on MT

mRNA levels could be another measurement of zinc status. However, mRNA levels

cannot be measured in mature erythrocytes because these cells are non-nucleated, and thus

do not produce new RNA. Alternatively, monocytes are nucleated blood cells and have

measurable amounts ofMT mRNA (Soumillion et al., 1992), but monocytes may not be

suitable for detection of MT protein levels because monocytes are much less abundant in

human blood (0.01% of total blood cells and 5% of white blood cells) and contain only

trace amounts ofMT (Morrison et al., 1988; Robertson et al., 1989). Neutrophils and

lymphocytes also contain negligable amounts of MT (Harley et al. 1989). Thus,

erythrocytes offer the best parameter to evaluate MT protein levels in humans while

monocytes are more suitable for measurement of MT at the mRNA level.









Traditional methods for assessing differences in mRNA levels, such as Northern

analysis, are not appropriate for the measurement ofMT mRNA in human monocytes

because unreasonably large quantities of blood are required for detection. Semi-

quantitative reverse transcriptase polymerase chain reaction (RT-PCR) allows detection

and amplification ofMT mRNA using small samples of blood. However, to determine

differences in mRNA expression, a quantitative RT-PCR method is required. Specifically,

there are two inherent problems that occur when semi-quantitative RT-PCR is used for

quantitative purposes. The first drawback is that due to the exponential nature of the PCR

reaction, small changes in amplification efficiency can lead to large differences in the

amount of end product cDNA produced. Thus two samples with the same amount of

starting RNA may show drastic differences in the amount of end product generated.

Secondly, the amount of product produced during the PCR reaction initially increases

exponentially, but then the rate of production plateaus. Ideally, the last cycle amplified in

the PCR reaction should fall in the exponential portion of the amplification curve before a

plateau is reached. However, the number of cycles required to reach the plateau phase

may vary between samples and may be affected by the efficiency of amplification.

Competitive RT-PCR controls for the intrinsic problems that occur with semi-

quantitative RT-PCR (Kohler et al., 1995). Because the competitor cDNA template is

amplified within the same tube as the template of interest, shares the same primers and is

very close in size, any variable influencing the amplification will affect both the competitor

and the template of interest. Thus the competitor cDNA template serves as an internal

control. Another important advantage of competitive RT-PCR is that the reaction can be

run beyond the exponential phase of the reaction curve well into the plateau phase because

the ratio of target to standard remains constant during the amplification. When a 1:1

relationship is reached between the competitor cDNA and the target cDNA, the amount of

target cDNA can be quantitated from the known amount of competitor template that has









been added to a specific reaction. Thus, a competitive RT-PCR assay could provide a

viable method for the detection of differences in human monocyte mRNA expression.

The specific aims of this project were as follows:

1) to perfect the current ELISA that was developed in our laboratory to

measure erythrocyte MT so that it may be used more effectively as an

evaluation method of zinc status in humans.

2) to develop a method for measuring MT mRNA levels in monocytes to be

used as another standard for definitive quantitation of zinc status in humans.

3) to test these methods in a human dietary supplementation study to evaluate

the comparative responsiveness of the two methods to an elevation of zinc

intake.

An improved and reliable method for the measurement of zinc status in humans

would improve our ability to define the level of dietary zinc that is essential for human

health. Metallothionein currently represents the most viable option towards the

development of an index for zinc status in humans because of the metal's role as a

transcriptional regulator of the MT gene.













CHAPTER 2
REVIEW OF THE LITERATURE


Zinc



Distribution and Functions

Zinc is a post-transitional element with an atomic weight of 65. It is present in all

cells, in concentrations typically between 10 and 100 pig/g wet weight. In humans, 60% of

total body zinc is present in skeletal muscle (as it comprises the largest amount of body

mass), while bone accounts for another 30% (reviewed by King, 1990). The highest zinc

concentration in tissues occurs in the choroid of the eye (Galin et al., 1962). Less than

0.5% of total body zinc exists in the blood, most of which (75-80%) is present in

erythrocytes (reviewed by Hambidge et al., 1986). Monocytes exhibit the highest zinc

content of the leukocytes (Goode et al., 1989), and small amounts of zinc are present in

lymphocytes. Normal plasma levels of zinc range from 10-18 pM.

Zinc has a multitude of diverse functions most of which can be categorized into the

general areas of catalytic, structural, and regulatory (reviewed by Cousins, 1996). Zinc is

essential for the catalytic activity of a wide range of enzymes which span all six enzyme

classes (reviewed by Vallee & Galdes, 1984). The zinc finger motif (repeated cysteine-

and histidine-containing domains of DNA-binding proteins that bind zinc in a tetrahedral

configuration) represents an important structural function of zinc. Zinc finger

transcription factors may play a role in the control of gene transcription (Chavrier et al.,

1988; reviewed by Cousins and Hempe, 1990;). It has been estimated that up to 1% of

the genome codes for proteins with the zinc finger motif (Klug et al., 1987), and zinc is









thought to be essential for the function of over 200 DNA-binding proteins (reviewed by

Vallee, 1991). Transcriptional regulation of genes is the third defined function. The

induction of MT by zinc is a principal example of this function. Specifically, there are

metal regulatory elements (MRE) on the MT promoter that interact with a metal-

dependent transcription factor (MTF) to initiate transcription of the gene (Tao et al.,
1992; Tennekoon et al., 1987).
The importance of zinc in immunity has been demonstrated by the fact that clinical

zinc deficiency leads to immune system dysfunction (reviewed by Chandra 1991). Zinc is

essential for the blast transformation of both T and B lymphocytes, for cell mediated

immunity, and for antibody mediated responses to T-cell dependent and T-cell

independent antigens. It is also necessary for normal development of lymphocyte subsets

and for proper function of phagocytic cells (Gershwin et al., 1985; Tapazoglou et al.,
1989). There is evidence that mitochondrial and microsomal membranes undergo lipid

peroxidation during zinc deficiency in mice (Burke and Fenton, 1985). Zinc and MT may
play a role in the reduction of oxidative damage caused by free radicals. However, the

underlying mechanism for an antioxidant role has not been clearly elucidated (reviewed by

Cousins and Hempe, 1990).


Absorption and Transport

In humans, about 20% of zinc consumed from the average mixed diet is absorbed

(reviewed by Hambidge et al., 1986). Zinc uptake across the brush border membrane

occurs by both a carrier mediated mechanism (which becomes saturated at high zinc

concentrations) and diffusion ( Steel and Cousins, 1985). The exact site of intestinal
absorption is unclear; however, it is thought that most absorption of zinc takes place in the
duodenum and jejunum (Lee et al., 1989). Once absorbed, zinc is bound to albumin for

transport in the blood (Smith et al. 1979). A functional storage reservoir for zinc does not









exist. However, there is a small exchangeable zinc pool probably derived from the bone,

liver and plasma that may be utilized in zinc deficiency (reviewed by King 1990). It

appears that this small exchangeable pool of zinc is not utilized until the dietary zinc intake

falls below 5-6 mg. In addition, efficient homeostatic mechanisms are in place for zinc

regulation (reviewed by Golden 1989). Zinc homeostasis is thought to be regulated by

higher absorption at low zinc intakes and increased excretion in response to high zinc

intakes (Cotzias et al., 1962; Baer and King 1984; Jackson et al., 1984).


Sources. Bioavailability. and Requirements

The most abundant dietary sources of zinc come from animal proteins, especially

muscle and organ meats. Generally, the zinc content of dark meats is higher than white

meats (Sandstrom, 1989). Certain types of shellfish such as oysters and shrimp, and dairy

products are also good sources of zinc. Whole grains are relatively high in zinc with most

of the zinc contained in the bran and germ portions. Thus, about 80% of zinc in refined

grains is lost in the milling process (Schroeder, 1971). Vegetables, fruits and fats are

among the poorest sources of zinc.

The bioavailability of zinc in foods varies widely mainly because various dietary

factors affect zinc availability. Phytate which is commonly found in whole grains can form

insoluble complexes with zinc and inhibits absorption (Sandstrom and Lonnerdal, 1989).

Other inhibitors of zinc absorption include dietary fiber, oxalate, tannin and caffeine

(reviewed by Solomons, 1982).

Evaluation of the minimal requirement for zinc is difficult in humans because of the

lack of a specific, sensitive indicator of zinc status. Estimates of human requirements for

zinc have been largely based on metabolic balance studies which have compared dietary

intake with urinary and fecal excretion. Since humans adapt to changes in zinc intake by

increasing or decreasing zinc absorption and/or excretion, balance studies may not









adequately reflect the true physiological requirement for zinc (King and Tumrnlund, 1989).
Requirements of zinc vary with age and physiological condition. Specific recommended

daily allowances are 3 mg zinc for infants < 6 months of age, 5 mg for older infants, 10 mg

for preadolescent children, 12 mg for adolescent and adult females, 15 mg for adolescent

and adult males, 15 mg during pregnancy, and 16-19 mg during the first and second 6

months of lactation (National Research Council, 1989).


Deficiency

Human zinc deficiency was first described in human populations subsisting on a

diet of unleavened breads and beans rich in phytic acid and devoid of animal proteins

(Prasad et al., 1961). Zinc deficiency has also been seen in alcoholism (Aggett 1989), in

chronic diarrheal diseases (Guerrieri et al., 1986; McClain et al., 1980; Golden), and in

parenteral nutrition devoid of zinc supplementation (Arakawa et al., 1976). The disease
acrodermatitis enteropathica is a genetic zinc deficiency syndrome that is transmitted in
humans as an autosomal recessive trait. Acrodermatititis enteropathica is associated with

decreased absorption of zinc (Moynahan, 1974). The clinical and physiological features of

zinc deficiency include lethargy, severe dermatological lesions, delayed sexual

development, alopecia, impaired reproductive function, dwarfism, anemia, depressed

immune function, diarrhea, and hepatosplenogmegaly (reviewed by Hambidge et al.,

1986). Signs of impaired reproductive function include poor pregnancy outcome(birth

defects and miscarriages), hypogonadism and undescended testes (reviewed by Apgar,
1985). Depressed immune function is exhibited as increased susceptibility to infection,

skin lesions, thymic atrophy, and reduced T-helper cell activity (Depasquale-Jardieu and
Fraker, 1980). Recently, several studies of malnourished children have shown that zinc

supplementation decreased the incidence, severity, and duration of persistent diarrhea and

dysentery ( Bhandari et al., 1996; Sazawal et al., 1995, 1996). This is important because









among children in developing countries, severe diarrhea accounts for 20% of all deaths

(Victora et al., 1993).

It is difficult to assess the occurrence and significance of mild zinc deficiency

because of the lack of a specific reliable marker for human zinc deficiency. However,

those most at risk include pregnant women, children and the elderly, especially in

developing countries mainly because of decreased intake of zinc rich foods (reviewed by

Hambidge et al., 1986).


Toxicity

Symptoms of zinc toxicity have been demonstrated at intakes in the range of 100-

300 mg Zn/d (reviewed by Fosmire, 1990). A documented toxic effect of zinc results

from its interference with copper absorption, leading to copper deficiency anemia and

neutropenia (Prasad et al., 1978). High intakes of zinc have also been associated with

increased serum cholesterol levels in rats (Klevay, 1974) and increased low-density-

lipoprotein and decreased high-density-lipoprotein cholesterol in humans (Hooper et al.,

1980). Other effects include depressed immune function with decreased lymphocyte

activation (Chandra, 1984), and severe gastritis (Brown et al., 1964). Bush et al., (1994)

found that high concentrations of zinc in vitro induce AP3 amyloid plaque formation, which

increases in abundance in the brains of Alzheimer's patients. Thus, there is some question

as to whether zinc may be involved in the neuropathogenesis of Alzheimer's disease.

Recent evidence suggests that zinc functions in synaptic signaling (Masters et al., 1994).

Assessment of Zinc Status


Many indices of zinc status have been proposed for use in humans through

measurement of the zinc concentration of skin, hair, blood, semen, sweat, saliva and urine









(reviewed by Golden, 1989). Physiological measurements including taste acuity, dark

adaptation and immune response have also been used as indices of zinc status.

Measurements of circulating zinc in blood plasma and serum have been used most

frequently to measure zinc status in humans (reviewed by King 1990). However, total

body zinc status is not fully represented by plasma zinc, because these levels are affected

by stress, pregnancy, infection, meals, short-term fasting, circadian variations,

complicating disease and hormones (reviewed by Golden, 1989). Use of plasma zinc as a

measurement of zinc status is further complicated by the lack of a physiological zinc

reserve to be drawn upon when zinc stores are insufficient. Instead, the body responds by

reducing excretion of zinc, or by a reduction in growth (reviewed by King, 1990). Thus,

in mild deficiency, zinc homeostasis may maintain the plasma level, while severe symptoms

are seen only with long term deficiency, concomitant with a reduction in plasma zinc.
Results from erythrocyte zinc and leukocyte zinc have shown little promise for the

assessment of total body zinc stores. Most of the studies to date have found that these

parameters are unreliable indicators of zinc status (Baer and King, 1984; Rabbani et al.,

1987; Crofton et al., 1983; Milne et al., 1985).

Urinary zinc may be decreased in long term zinc deficiency (Prasad et al., 1983).

Other studies have shown no difference in urinary zinc excretion with changes in zinc

status (Wada et al., 1985). A potential limitation to urinary zinc as a measurement of zinc

status is that increased urinary excretion of zinc has been reported to occur with zinc

deficiency in specific disease states such as hepatic cirrhosis, diabetes and sickle cell

disease (Sullivan and Heaney, 1970; Kumer and Rao, 1974; Prasad et al., 1975).
The results from measuring hair zinc for the analysis of zinc status have been

inconclusive (Klevay 1974; Baer and King, 1984). In addition, hair zinc concentrations

can be affected by environmental contaminants, gender, age, hair products, and rate of hair
growth (reviewed by Hambidge et al., 1986).









Specific zinc metalloenzymes have been investigated as possible parameters for the

measurement of zinc status. Serum alkaline phosphatase has been the most thoroughly

investigated enzyme. While some studies have shown this measurement to be useful in

determining changes in zinc nutriture (Kay et al., 1976), others have found no consistent

changes in plasma alkaline phosphatase levels with zinc depletion or supplementation (Ruz

et al., 1991). Angiotensin I-converting enzyme has also been investigated, but results have

been inconclusive limiting the value of this enzyme for the measurement of zinc status

(White et al., 1984; Ruz et al., 1991).

Metallothionein


Structure. Distribution, and Function. Metallothionein is a ubiquitous cysteine-rich

61 amino acid protein of low molecular weight (6000 7000 Daltons). The absence of

disulfide bonds and the hydrophilic nature of this protein allow for specific binding of

heavy metals within the range of 7-12 g atoms ofmetal/mol (Dunn et al., 1987). In

humans, MT is encoded by a multigene family that exits as a gene cluster on chromosome

16q22 (Karin et al., 1984). Of the 14 MT genes that have been identified in humans,

there are 8 that are currently considered to be functional (Stennard et al., 1993). Two

distinct yet very similar MT isoforms termed MT-1 and MT-2 are found in rats (and other

species). In humans, microheterogeneity (a group of closely linked forms of the gene

product) is found in MT-1 but not MT-2 isoforms (Kagi and Kojima 1987). The structure

of MT is highly conserved among species and it is ubiquitous among eucaryotes.

Metallothionein is primarily found in the cell cytosol but may also be seen in the

nucleus in specific tissues (Panemanangalore 1983). While variable amounts are found in

most tissues, MT levels are particularly high in the liver, pancreas, kidney, and intestinal

mucosa (Kagi and Kojima 1987). MT has been proposed to play a role in the control of

metal absorption, tissue uptake, transport, storage and detoxification (reviewed by Dunn









et al., 1987; reviewed by Cousins, 1985). In addition to metals, many other factors are

thought to induce MT synthesis. Factors that induce MT synthesis include starvation,

bacterial infection, irradiation, oxidative challenge, and physical and inflammatory stress.

These MT inducers are regulated by hormone mediators such as glucocorticoids,

cytokines and other factors related to the stress response (reviewed by Cousins 1985;

reviewed by Dunn et al., 1987; Schroeder & Cousins, 1990).

Zinc and Metallothionein Gene Regulation. The metallothionein genes are

transcriptionally regulated by zinc (reviewed by Shay and Cousins 1993; reviewed by

Cousins 1994). Zinc regulates gene expression by binding to a metal responsive element

binding protein (MRE-BP). The MRE-BP/zinc complex is thought to have a higher

affinity for metal responsive element (MRE) sequences of specific promoters, and to

enhance transcription of sequences on the metallothionein gene (reviewed by Shay and

Cousins 1993). Several studies in rats have shown that dietary levels of zinc regulate

levels ofMT mRNA, especially in the kidney, liver and intestines (Blalock et al., 1988;

Cousins and Lee-Ambrose 1992). Human studies have also implicated dietary zinc in

regulation oferythrocyte MT levels (Grider et al., 1989; Thomas et al., 1993). This has

not been investigated at the transcriptional level through measurement of mRNA in

humans, however, since erythropoiesis takes place in the bone marrow (Huber and

Cousins 1993a).


Metallothionein for the Measurement of Zinc Status

Since the initial discovery of metallothionein (Kagi and Vallee 1960), several
methods have been developed to quantify this protein in tissue and biological fluids. The

first assays to offer a precise and efficient measurement of MT were metal binding assays

(Piotrowski et al., 1973; Onasaka et al., 1978). Later, radioimmunoassays (RIAs)

provided a more sensitive and specific measurement ofMT (Vander Mallie and Garvey









1979; Mehra and Bremner 1983). Sato et al. (1984) were the first to demonstrate that

plasma MT decreased in rats fed a zinc deficient diet. They used an RIA specific for

MT-1. An ELISA developed by Grider et al. (1990) has shown promise for the

measurement of MT in human erythrocytes. Based upon experiments with rats, an

advantage of erythrocyte MT evaluation is that it appears much less responsive to stress

and infection than plasma MT. While endotoxin treatment of rats has been shown to

produce a large increase in plasma MT-1 concentrations, only slight increases are seen in

erythrocyte MT-1 (Bremner et al., 1987). However, there are other physiological and

nutritional factors that affect erythropoiesis. Erythrocyte MT-1 levels have been shown to

be increased in rats that are deficient in both iron and zinc (Robertson et al., 1989). Huber

and Cousins (1993a,b) demonstrated that dietary zinc has a direct effect on MT in bone

marrow of rats. These results from animal experiments, plus the limited experimentation

with the ELISA in humans, suggest that erythrocyte MT has potential as a monitor of zinc

status. Other blood cells may also have potential in this regard. Monocytes have the

highest concentration of MT among white blood cells (Harley et al., 1989). Soumillion et

al. (1992) showed that MT mRNA in human monocytes could be detected using RT-PCR.

Thus, the measurement ofMT mRNA levels in monocytes, can offer a comparison of the

response of MT to dietary zinc changes at the transcriptional level (mRNA). The

monocyte population can expand during infection and this could represent a complicating

factor in the use of monocyte MT as a diagnostic aid in some situations. However,

monocyte numbers could be monitored by flow cytometry to correct for this complication.


Reverse Transcriptase Polymerase Chain Reaction

The polymerase chain reaction, which was first developed in 1985 (Saiki et al.,

1985), is a method that has a dramatic impact on scientific progress in many areas. The

enhancement of this method through the use of thermostable DNA polymerases and









automation of the PCR cycling protocol (Mullis and Faloona, 1987) made the method

available to most scientists as a simple and inexpensive method for the amplification of a

single fragment of DNA. Another important advance was made by coupling RNA

microisolation with reverse transcription and PCR to amplify cDNA from less than 100

copies ofmRNA (Rappolee et al., 1989). Recently, RT-PCR has been used to amplify

mRNA from less than 250 cells (Klebe et al., 1996). Although RT-PCR is extremely

sensitive for detecting small amounts of mRNA, there are limitations to using this method

for quantitative purposes. The two main obstacles to RT-PCR involve the exponential

nature of PCR. The first is the plateau effect. Under theoretical conditions, the PCR

increases exponentially, with the amount of product doubling with each cycle of the

reaction. However, in practice, the amount of product increases exponentially only up to

a certain point and then a leveling off of the rate of amplification occurs (Saiki, 1990).

Thus it is practical to compare samples only during the exponential portion of the reaction

curve before a plateau is reached. The plateau effect may occur for the following reasons:

1) accumulation of product which competes with primer annealing and extension, 2) the

molar ratio of polymerase to template falls below a critical level, 3) the accumulation of

polymerase inhibitors such as pyrophosphates, and 4) depletion of one or more of the

necessary reaction components (Siebert, 1993).

The second limitation to RT-PCR for quantitative purposes is the efficiency of

amplification. The amplification efficiency is the consistency of the quantity of template

that is replicated during each reaction cycle. The length and composition of the sequence

being amplified and the sequence of the primers can affect the amplification efficiency and
result in interference with primer binding and reduction of polymerase processivity.

Because of the exponential nature of PCR, a very small change in the amplification

efficiency can lead to dramatic differences in the amount of end product generated. For

example, if the same amount of starting sample is amplified in two separate tubes, and

there is a change in amplification efficiency of 0.05 in one tube, there would be a twofold









difference in the amount of end product generated (Siebert, 1993). Therefore, RT-PCR

alone is not suitable for quantitating differences between samples.

Several techniques have been developed for the quantitation of RT-PCR. Addition

of an endogenous standard such as B-actin has been used to quantitate RT-PCR

(Kinoshita et al., 1992). Another method involves biotinylation of an internal standard and

hybridization of the products on microtiter plates (Jalava et al., 1993). A competitive RT-

PCR ELISA has also been developed (Taniguchi et al., 1994). PCR products have also

been quantified using high performance liquid chromatography (HPLC) (Katz and Dong,

1990).

Competitive RT-PCR eliminates the inherent problems that limit the use of RT-

PCR for quantitative purposes. Competitive RT-PCR uses an exogenous DNA template

as an internal standard (Wang et al., 1989). In competitive RT-PCR, a series of known

dilutions is made of the competitor sequence, while a constant amount of the template is

added to each reaction. The advantage of using competitive RT-PCR over other

quantitative methods is that during amplification, the standard and the target sequence

compete for the same primers and reagents in the reaction mix. Thus any changes that

affect the template, such as a change in amplification, will also affect the competitor. If

the competitor template and the target template are amplified with the same efficiency, the

amount of initial target cDNA can be determined by determining the point at which there

is a 1:1 ratio of the competitor to target template. Another important advantage of

competitive RT-PCR is that, because the ratio of target to standard remains constant

during the amplification, it is not necessary to run the reaction in the exponential phase
before a plateau is reached. Quantitation of the products may be determined by

differences in size of the cDNA bands based on hybridization properties when the cDNA is

run out on a polyacrylamide gel. Alternatively, differences in the size of cDNA bands can

be determined using HPLC (Katz and Dong, 1990). Recently, this method has shown

promise for the measurement ofMT mRNA levels in human monocytes in response to





16


dietary zinc (Sullivan and Cousins, 1997) and in human lymphocytes for determination of

exposure to cadmium (Ganguly et al., 1996).














CHAPTER 3
MATERIALS AND METHODS


Refinement of the Human Metallothionein ELISA



Competitive ELISA

A variety of tests were done in an attempt to modify and improve the hMT ELISA

which was previously developed in our lab (Gider et al., 1989; 1990). The previous

method was a competitive assay using purified hMT-1 as a plate coating antigen, a sheep

anti-hMT-1 primary antibody and a donkey anti-sheep/alkaline phosphatase conjugated

secondary antibody (Sigma, Chemical Co., St. Louis, MO). The following parameters of

the previous hMT ELISA assay were evaluated in an attempt to make this a simpler, more

reliable assay.

ELISA Plates

A number of different ELISA immunoassay plates were tested including Xenobind

(Xenopore, Saddle Brook, NJ.) covalent binding microwell plates, copper chelate

microwell plates (Xenopore), Coming (Coming, NY) ELISA plates, Nunc-Immuno plate

(Fisher Scientific, Pittsburgh, PA), and Costar (Cambridge, MA) carbohydrate binding

plates.

Source of Metallothionein

Human MT that was used for coating ELISA plates and for the development of a

standard curve was purified from human liver obtained from the pathology and laboratory

medicine department of the College of Medicine, University of Florida, and from the NIH-









supported Liver Tissue Procurement and Distribution System (University of Minnesota).

The human liver MT was purified by FPLC gel filtration and FPLC ion exchange. Briefly,

human liver was homogenized (Polytron; Brinkmann Instruments, Westbury, NY) in 3
volumes of 1 mM Tris HCL; 0.02% NaN3 pH 8). The homogenate was centrifuged at

40,000 x g for 10 min at 4 C. The supernatant was heated in a 100 C water bath for 5

min, and re-centrifuged for 30 min at 40,000 x g. The supernatant was then applied to a

Hi-Load 16/60 Superdex G-75 column (Pharmacia Inc.,Piscataway, NJ), equilibrated with

1 mM Tris HCL; 0.02% NaN3 (pH 8) at 4 C. The zinc containing peaks were pooled

after detection by air acetylene flame atomic absorption (FAA). The pooled fractions

were then concentrated to 5 mL under nitrogen using an Amicon ultrafiltration cell and a

PM10 membrane (Amicon Division, W.R. Grace and Co., Danvers, MA). The

concentrated solution was filtered (0.45 ptm), and applied to an Econo-Pak Q ion

exchange cartridge (BioRad, Hercules, CA). Metallothionein isoforms were separated by

using a step gradient of 20 mM Tris HCL (20 min) followed by 50 mM Tris HCL (20

min). The flow rate was 1 mL/min and 1.5 mL fractions were collected. Fractions

containing human MT-1 and MT-2 were identified by zinc content using FAA. Each

isoform was pooled, concentrated by ultrafiltration and stored at 80 C.

Cell Culture. A human hepatoblastoma cell line (HEP-G2) was tested to ascertain

whether high enough quantities ofhMT could be obtained from cultured cells. Cells were

incubated in Eagle's Minimum Essential Medium supplemented with non-essential amino

acids, 90% sodium pyruvate with Earle's BSS, 10% Fetal Calf Serum (FCS), and penicillin

G (0.1 U/L)/ gentamycin (65 tg/L). Twenty-four hours prior to harvesting, HEP-G2 cells
were cultured with 100 jiM zinc (as ZnSO4) and 0.1 [tM dexamethasone to increase the

hMT yield (Failla and Cousins 1978; Schroeder and Cousins 1991). Harvesting of cells

involved scraping the plates after the addition of Trypsin solution (0.25% trypsin,0.02%

EDTA), washing the cells three times with 10 mL of 1 x PBS (pH 7.4) buffer, and









pelleting by centrifugation at 500 x g for 10 min. The cells were re-suspended in 2 mL 1 x
PBS and homogenized (Polytron) at 70% full speed for 15 sec.
Metallothionein levels in HEP-G2 cells were measured using a g09Cd binding assay

(Eaton and Toal 1982) to assess the MT yield. Briefly, a known specific activity of the
109Cd isotope was added to heat-denatured protein homogenates and incubated for 10 min

(because MT has a stronger affinity for cadmium, zinc is displaced). The remaining free
l9Cd was then precipitated with hemoglobin (which also has a strong affinity for

cadmium) in two additional heat-denaturation steps. A gamma ray spectrometer was used
to quantitate the radioactivity in 100 pL of sample, and gg MT/mg protein was calculated

based on the l09Cd specific activity.
Metallothionein from other species. Because there is cross-reactivity among

species for hMT, commercially purchased rabbit liver MT and horse kidney MT were
tested as sources for coating ELISA plates. Chicken anti-human MT-1 and MT-2, and

sheep anti-human MT-1 antibodies were used to test these sources of MT by direct

ELISA.

Primary Antibody
Animal care. The animals used for the production of antibodies were handled

following guidelines established by The University of Florida Institutional Animal Care and

Use Committee (IACUC).
Sheep antibodies. In an attempt to increase specificity, following Protein G

purification, sheep antibodies were affinity purified using cyanogen bromide affinity

chromatography. First protein G Sepharose 4 Fast Flow (Pharmacia) was used for
purification of total Sheep IgG. Fifty milliliters of sheep serum was adjusted to pH 7.0
with binding buffer (20 mM sodium phosphate, pH 7.0). The serum was applied to a 5 mg

column of Protein G after equilibration with 25 mL binding buffer. The IgG fraction was

eluted with 100 mM Glycine-HCL, pH 2.7 at 0.8 mL/min collecting 1 mL fractions. Two









drops of IM Tris were added to test tubes before eluting IgG to neutralize the antibody
solution upon collection of each fraction. The IgG peak (detected by absorbance at 280

nm) was pooled, dialyzed and concentrated by ultrafiltration using an XM50 filter (Diaflo,

Amicon). Secondly, for affinity purification, rabbit MT (Sigma) was coupled to Cyanogen

Bromide Sepharose (Pharmacia) according to the manufacturer's directions. Sheep IgG
(100 mg) was diluted to 10 mL with 100 mM sodium phosphate buffer (pH 8.0). The
sample was applied on a 15 mL column at a flow rate of 0.5 mL/min and left on the
column overnight (4 C). The column was then washed with 10 bed volumes of 100 mM
sodium phosphate, or until absorbance at 280 nm was zero. Sheep IgG fractions were

eluted with 100 mM sodium phosphate; 1 M sodium chloride (pH 8.0). Pooled fractions

were dialyzed in I x PBS and concentrated by ultrafiltration (XMN450 filter, Amicon).
Chicken antibodies: production purification, and conjugation. Anti-hMT-1 and

anti-hMT-2 antibodies derived from egg yolks were developed and tested as a possible
source of primary antibody. Human MT was purified as previously described (Source of
Metallothionein). Chickens were initially inoculated with 100 .ig hMT-1 or hMT-2
suspended in complete Freund's adjvant (1 mg/mL). Two additional 100 ug injections
were given in incomplete Freund's adjvant at two week intervals (Gassman et al., 1990).

Eggs were collected and stored at 4 C. Using a method adapted from Gaussman et

al.,(1990) and Hassl and Aspcok (1988), hydrophobic interaction chromatography was

used to purify the chicken IgG from egg yolks. Briefly, egg yolks were separated from
whites, mixed with 120 mnL polyethylene glycol (4.4%) solution and incubated for 30 min
at room temperature. The mixture was centrifuged at 3600 x g for 1 hr at 4 C. The
supernatant was filtered (0.45 tim) and applied on a 1.5 x 70 cm Sepharose CL4B
(Pharmacia) hydrophobic interaction column. The chicken IgG was eluted with glass-

distilled deionized water (dd H20). IgG containing fractions (which were determined
spectrophotometrically by measuring absorbance at 280 nm) were pooled and
concentrated on an Amicon ultra-filtration unit using a Diaflo XM50 membrane.









In an attempt to simplify the sandwich ELISA and eliminate an additional step,

chicken antibodies were conjugated to alkaline phosphatase (immunoassay grade,

Boehringer Mannheim, Indianapolis, IN). Anti-hMT chicken antibodies (5 mg) were

dialyzed against 2 L PBS with three changes ofPBS. The antibodies were then diluted

with PBS to 3 mg/mL. The dialized chicken antibodies (100 4L) were combined with 90

tL of 10 mg/mL alkaline phosphatase and 5 gxL glutaraldehyde (25%) in a 1.5 mL

microcentrifuge tube, and the solution was gently mixed. At specific time points (0, 5, 10,
15, 30, 60, and 120 min), 25 pL samples were removed and placed in separate 1.5 mL

microcentrifuge tubes. PBS (125 tL) was added to each sample, and then 1.1 mL

Tris/ovalbumin solution (0.05 M Tris-CI, pH 8.0, 5% ovalbumin, 5mM MgCI2, 0.5%

NaN3, 0.5% mertiolate) was added to consume any excess substrate and prevent further

conjugation. The samples were stored on ice until each time point was completed. The
samples were once again dialyzed against PBS and tested for alkaline phosphatase activity

using a direct ELISA assay (Ausubel et al., 1987).
To increase sensitivity and decrease steric hindrance, chicken anti-hMT antibodies

were biotinylated using the Protein Biotinylation System (Gibco BRL, Grand Island, NY)

according to the manufacturer's directions. Ten milligrams chicken IgG was dialyzed

against 1 x PBS, pH 7.4 overnight, with 3 one L changes of solution. The chicken IgG

was diluted to 1.5 mg/mL in 1 x PBS and 660 4L sodium carbonate (500 M, pH 9) was

added to the dialyzed IgG for a final sodium carbonate concentration of 5 mM. Twenty-
six microliters of &- ester 2 -(4'-hydroxyazobenzene) benzoic acid (CAB-NHS) were

added (50 mg/mL) to the chicken IgG solution. The mixture was vortexed for 10 sec and
incubated for 1 h at room temperature with gentle shaking. The reaction was stopped by

the addition of 50 pL of 1 M ammonium chloride to the solution. To separate the

biotinylated from the non-biotinylated antibodies, the chicken IgG solution was then run

through a Sephadex G-25 gel filtration desalting column (9 mL bed volume) which was
equilibrated with 40 mL 1 x PBS. The protein was eluted with 20 mL PBS (1 mL









fractions). Biotinylated IgG containing fractions were determined spectrophotometrically

by measuring absorbance at 280 nm. The ratio of moles biotin to moles protein was

determined according to the manufacturer's directions: The biotin to IgG ratio was

determined to be 30 moles biotin per mole of IgG.
Monoclonal antibodies. In an attempt to increase the sensitivity of this assay, a

commercially available mouse monoclonal anti-MT antibody (Dako), Carpinteria, CA was

tested as an alternative to the polyclonal sheep anti-human primary antibody currently in

use. The commercially made monoclonal antibody showed very low reactivity against

hMT. Therefore, two monoclonal antibodies specific for hMT were made at the

University of Florida Interdisciplinary Center for Biotechnology Research (ICBR)

Hybrodoma Core Facility. Human MT-1 and 2 were purified and separated as described

above (Source of Metallothionein). To enhance immuno-genicity, the purified hMT was

conjugated to a carrier protein, keyhole limpet hemocyanin (KLH), according to the

manufacturer's directions (Pierce, Rockford, IL). Briefly, 2 mg hMT was dialyzed in dd

H20 at 4 C overnight with 3 changes of water overnight. The hMT was then lyophilized,

and diluted in 500 iL conjugation buffer (100 mM 2-N-morpholino-ethanesulfonic acid,

150 mM NaCI, pH 4.7). A solution of 1-ethyl-3-dimethylaminopropyl-carbodiimide was

then added (50 utL) and the sample was incubated for 2 h at room temperature.

Five Balb C mice were immunized with 50 uL of the hMT/KLH conjugate in

Freund's complete adjvant (1 mg/mL) for the initial injection and Freund's incomplete

adjvant (100 uL) for 4 more consecutive injections over a 5 month period. The animals

were handled according to guidelines established by IACUC. Two animals with the

highest titers were then sacrificed for a fusion of spleen B lymphocytes with myeloma

cells. Two hybridoma clones were selected for the production of monoclonal antibodies,

one specific for hMT-2 (HL 1158) and one that cross-reacted with both hMT-1 and hMT-

2 (HL 1153). These clones were cultured in DMEM with 10% ultralow IgG fetal bovine

serum (FBS). The supernatants were removed and purified using Protein G Sepharose 4









fast flow (Pharmacia), and Gentle Bind binding and elution buffers (Pierce). Briefly, the

supernatant was diluted 1:3 in Gentle Binding Buffer and passed through the Protein G

column twice at 0.8 mL/min. The IgG fraction was detected by absorbance at 280 nm and

stored at -20 C. A range of concentrations of the monoclonal antibodies (1:50 1:5000)

were tested using a checkerboard ELISA approach to determine the optimal concentration

of antibody for the ELISA.


Erythrocyte Lysate Preparation

Erythrocytes were washed and lysed in preparation for detection of hMT by

ELISA. Ten parts whole blood was mixed by inversion with 1 part 6% Dextran 500

(w/v), 0.9% NaCI for 40 min. The plasma layer and buffy coat were then removed for

monocyte enrichment, and the erythrocytes were mixed with an equal volume of ice cold

0.9% NaCI and centrifuged at 600 x g for 5 min. The supernatant from the crude

erythrocyte preparation was removed, and the process was repeated two more times.

After the final wash, the packed red blood cells were lysed by the addition of ice cold dd

H20O (1:1.4). The erythrocyte lysate was aliqouted into 0.5 mL microcentrifuge tubes and

stored at -80 C.

As an alternative to the original method for erythrocyte lysate preparation, an

additional centrifugation step was added to clear the lysate of cell membranes and debris.

After lysing the cells with ice cold water, cell lysates were centrifuged at 21,000 x g for 30

min. The clear supernatant was removed and stored at -80C.

An experiment was conducted to test the stability of erythrocyte hMT stored at -
80C over time. Erythrocyte lysate was prepared using the original method and split into

two aliquots (both from the same subject). The first aliquot was treated with a mixture of

protease inhibitors (0.2 mM phenylmethyl sulfonyl fluoride, 0.1 mM pepstatin A, and 0.1

mM leupeptin) (Sigma) to inhibit protease activity. The second group received no









treatment. The erythrocyte lysates were then used for MT measurement by ELISA on the

following dates after lysate preparation: day 1; day 60; day 120; day 180 and day 240.


Protein Ouantitation

Protein measurements for erythrocyte lysate, hMT and antibody concentrations

were done by the Folin phenol reagent method of Lowry et al., (1951). Dilutions of

samples were made and compared to a bovine serum albumin (BSA) standard curve by

linear regression analysis of absorbance at 500 nm after 30 min. Because sulfhydryl

groups interfere with the Lowry assay by increasing the absorbance (Vallejo & Lagunas,

1970), a correction factor (25% of the measured concentration) was used for all MT

samples (Vallejo and Lagunas, 1970).


Plasma Zinc and Copper

For the preparation of plasma zinc and copper, fasting blood samples were drawn

into trace element-free tubes (Fisher Scientific; Vacutainer No. 369735) containing

heparin. Plasma was diluted 1:5 with glass dd H20. Zinc and copper concentrations were

measured by air acetylene flame atomic absorption.


Sandwich ELISA Assay

In an attempt to improve the hMT ELISA, a sandwich ELISA was developed as

an alternative to the competitive ELISA. After testing all combinations of the antibodies

available (sheep anti-human MT-1 and MT-2; chicken anti-human MT-1 and MT-2;

mouse monoclonal anti-human MT-2, and mouse monoclonal anti-human MT-1/MT-2),

and the previously mentioned ELISA plates, the following protocol was developed.

Nunc-immuno plates were coated with goat anti-mouse FAB antibody (Pierce) at a

concentration of 8 ug/mL diluted in carbonate buffer (100 mM sodium carbonate, 0.1%









sucrose, 0.02% NaN3,pH 9.5) and incubated overnight at 4 C. Nonspecific sites were

then blocked with blocking buffer (1 x PBS, 3% BSA, 0.02% NaN3) for 2 h at room

temperature. The BSA was removed and the plates were frozen at -20 C. All plates

were prepared through the blocking step on the same day to minimize variation. All
incubations were done at room temperature.
When assays were performed, plates were thawed and 100 pL of monoclonal anti-

human MT-1/MT-2 (2 mg/mL) diluted in antibody buffer (1 x PBS, pH 7.4, 1% BSA,

0.01% Tween 20, 0.1% sucrose) were added (1:600) for 2 h. The plate was washed 3

times with wash buffer (10 mM phosphate buffered saline; 0.5% Tween 20; 0.02% NaN3,
pH 7.4). Purified hMT or samples of red blood cell lysate (100 4L) were then added in
duplicate to the plate, and serial dilutions (1:2) were performed. Plates were washed 3

times with wash buffer and biotinylated chicken anti-human MT-1 antibody was added and
incubated for 2 h. Plates were washed 3 times with wash buffer and ExtraAvidin alkaline

phosphatase conjugate (Sigma) was added to the plates (1:4000) for 1 h. The plate was
washed 5 times with wash buffer and, an alkaline phosphatase substrate, p-nitrophenyl

phosphate (Sigma) was applied in carbonate buffer (160 mM NaHCO3; 140 mM

Na2CO3; 19 mM MgCl2; 3 mM NaN3, pH 9.6). After a 20-30 min incubation period,

absorbance was read at 405 rnm Total protein values for erythrocyte lysate were

determined using the Lowry method. Erythrocyte values were expressed as ig MT per
gram of total protein.
The sandwich ELISA was used to assess differences in hMT erythrocyte levels

between zinc supplemented and normal subjects in the preliminary and longitudinal human
studies which are described in following sections.
To test the specificity of the sandwich ELISA for hMT, a sandwich ELISA was
performed to compare erythrocyte lysate samples (run independently) with erythrocyte

samples to which a specific amount (1.5 pL) of purified hMT had been added.








Development of Competitive Reverse Transcriptase Polymerase Chain Reaction Assay



Cell Culture

THP-1 cells, a human monocytic leukemic cell line, were obtained from American

Type Culture Collection (Rockville, MD). The cells were grown in RPMI 1640 medium

(Gibco BRL) with 5 !M 2-mercaptoethanol and 10% fetal bovine serum. Twenty-four

hours prior to harvesting the cells, the medium was supplemented with 100 tM zinc

sulfate, while cells incubated in medium without zinc served as a control.


Preliminary Human Study

Eight subjects (4 male; 4 female) were randomly divided into a treatment group

that received 50 mg of zinc gluconate (Healthline, Clearwater, FL) for 10 days, or a

control group that received no treatment. On day 10 blood samples were drawn into

tubes (Fisher Scientific; Vacutainer No. 6457) containing EDTA for preparation of

erythrocytes and monocytes. Tests for significance were performed using a one tailed T-

test (Excel 4.0). Significance was established at P < 0.05. A coefficient of variation of

10% was used when analyzing MT competitor and MT cDNA bands for which a 1:1

relationship in intensity was established.


Isolation of Monocytes

Monocytes were isolated using NycoPrep 1.068 (Gibco BRL) according to the

manufacturer's directions. The blood was first mixed 1:10 with 6% (w/v) Dextran 500 in

154 mM NaCl. The plasma was removed, layered over NycoPrep, and centrifuged at 600

x g for 15 min. The monocytes were collected at the interface and the cells were washed

twice with 154 mM NaCl containing 13% EDTA and 1% BSA.
0'1,









Flow cytometry. Monocyte purity using Nycoprep 1.068 was determined by flow

cytometry (ICBR Flow Cytometry Core Laboratory). Monocyte cell suspensions were

diluted to 1 x 106 cells/mL in Hanks BSS and centrifuged at 400 x g for 20 min. The cells

were re-suspended in DMEM (Gibco BRL) and incubated with 20 uiL monoclonal mouse

anti-human monocyte CD 14 fluorescein isothiocyanate(FITC)-labeled antibody (Becton

Dickinson, San Jose, CA) for 30 min prior to flow cytometry. A mouse IgG2b kappa

antibody was used as a control (Sigma).


Extraction and Purification of RNA

Total RNA was extracted from human monocytes using TRIzol Reagent

(GibcoBRL) according to the procedures outlined by the manufacturer. Following

enrichment using NycoPrep, the monocyte pellet was homogenized for 12 sec at 60 rpm in

1 mL TRIzol Reagent. RNA was either immediately extracted or stored overnight at -70

and extracted the following day. For RNA extraction, the homogenized samples were

incubated at room temperature for 5 min. Chloroform (0.2 mL) was added and the tubes

were shaken by hand for 15 sec, then incubated at room temperature for 2-3 min.

Samples were then centrifuged at 12,000 x g for 15 min at 2-4 C. The clear aqueous

layer (top) was then transferred to a new 2 mL microcentrifuge tube. RNA was

precipitated by mixing the aqueous phase with 0.5 mL isopropyl alcohol. Samples were

incubated at room temperature for 10 min and then centrifuged at 12,000 x g for 10 min at

4C for 10 min. The supernatant was then removed from the remaining RNA pellet. The

RNA pellet obtained from this extraction procedure was washed 3 times with 75% EtOH

and then dissolved in sterile diethylpyrocarbonate (DEPC) treated water. Concentration

and purity of the RNA samples were determined spectrophotometrically by measuring the

absorbance of 5 A of sample diluted in 50 jiL TE (10 mM Tris-HCL, pH 8.0, 50 mM









EDTA) at 230, 260 and 280 n. Erythrocytes were processed as described previously

(Erythrocyte Lysate Preparation),and stored at -70C.


Reverse Transcriptase Polymerase Chain Reaction Assays

Figure 3-1 outlines the competitive RT-PCR procedure beginning with reverse

transcription ofmonocyte RNA. Methods for RT-PCR were modified from previously

published protocols (Leibbrandt and Koropatnick, 1994; Soumillion et al., 1992). The

following PCR primers were synthesized by the University of Florida ICBR DNA

Synthesis Core Facility: 5'ATG GAT CCC AAC AAC TGC TCC TGC G 3' (designated

as MT5') which contains a sequence complementary to the 3' end of human MT-2 mRNA,

and 5' AGG GCT GTC CCA ACA TCA GGC 3' (designated as MT3'), which contains a

sequence complementary to the 5' end of human MT-2 mRNA. The GeneAmp RNA

PCR kit (Perkin Elmer Cetus, Foster City, CA) was used for PCR amplification according

to the manufacturer's instructions. Reverse transcription was performed with 200 ng RNA

and 300 U reverse transcriptase (20 g.L reaction volume) for 60 min at 37 C followed by

heat inactivation for 5 min at 95 C (Ausubel et al., 1995).
Human MT-2 genomic DNA (hMT-IIa; American Type Culture Collection) was

used as a control for PCR. Because the sequence of the human MT-2 genomic DNA that

is amplified during PCR contains an intron, this control template results in a 500 bp cDNA

product which can easily be distinguished (on an acrylamide gel) from the 201 hMT cDNA

product produced by PCR The PCR reactions were performed using 2 4i 10 x PCR

reaction buffer, 2 gL MT 5' primer, 2 uL MT 3' primer, 1.6 4L 10 mM dNTPs, 0.1 IL

AmliTaq DNA polymerase, 2 pL target cDNA, and 10.3 iL ddH20. The following

cycle was used for PCR: denaturation for 30 sec at 95 0 C, annealing for 1 min at 55 0 C,

extension for 1 min at 72 C. These steps were repeated for 25 cycles for THP-1 cell

RNA (semi-quantitative RT-PCR), and 30 cycles for human monocyte RNA (competitive









RT-PCR). The same PTC-100 PCR thermal cycler (MJ Research, Inc. Watertown, MA)

was used to perform all RT-PCR reactions.


Competitive RT-PCR

A competitor MT cDNA was constructed (Figure 3-2) using as a template human

MT cDNA derived from reverse transcribed monocyte RNA. A 180 bp segment of the

hMT-2 cDNA was amplified using MT5' (described above) and the following specially

designed composite 3' primer: 5' GGG CTG TCC CAG CAT CAG GCC CCT TTG CAG

ATG CAG CCT TG 3' (MTC) (Celi et al. 1993). This design created a 21 bp internal

deletion of the hMT-2 cDNA and yielded a 180 bp competitor cDNA template upon PCR.

To first amplify the competitor cDNA, the MTC and the MT5' primers and the following

PCR cycling protocol were used: denaturation for 30 sec at 95C, annealing for 1 min at

60C, extension for 1 min at 72C. These steps were repeated for 30 cycles. For long

term storage the MT cDNA competitor template was ligated into the pCRII vector using

the TA Cloning Kit (Invitrogen, San Diego, CA).

To obtain a purified competitor template, the competitor cDNA was excised from

the pCRII vector using the EcoRI restriction enzyme (Gibco BRL). The insert was

separated from the vector by electrophoresis on a 1.5 % agarose gel at 60 V for 1.5 hours.

The gel was stained with I ng/mL ethidium bromide and visualized under UV light. The

competitor band was excised from the gel with a razor blade, and purified from the

agarose using a QIAquick Gel Extraction column (Qiagen, Chatsworth, CA) according to

the manufacturer's directions. The competitor cDNA in 200 mg of gel and 200 gL of QXI

buffer (Quiagen) was added to the column, and incubated at 50C for 10 min. The tube
was tapped every 2-3 min during the incubation. Isopropanol (200 pL) was added and the

mixture was loaded on a QIAquick spin column, and centrifuged at 10,000 x g for 1 min.

The column was centrifuged after addition of 0.75 mL of wash buffer (Qiagen) for 1 min









at 10,000 x g followed by a 1 min centrifugation at 10,000 x g to remove excess buffer.

The spin column was transferred to a 1.5 mL collection tube and 50 .iL of TE buffer (10

mM Tris-HCL, pH 8.0, 1mM EDTA) was added to the column, incubated for 1 min at

room temperature and centrifuged at 10,000 x g for 1 min. The concentration of the

competitor cDNA was determined by spectrophotometry at 260 nm.

Competitor cDNA templates that were amplified by PCR (that were not ligated

into the pCRII vector for storage) were also purified by electrophoresis and QIAquiek Gel

Extraction columns exactly as described above.

The competitive RT-PCR assay, beginning with reverse transcription of monocyte

RNA, is outlined in Figure 3-1. In practice, for this competitive RT-PCR approach, the

original MT5' and MT3' primers were used to simultaneously amplify both the competitor

MT cDNA and the target MT cDNA templates. In these experiments, ten twofold

dilutions of the competitor MT cDNA template were made with a range from 0.58 pg/gL

(1 gJL per reaction) to 37.6 pg/uL. Five initial dilutions were run for each hMT mRNA

sample. A range of dilutions between 0.58 pg/IL and 9.4 pg/iuL was run for the control

samples, and a range of dilutions between 2.3 pg/uL and 37.6 pg/uL was run for the zinc

supplemented samples. Additional reactions were run on samples that required a wider

range of dilutions. These dilutions were added to a constant amount of the monocyte MT

cDNA in a 20 uL reaction volume and co-amplified using the cycling protocol for RT-

PCR described above.

The RT-PCR products were separated on an 8% polyacrylamide gel at 40 mV, and

stained with ethidium bromide (Sambrook & Maniatis, 1989; Ausubel et al., 1995). As

diagrammed in Figure 3-3, the competitor MT cDNA can be distinguished from the MT

cDNA by the difference in size when separated using polyacrylamide gel electrophoresis

(PAGE). The gels were then photographed and analyzed by densitometric scanning of

photographs. The concentration of competitor cDNA which gives a 1:1 signal with the

target cDNA was used to calculate the concentration of the latter, basically as described








MonocyteRNA

Reverse Transcription


Copetitor cDNA
Target cDNA added at known
SPrime(MT) dilutions
5 Primer (VTT15)
3 Primer (MT3') Polymerase Chain Reaction

cDNA Products




Polyacrylamride Gel Bectrophoresis

Target cDNA -- 201 bp
Competitor cDNA 180 bp


competitor dilutions




*Ethidium Bromride Staining
*Photography
*Densitometry

Figure 3-1. Diagram of competitive reverse transcriptase polymerase chain reaction
(RT-PCR) assay. Total monocyte RNA is extracted and reverse transcribed to cDNA.
For each human monocyte sample, a series of known dilutions of the competitor cDNA is
added to a constant amount of the human monocyte cDNA. PCR is performed for 30
cycles, and the cDNA products are resolved on an 8% polyacrylamide gel, stained with
ethidium bromide and photographed under UV light. Densitometric scanning of film
negatives establishes the competitor dilution at which a 1:1 relationship occurred between
the competitor cDNA and monocyte cDNA bands. The competitor concentration is used
to calculate the relative mRNA abundance.














3' Primer
(1) t m late

201 bp MT cDNA template


180 bp competitor cDNA


5' Primer


3' composite primer


05'


21 bp match upstream


MT cDNA temF


I
3'4 ;A4. " 3' primer sequence



n In n ==='=3' 1-0

)late


21 bp deletion


5' Primer


Figure 3-2. Overview of metallothionein (MT) cDNA competitor template construction.
1) The MT cDNA competitor was amplified using the MT cDNA template. 2) The MT 5'
primer and a 41 bp composite primer (MTC), which was designed to eliminate a 21 bp
region within the MT cDNA template, were used for polymerase chain reaction (PCR).
3) This resulted in a 180 bp competitor cDNA product.


o















00
1 0

^- A










180 bp 201 bp








Figure 3-3. The metallothionein (MT) cDNA and competitor cDNA were resolved on an
8% polyacrylamide gel stained with ethidium bromide. The 201 bp MT cDNA is visually
distinguished from the 180 bp competitor cDNA when viewed under UV light.

















DNA size
markers


hMT cDNA
MT Competitor


600 60 6 0.60 0.06


Competitor Dilutions (pg)









Figure 3-4. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay
to quantify metallothionein (MT) mRNA. As outlined in Figure 3-2, 200 ng of monocyte
total RNA was reverse transcribed. Ten-fold dilutions of the competitor cDNA and a
fixed quantity ofmonocyte cDNA were amplified by PCR for 30 cycles. The cDNA
products were resolved on an 8% polyacrylamide gel, stained with ethidium bromide, and
photographed under UV light. Relative intensities of bands were determined by
densitometric scanning of film negatives. The amount of MT cDNA was established as the
point at which a 1:1 relationship occurred between the competitor cDNA band and the
MT cDNA band (6 pg in this example).









by Gilliland et al. (1990). A coefficient of variation of the densitometric intensity was

determined for each competitor and MT cDNA dilution for which a 1:1 relationship could

be visually established. Samples with a coefficient of variation of 10 % or less between

the competitor and MT cDNA bands were included. Samples with a coefficient of

variation greater than 10% were repeated or excluded. Figure 3-4 shows a typical

competitive RT-PCR dilution series used to quantitate hMT cDNA for the preliminary

studies.


Longitudinal Human Study

Twenty-five male subjects between the ages of 19-35 (mean = 24) with an average

weight of 72 Kg were selected from approximately 50 candidates. The study was

approved by the Institutional Review Board of the University of Florida. Candidates were

recruited through the use of flyers posted across University of Florida campus and through

adds placed in the campus newspaper (The Alligator). Following an initial screening

interview, subjects completed an in-depth questionnaire which addressed health status and

medication records, eating habits, and activity status. Food records were reviewed to

ensure that all subjects consumed a typical non-vegetarian diet. Blood chemistry profiles

(SMAC 25) were performed on all subjects that met the selection criteria (SmithKline
Laboratories, Gainesville, FL). Candidates with blood chemistry measurements that were

substantially outside of the normal range were excluded. All subjects signed informed

consent forms in which they agreed to refrain from drinking alcohol, to avoid eating

specific zinc rich foods (such as oysters and shellfish), and to limit their intake of caffeine
to no more than three servings per day. Subjects were also asked to inform the researcher

if they became ill and or consumed any type of medications during the study. During the

zinc supplementation phase, subjects were required to come in daily to take the

supplement under supervision. They were advised that non-compliance would result in








their elimination from the study without remuneration. Upon completion of the study and

adherence to all requirements, subjects received a $200 honorarium for their participation.


Experimental Design

The 36 day study was divided into three phases as shown in Figure 3-5. The first

phase was a 7 day acclimation phase (day -7 day 0) that was used to evaluate any

alterations in blood parameters being measure over time. During this acclimation phase,

subjects were required to adhere to the requirements of the study (such as refraining from

alcohol, zinc rich foods, excessive caffeine, and dietary supplements). For the zinc

supplementation phase (day 1 day 18), subjects were randomly divided into either a

treatment group which received 50 mg of zinc gluconate per day or a control group which
received 50 mg sodium carbonate (J.T. Baker, Phillipsburg, NJ), per day. Following the

supplementation phase, subjects received no supplement or placebo for 12 days (day 19 -

day 30) and blood was drawn every four days to evaluate post-treatment trends.


Analytical Procedures

All blood samples were drawn by a trained phlebotomist between 0700 and 0745 h

after overnight fasting. Venous blood samples (20 mL) were drawn into tubes (Fisher

Scientific; Vacutainer No. 6457) containing EDTA for preparation of erythrocytes and

monocytes on days -7, 0, 2, 4, 6, 8, 10, 15, 18, 22, 26, and 30. For preparation of plasma

zinc and copper, blood samples were drawn on days -7, 0, 8, 15, 22 and 30. Blood

samples were processed as previously described under the sections Erythrocyte Lysate
Preparation, Isolation of Monocytes, and Plasma Zinc and Copper. Briefly, blood samples

were mixed 1:10 with 6% (w/v) Dextran 500 in 154 mM NaCl. The plasma was then

removed and layered over Nycoprep 1.068, and the red blood cells were washed with









0.9% NaCI, and lysed with ddH20. An additional blood sample was taken for plasma

copper and zinc measurements.
Erythrocyte hMT was analyzed by sandwich ELISA and monocyte hMT mRNA

levels were analyzed by competitive RT-PCR. The dilution series for the competitor was

modified from a tenfold series (used in the preliminary human study) to a twofold dilution

series to better quantitate the cDNA end products. The modified two fold dilution series

is demonstrated in Figure 3-6. A graphical representation of the integrated intensity of the

competitor and MT cDNA bands as determined by densitometric scanning of acrylamide
gel photographs is shown in figure 3-7.


Statistical Analysis

Linear regression (Excel 4.0 software; Microsoft, Redmond, WA) was used to

determine concentrations for monocyte hMT cDNA, erythrocyte hMT, and plasma zinc.

Tests for significance were performed by repeated measures ANOVA (SAS, 1996; Littel

et al., 1996). Significance was established at P<0.05. Sample size was determined using
the following formula: N = 2x(S / D)2 x(zaiph +zb)2 (Lameshow et al. 1990). In this

formula, N = number of subjects in each group, S = estimated standard deviation (S =15),

D = the minimum anticipated change in a variable (D= 25), alpha coresponds to a two

sided test with a = 0.5, and zbeta corresponds with the power (0.95). These power

estimates indicated each treatment group needed an n of 10.








Study Design


Zinc Supplementation
or Placebo


-7 0 2 4 6 8 10 15 18 22 26 30
Days of Blood Draws During Study



Figure 3-5. Overview of protocol for longitudinal human study. Numbers indicate days of study on which blood samples were drawn.
The protocol consisted of a 7 day acclimation period, an 18 day treatment phase and a 12 day post treatment phase. Subjects were
randomly divided into either a treatment group which received 50 mg of zinc gluconate per day or a control group which received 50
mg sodium carbonate.


















DNA size
markers


hMT cDNA
MT Competitor


1.25 2.5 5 10 20


Competitor Dilutions (pg)










Figure 3-6. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay
to quantify metallothionein (MT) mRNA. Monocyte total RNA (200 ng) was reverse
transcribed. Two-fold dilutions of the competitor cDNA and a fixed quantity ofmonocyte
cDNA were amplified by PCR for 30 cycles. The cDNA products were resolved on an
8% polyacrylamide gel, stained with ethidium bromide, and photographed under UV light.
Relative intensities of bands were determined by densitometric scanning of photographs.
The amount of MT cDNA was established as the point at which a 1:1 relationship
occurred between the competitor cDNA band and the MT cDNA band (5 pg in this
example).











Integrated Intensity for Competitve RT-PCR


120

100

80

60

40

20


1.25


2.5 5 10 20
ng Competitor cDNA

hMTcDNA

Competitor cDNA


Figure 3-7. Percent integrated intensity for cDNA bands after competitive RT-PCR.
Two-fold dilutions of the competitor were co-amplified by PCR with a fixed quantity of
monocyte cDNA. The cDNA products were resolved on an 8% polyacrylamide gel,
stained with ethidium bromide, and photographed under UV light. Integrated intensity
was determined by densitometric scanning.













CHAPTER 4
RESULTS


Refinement of Human Metallothionein ELISA



ELISA Plates

After evaluation of numerous brands of ELISA plates, Nunc-immuno plates were

determined to be the most reliable based on the evaluation of direct ELISA standard

curves of hMT antibodies and hMT protein, consequently, these plates were used for the

Sandwich ELISA.


Antibody Specificity

Further purification of the sheep anti-human MT-1 antibodies using cynogen

bromide affinity column chromotography did not increase the specificity of the antibody

response. The chicken anti-hMT-2 antibodies showed a higher titer than chicken anti-

hMT-1 antibodies (Figure 4-1), although both antibodies cross-reacted with both isoforms

of MT. Chicken anti-human MT antibodies were superior to sheep anti-human MT

antibodies for the sandwich ELISA. Biotinylation of chicken antibodies produced a strong

specific signal, and a linear standard curve was developed upon dilution of the antibodies.

To further test the specificity of the sandwich ELISA for hMT, purified hMT was added

to erythrocyte lysate. There was an increase in the signal which was approximately equal

to the sum of the purified hMT and erythrocyte lysate hMT samples when run












independently (Figure 4-2). Conjugation of chicken antibodies with alkaline phosphatase
produced a weak signal that could not be used to develop a linear standard curve. The

commercially available monoclonal anti-human MT antibody also produced a weak signal

that could not be used for the ELISA. Of the two monoclonal antibodies that were
developed by the ICBR hybridoma core laboratory, the antibody that cross reacted with

hMT-1 and hMT-2 produced a better standard curve (coefficient correlation r = 0.94) than
the hMT-2 specific monoclonal (r = 0.78) (Figure 4-3) when combined with the
biotinylated chicken anti-human antibody in the sandwich ELISA.


Source of Metallothionein

Human MT was superior as a source of MT for developing a standard curve for

the ELISA when compared to commercially available rabbit liver MT-1 and MT-2, or

horse kidney MT (both isoforms) (Figure 4-4). These results suggest that the ELISA

exhibits a degree of specificity.


Erythrocte Lysate Preparation

When compared to the original method for preparation of erythrocyte lysate, there

was no difference when an additional centrifugation step was added to clear the lysate of

cell membranes and debris. In addition, the stability of the hMT in erythrocyte lysate was
not different from day 1 through day 240, when stored at -80C. Addition of protease
inhibitors did not have an effect on the stability of hMT from erythrocyte lysate as there
was no difference between protease inhibitor treated samples when compared to samples

that did not contain protease inhibitors (4-5).















Titration Curve for Anti-Human

Metallothionein Chicken Antibodies


J1.4


StO.
Z 1.2


0
S15
aO8
g.0.8

0.6

0.4
C


0 1 I 1i-
1:400 1:600 1:1000 1:2000 1:4000
Dilutions of Chicken Antibody


1:6000


Figure 4-1. Titration curve for chicken anti-human metallothionein (MT) antibodies.
Chicken antibody titer was measured by direct ELISA. Dilutions were performed of either
anti-human MT-1, or anti-human MT-2 chicken antibodies (10 mg/ ml). Values are
expressed as the absorbance at 405 nm.


Anti-human MT-1 Chicken Ab

Anti-human MT-2 Chicken Ab
-UW













Sandwich ELISA to Test for Human
Metallothionein Specificity


0.6


0.5


Purified hMT
(1.5 ug)


Erythrocyte
Lysate


Erythrocyte
Lysate + hMT(1.5 ug)


Figure 4-2. Sandwich ELISA for hMT specificity. Purified hMT (1.5 ;ig) was added to
erythrocyte lysate to evaluate the specificity of the sandwich ELISA for hMT.
Absorbance was measured at 405 nm.


E 0.4

0
*t-

S0.3
U)
C)
o0
I0.2
C<
.0


0.1


0















Sandwich ELISA for Comparison of
Monoclonal Antibodies


Monoclonal MT-1

Monoclonal MT mix


/




I I I I


15


7.5 3.8


1.9 0.9 0.5 0.3


Metallothionein (ug/mL)


Figure 4-3. Sandwich ELISA for the comparison of monoclonal antibodies against hMT-2
and a 1:1 mixture of monoclonal antibodies against hMT-1 and hMT-2 (MT-mix).
Several different concentrations of monoclonal antibodies were tested to determine the
optimal concentration for the Sandwich ELISA. In this example, antibodies were diluted
1:600. Absorbance at 405 nm was measured after a 30 min incubation period.


1.4

E 1.2
10
C
0
4-1
(0

0 0.8
0)
a

0.6
0.4
0.4


0.2


3o














Direct ELISA for Comparison of Metallothionein
from Different Species


SI I..........


120 60 30 15 7.5 3.8
Metallothionein (ug/mL)


1.9 0.9


Figure 4-4. Direct ELISA for the comparison ofmetallothionein (MT) from different
species. Chicken anti-human MT-2 antibodies were used to evaluate human liver MT,
rabbit liver MT and horse kidney MT as a source of MT for the development of standard
curves. Absorbance was measured at 405 nm


1.5



1



0.5

















Effect of Protease Inhibitors on Erythrocyte Lysate MT
Stability Over Time


0.5

0.48

0.46

S0.44

|0.42

0.4

0.38


240


1 60 120 180
Time (Days)


Figure 4-5. Effect ofprotease inhibitors on erythrocyte lysate stability over time.
Erythrocyte lysate preparations were devided into two aliquots; one that was treated with
protease inhibitors and one that received no treatment. Erythrocyte lysate MT levels were
then measured by direct ELISA. Absorbance was determined at 405 nrun.


Protease Inhibitors
-U-
No Protease Inhibitors
-4-*














0
$
I


-201 bp








Figure 4-6. Zinc-stimulated increase in metallothionein mRNA levels in THP-1 cells using
semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA
was extracted from THP-1 cells and reverse transcribed. PCR was then performed for 25
cycles, and cDNA products were resolved on an 8% polyacrylamide gel. Treated THP-1
cells were cultured with medium containing 100 BM zinc 24 h prior to harvesting, and
control cells received no zinc treatment.



























1_ -






-A' t::'^ .C 2 i


Forward Scatter


CD-14 FITC Fluorescent Intensity


Figure 4-7. Flow cytometry profile of human monocytes. Panel A) Monocyte (R2) and
lymphocyte (RI) population profiles after enrichment using Nycoprep 1.068. Panel B)
Fluorescent intensity of gated monocyte population (R2 from panel A) using fluorescein
isothiocyanate (FITC)-labeled anti-CD14 antibodies. Monocyte enrichment was 80%
when the cells were gated, and enrichment was 64% for the ungated profile.















Preliminary Experiments for Competitive RT-PCR Assay and Sandwich ELISA


THP-1 Cells

The hypothesis that zinc treatment up-regulates MT mRNA was first tested in

vitro using THP-1 cells. Figure 4-6 shows a visual comparison ofMT cDNA levels in

zinc treated THP-1 cells and control cells after semi-quantitative RT-PCR. Clearly, zinc

treatment up-regulated mRNA levels in THP- 1 cells as demonstrated by differences in the

cDNA yield after 25 cycles of RT-PCR.

Flow Cytometry

The results of flow cytometry for human monocyte purity are presented in Figure 4-7.

Panel A shows that the gated monocyte purity as confirmed by FITC labeled anti-CD14

antibodies was 80% ( 2). Panel B shows the distribution of CD14 marker across the

gated monocyte population. The ungated profile showed a 64% ( 5). monocyte purity

based on the fluoresently labeled anti-CD 14 antibody population.

Competitive RT-PCR

Competitive RT-PCR showed that there was a significant increase (P = 0.048) in

human monocyte MT mRNA when zinc supplemented subjects were compared to

controls. Figure 4-8 shows that monocyte MT mRNA levels were significantly higher in

the group that were given a zinc supplement for 10 days when compared to the control

group (P<0.05). For statistical analysis, the data were transformed to the logio to achieve

homogeneous variance between the groups.














Human Monocyte MT cDNA
after Competitive RT-PCR


25 -

20 -


5-

0 -


Group


Figure 4-8. Effects of dietary zinc supplementation on human monocyte metallothionein
(MT) mRNA levels. Monocytes were obtained from subjects that either received
supplemental zinc (Zn Supplement, 50 mg zinc per day) or received no zinc (Control) for
10 days. Monocytes were enriched by gradient centrifugation, total RNA was isolated
and competitive reverse transcriptase polymerase chain reaction (RT-PCR) was performed
(as outlined in Figure 3-4). Metallothionein cDNA is expressed pg hMT cDNA produced
per ng monocyte RNA from zinc treated and control subjects. Means SEM are shown
(n = 8). The difference between zinc supplemented and control subjects is significant at P
<0.05.














Sandwich ELISA for

Erythrocyte Metallothionein


* Zinc
I" Control


120

100

80

60

40

20

0


Groups




Figure 4-9. Erythrocyte hMT as analyzed by sandwich ELISA. Erythrocytes were
obtained from subjects that either received supplemental zinc (Zn Supplement, 50 mg zinc
per day) or received no zinc (Control) for 10 days Erythrocyte MT is expressed as gg
MT/ g protein. Means SEM are shown (n = 8). The difference between zinc
supplemented and control subjects is significant at P < 0.05.















Standard Curve for Sandwich ELISA


E25
In
2O
CU
0
5 20


c15
(0
I..
0


0o
C)
o 5


0.5 0.9


60


1.9 3.8 7.5 15 30
Metallothionein (ug/mL)


Figure 4-10. Standard curve for hMT sandwich ELISA. Serial dilutions of purified hMT
were used to produce a standard curve. Absorbance was measured after a 30 min
incubation period and is expressed as logo absorbance at 405 nm.













Sandwich ELISA

Figure 4-9 shows the results of the sandwich ELISA for the measurement of erythrocyte

lysate. There was a two fold increase in erythrocyte lysate values (P0.05) after 10 days

of zinc supplementation. No gender difference were found between the four subjects in

the two groups. The intra-assay coefficient of variation for the sandwich ELISA (which

was determined by evaluating 8 repetitions of the same concentration ofhMT on two

different ELISA plates on the same day) was 6 %, and the inter-assay coefficient of

variation (determined by comparing the same concentration of hMT on 8 different days)

was 17.5 %. The detection limit for this assay was 240 ng/mL. A standard curve for the

sandwich ELISA is shown in Figure 4-10.


Longitudinal Human Study



Competitive RT-PCR

Competitive RT-PCR showed that there was an increase in monocyte hMT cDNA

generated when RNA from zinc supplemented subjects was compared to RNA from

controls. Monocyte hMT mRNA levels (measured as metallothionein cDNA in pg

cDNA/ng of total monocyte RNA) were significantly greater (P<0.05) in zinc

supplemented subjects when compared to control groupsubjects by day 2 of

supplementation. These levels remained significantly elevated through day 18 of

supplementation (Figure 4-11). Furthermore, monocyte hMT mRNA levels returned to

baseline values by day 22, four days after supplementation was discontinued. The

interassay coefficient of variation for this competitive RT-PCR method was 6% (Figure 4-












12) whereas the intra-assay coefficient ofvaration was 9%. While the intra-assay

variation (within assays) is slightly greater than the interassay variation, the percent

difference (between 6% and 9%) is very similar and is subject to slight variations

depending upon the time the assay is performed and experimental variations such as

pipeting differences and differences in sample handling.


Sandwich ELISA

The sandwich ELISA showed that there was an increase in erythrocyte hMT (P=0.053) by

day 6 of zinc supplementation when compared to control values, and a significant increase

(P<0.05) in erythrocyte hMT levels by day 8 of zinc supplementation (Figure 4-13).

Erythrocyte hMT levels were also significantly different on days 10, 15, and 22 when

compared to control values. When zinc supplementation was discontinued, erythrocyte

hMT levels returned to baseline by day 26, and were no different than control values

through day 30. The control group showed significant day to day variation across time.

Day -7 was significantly different from days 10, 18 and 22. compared to control values.

When zinc supplementation was discontinued, erythrocyte hMT levels returned to baseline

by day 26 in the supplementation group, and were no different from control values

through day 30. The control group showed significant day to day variation across time.

Significant differences between the control group are designated by lower case letters in
Figure 4-13.


Plasma Zinc

Plasma zinc concentration was increased 80% (P<0.01) at 6 day of

supplementation when compared to day 0 (Figure 4-14). By the 15 d of supplementation












plasma zinc had markedly decreased compared to that at 6 d, but was still significantly

elevated (20%; P<0.05) compared to controls. Plasma zinc levels in zinc supplemented
subjects were no different from control levels by day 22, and remained within normal

levels through day 30. Plasma zinc values in control subjects remained within the normal

range (7-15 pM) throughout the study period.


Plasma Copper

Plasma copper levels remained within normal levels (10-22 AM) in the control and

zinc supplemented groups. There were no differences between control and zinc

supplemented subjects at any time during the study (Figure 4-15).












Competitive RT-PCR:

Monocyte Metallothionein cDNA


**
30 **
U Zinc
25 0 Control
7 SEM
20
< ~** ** ** *
Z 15
E

< 10
Z





-7 0 2 4 6 8 10 15 18 22 26 30
A Zinc Supplementation A
Days of Study


Figure 4-11. Effects of dietary zinc supplementation on human monocyte metallothionein
(MT) mRNA levels. Human monocytes were obtained from subjects that either received
supplemental zinc (Zn Supplement, 50 mg zinc as zinc gluconate per day, n = 7-12) or
received 50 mg sodium carbonate (Control, n = 8-13) for 18 days. Prior to the
supplementation period, subjects were acclimated for 7 days. Zinc supplementation was
discontinued on days 19-30. MT cDNA is expressed as pg MT cDNA produced per ng
monocyte RNA from zinc treated and control subjects. Means SEM are shown. The
difference between zinc supplemented and control subjects is significant at (**) P < 0.01.

















Intra-assay Variation
for Competitive RT-PCR


hMT cDNA
MT Competitor


Figure 4-12. Polyacrylamide gel electrophoresis showing intra-assay variation for the
competitive RT-PCR assay. In this example, a constant concentration of monocyte hMT
and competitor cDNA were added to six separate reactions that were all amplified on the
same day. The intra-assay variation for the competitive RT-PCR assay was 9%.

























Figure 4-13. Erythrocyte human metallothionein (hMT) as analyzed by sandwich ELISA. Erythrocytes were obtained from
subjects that either received supplemental zinc (Zn Supplement, 50 mg zinc as zinc gluconate per day n = 10-12) or received 50
mg sodium carbonate (Control n = 9-13) for 18 days. Prior to the supplementation period, subjects were acclimated for 7 days.
Zinc supplementation was discontinued on days 19-30. Means SEM are shown. The difference between zinc supplemented
and control subjects is significant at (**) P < 0.01 or (*) P < 0.05. Lower case letters represent differences (P < 0.05) between
the control group across time. a = significantly different from control group day -7; b = significantly different from control
group day 0; c = significantly different from control group day 2; d = significantly different from control group day 4; e =
significantly different from control group day 6; f= significantly different from control group day 8; g = significantly different
from control group day 10; h = significantly different from control group day 15; i = significantly different from control group
day 18; j = significantly different from control group day 22.










E ryth


S andw
rocyte


ich ELISA:
M eta Ilothionein


- ** M Zinc
r-- Control

a x SEM



B a,b,c,hi
b.d. eh **


Days of Study


120

100

80


60


40


-7 0 2 4 6 8 10 15 18 22 26 30
A Zinc Supplementation A






60




Plasma Zinc


*Zinc
L-Control

SEM


*


0


15


22


30


Days of Study

Figure 4-14. Effects of dietary zinc supplementation on plasma zinc concentrations.
Plasma was obtained from subjects that either received supplemental zinc (Zn
Supplemented, 50 mg zinc as zinc gluconate per day, n = 10-12) or received sodium
carbonate (Control, n = 11-13) for 15 days. Prior to the supplementation period, subjects
were acclimated for 7 days. Zinc supplementation was discontinued on days 19-30.
Plasma zinc levels were determined by air acetylene flame atomic absorption. Means
SEM are shown (n = 25). The difference between zinc supplemented and control subjects
is significant at (**) P < 0.01 or (*) P < 0.05.


25



20


15


10


-7






61




Plasma Copper


-7


6 15 22


-T


30


Days of Study




Figure 4-15. Effects of dietary zinc supplementation on plasma copper concentrations
Plasma was obtained from subjects that either received supplemental zinc (Zn Supplement,
50 mg zinc as zinc gluconate per day, n = 10-12) or received sodium carbonate (Control,
n = 11-13) for 18 days (n = 25). Prior to the supplementation period, subjects were
acclimated for 7 days. Zinc supplementation was discontinued on days 19-30. Plasma
copper levels were determined by air acetylene flame atomic absorption. Means SEM
are shown. None of the means were significantly different.













CHAPTER 5
DISCUSSION AND CONCLUSIONS


With the abundance of new information about the benefits of zinc for human health

and well being, the necessity for a specific reliable marker of zinc status becomes obvious.

However, several factors have made the identification of such a marker quite difficult.

Because zinc metabolism in the body is under tight homeostatic control, the most

commonly used parameters for measuring nutrient status in humans, such as body fluids

and enzyme markers, have not been reliable for the measurement of zinc. Marginal zinc

deficiency, which is especially difficult to detect, may be prevalent in several populations

throughout the US and the world (Golden, 1989). However, until a definitive marker for
zinc status in humans is established, the optimal amount of zinc required for the

maintenance of proper health will also be uncertain.

Metallothionein has shown great promise as a potential marker for the

measurement of zinc status in animals and humans. Blalock et al.(1988) showed that MT

mRNA expression is regulated by dietary zinc in rat liver, intestine and kidney.

Metallothionein protein regulation by zinc has also been demonstrated in rat plasma and

erythrocytes (Sato et al., 1984). Grider et al. (1990) showed that erythrocyte MT in

humans also reflected alterations in dietary zinc using a competitive ELISA. The

development of the competitive ELISA for the measurement of MT levels in erythrocytes
was a major accomplishment in the search for a method for assessing zinc status in

humans. However, the competitive ELISA was in need of further refinement.
The first aim of this study was to refine and improve the former ELISA method for

the measurement of zinc status in human erythrocytes. A sandwich ELISA was developed

as an alternative to the competitive ELISA for the following reasons. The competitive









ELISA required the use of large quantities ofhMT to coat the ELISA plate and serve as a

competitor. Because hMT is not commercially available, human liver must be obtained for

extraction and purification of the MT protein. However, human liver may be difficult to

obtain and its processing represents a biohazard. The sandwich ELISA eliminated the

need for large quantities of hMT because for the sandwich ELISA, plates are coated with

a sheep anti-mouse antibody followed by a monoclonal anti-human antibody for detection.

Human MT is then only needed for the standard curve. The antibodies for coating the
plate are commercially available and the monoclonal antibodies are easily obtained from

hybridoma cells in culture.

Although the polyclonal sheep anti-human antibody used in the competitive ELISA

had a high titer against hMT, it also exhibited considerable non-specific binding. An

attempt was made to further purify the sheep antibody using a cyanogen bromide affinity
column. However, this procedure led to a decreased response to hMT and did not

improve non-specific binding. For the sandwich ELISA a monoclonal antibody was used

that did not exhibit non-specific binding and showed greater specificity for hMT which

was a significant advantage over previously used antibodies. In addition, a chicken anti-

hMT antibody that had been developed earlier was used for the sandwich ELISA.

Antibodies produced in avians offer several important advantages over antibodies

produced in mammals. Because of the phylogenetic divergence of avians and mammals,

there is greater potential of producing a higher percentage of specific antibodies against
humans in chickens, when compared to other mammalian species. This is of particular

relevance for a protein such as MT that is highly conserved between closely related

species. Chicken antibodies also offer the convenience of collecting antibodies from egg
yolks as opposed to bleeding animals for the extraction of antibodies. A single egg yolk

will typically yield as much antibody as a single bleed from a rabbit, and a laying hen

produces approximately five to six eggs per week (Larsson et al., 1993). In addition, the

cost of feeding and housing for hens is considerably less than that of mammals.









Another advantage of the sandwich ELISA is the ability to prepare a third of the

assay well in advance. The competitive ELISA procedure required that the plates be

coated with hMT the night before the assay was performed. The plates used for the

sandwich ELISA can be coated with antibody, blocked and coated with the primary

monoclonal antibody in large batches well in advance of performing the assay. The ability

to prepare the plates in batches also decreases plate to plate variation. The plates can be
prepared and stored in advance at -20 for up to 1 year. The ability to prepare and store
plates in advance also reduces preparation time during an experimentation period. The

competitive ELISA requires an overnight incubation and a seven hour block of time the

following day while in comparison, the sandwich ELISA requires a 6 hour block of time,

using plates that have been prepared well in advance.
The results of the preliminary study using the sandwich ELISA were similar to the

findings of Hooker (1993), who also found an approximately two-fold increase in
erythrocyte hMT after 10 days of 50 mg /mL supplemental zinc. While no differences
were observed between male and female subjects in the preliminary study, the small
sample size does not allow for any firm conclusions on gender differences in MT

expression. In the longitudinal human study, erythrocyte hMT was significantly increased

by day 8 in the zinc supplementation group, and remained significantly elevated through

day 15. The linear increase up to day 10, and then plateau in erythrocyte hMT seen in the
zinc supplemented group reflects the results of both Grider et al. (1990), and Hooker
(1993). Grider et al. (1990) found that increased levels in erythrocyte hMT were
maintained through day 63 of zinc supplementation.
The plateau in erythrocyte hMT seen after day 10 of supplementation suggests that

a steady state in turnover in hMT follows the initial induction of hMT in the marrow cells.
The gradual decline in erythrocyte hMT (over 8 days) is also similar to the findings of

Gider et al. (1990) who saw a decrease back to baseline by day 7 after zinc

supplementation was terminated. The mean erythrocyte hMT value for the control group









during the acclimation period was very close to the results of Hooker (1993), (26 pg

MT/g protein), and slightly lower than the results of Grider et al. (1990), which was 37 ug

MT/g protein. By day 7 of zinc supplementation Grider et al. (1990) reported erythrocyte

hMT values of 273 ug MT/g protein with supplementation of 50 mg of zinc per day. In

the current study, erythrocyte hMT levels were 55 jg MT/g protein by day 8, with a

maximum high of 104 ug MT/g protein. The inter- and intra-assay variation for the

sandwich ELISA (17.5% and 6%) were similar to those reported for the competitive

ELISA (15.4% and 4.2%). Although the sandwich ELISA appears to be more sensitive

than the competitive ELISA, based on the detection limit (240 ng/mL compared to 300

ng/mL), there is still some question as to why supplementation values reported by Grider

et al. (1990) are higher. The higher erythrocyte hMT values may relate to the non-specific

binding observed with the sheep antibody that is used for the competitive ELISA. Further

studies are needed to determine whether the sandwich ELISA is able to measure

erythrocyte hMT during zinc depletion.
The sandwich ELISA did not detect differences in plasma hMT levels (results not

shown) in response to zinc supplementation in subjects from the preliminary or the

longitudinal study. Akintola et al. (1995) reported the development of a competitive

ELISA using a rabbit anti-human MT-1 antibody for the measurement of hMT in plasma.

However, it has been shown that endotoxin causes much larger increases in plasma MT

when compared to erythrocyte MT, suggesting that erythrocyte MT is much less

responsive to stress and infection (Bremner et al., 1987).

In designing the present study, there was some concern about the physiological

consequences of the total amount of blood loss over a relatively short period of time.
However, the decision to include numerous time points ( a total of 12) was made in order

to evaluate the complete time course for the response of monocyte hMT mRNA to

increased dietary zinc in humans (which had never been done). A significant increase in

erythrocyte hMT values in the control group occurred over time, with the increasing









number of blood draws. Robertson et al.(1989) showed that most of the MT in the

erythrocyte population of rats is contained within the reticulocytes. Huber and Cousins

(1993b) showed that the source of erythrocyte MT is early progenitor cells from the bone

marrow in rats, and that marrow MT induction is the result of increased cellular

proliferation associated with acute blood loss. Thus, the increased erythrocyte hMT in the

control group can most likely be attributed to an increased erythropoesis. However, the

zinc supplemented group values for erythrocyte hMT were still significantly different from

the control group (with the exception of day 18) for most of the supplementation period.
The design of the longitudinal study may have contributed to the day to day

variation in erythrocyte hMT that occurred in the control group. The subjects were free
living, thus differences that occurred within the control group may have been due to

differences in diet, stress, and/or infection. While compliance for supplementation was

monitored, compliance for alcohol, caffeine and refraining from zinc rich foods could not

be followed. Subjects were asked to report any illness during the study, and any occasions

for which they were required to take medications. Three subjects reported having colds
during the study. Two of these subjects had increased monocyte hMT mRNA (one

control subject on day 10, and one zinc supplemented subject on day 8). The control

subject also showed increased erythrocyte hMT levels on days 10 and 15. Two subjects

reported taking Tylenol during that period.

Subjects from several different ethnic backgrounds were chosen to participate in

the study to simulate a true population. Two subjects were Hispanic, two were Afro-
American, two were Indian, and one was of Asian descent. This may have accounted for
within group variation on specific days. Consequently, population differences in MT

expression need to be considered in future studies.
The second aim of this study was to develop a method for evaluating MT mRNA

levels in humans. Prior to this study, the response of MT mRNA levels to changes in

dietary zinc status had never been evaluated in humans. Monocytes were chosen for the









measurement of hMT mRNA because, of the white blood cells, monocytes have the

highest level of MT (Harley et al., 1989). While hMT protein levels had previously been

evaluated in erythrocytes, these cells are non-nucleated and cannot be used for the

detection of hMT mRNA. We were not able to detect hMT protein levels in monocytes

using the ELISA method in control subjects or subjects that received 50 mg/day zinc for

10 days. However, because monocytes are nucleated, and hMT mRNA is inducible by

zinc in vitro in these cells (Pauwels et al., 1993), monocytes represented the most viable

parameter for the evaluation ofMT gene expression.

We first attempted to visualize hMT mRNA levels using northern analysis, but

were unable to detect mRNA levels in monocytes isolated from 50 mL of blood. Thus, it

was necessary to use a more sensitive measurement, such as RT-PCR to detect hMT

mRNA levels in monocytes.

Pauwels et al. (1993) successfully used RT-PCR to detect hMT in monocytes.

Using primers for hMT modified from that study, THP-1 cells were used to develop a

semi-quantitative RT-PCR method to detect an up-regulation of monocyte mRNA with

zinc treatment. This method involved running a series of PCR cycles to determine the

optimal number of cycles within the exponential phase of the amplification curve necessary

for the detection of differences in mRNA levels. A visible difference between MT cDNAs

produced from RNA of zinc treated and control cells was evident on an ethidium bromide-

stained polyacrylamide gel after semi-quantitative RT-PCR. However, differences

between zinc-supplemented human subjects and controls using monocyte RNA were not

evident when semi-quantitative RT-PCR was used. The zinc concentration to which the
THP-1 cells were exposed to (100 WM Zn) is high compared to the zinc concentrations to

which monocytes would be exposed to in human subjects. Consequently, the zinc-induced

increase in MT mRNA in the cells would be expected to be high.

The preliminary human study showed that differences in human monocyte MT

mRNA levels after 10 days of supplementation with dietary zinc could be detected using









competitive RT-PCR. To improve quantification, the competitive RT-PCR method was

further modified after the preliminary study by decreasing the dilution factor of the

competitor template from ten-fold dilutions to two-fold dilutions for the longitudinal

study.

While there was a significant increase in monocyte hMT mRNA levels in the zinc

supplementation group, days 8 and 18 were elevated when compared to the other days of

supplementation (day 8 was significantly different from day 6; day 18 was significantly

different from days 2, 4 and 6). Several factors may have been involved in this increase.

One subject was undergoing a high level of stress during the last part of the

supplementation period which may have explained the increased monocyte hMT mRNA

levels on days 10 and 15. Another subject within the zinc supplementation group who had

a high mRNA level on day 8, and a high erythocyte hMT mRNA level on day 10, reported

the onset of a cold on day 10 of supplementation. Three of the five subjects that had

exceptionally high monocyte mRNA levels on day 8 also had high levels on days, 2, 15

and 18. Erythrocyte hMT levels were comparatively high on day 8 for two of these

subjects. Again, a lack of compliance may have occurred in this free living group of

subjects. However, none of the control subjects exhibited high MT mRNA levels

suggesting that they complied with the restrictions on zinc rich foods and did not

experience illnesses that would increase monocyte MT levels. The combination of these

extraneous factors with zinc supplementation may account for the increased levels of hMT

mRNA on specific days of zinc supplementation.

While erythrocyte MT levels have been shown to be less responsive to stress and

infection than plasma MT levels (Bremner et al., 1987), monocyte MT mRNA levels may

be up-regulated by these factors. Monocytes in vivo and in culture have been shown to

respond to host defense mediators. For example, lipopolysaccharide may produce a

transient increase in MT mRNA levels in THIP-1 cells (Leibbrandt and Koropatnick,

1994). The MT promoter has response elements for cytokines, glucocorticoid hormones,









and oxidative stress mediators (Dunn et al., 1987). This suggests that illness that increases

monocyte MT expression could be a complicating factor in the use of monocyte MT

mRNA for zinc status assessment. An assay that would be an indicator of a complicating
factor, e.g., increase in an acute phase protein, could address this question in field studies.

A fluorescence-activated cell sorter (FACS) analysis of the leukocyte population would

also be an indicator of elevated monocyte production. Further use of this RT-PCR

method may clarify the potential response of monocyte MT mRNA to disease variables.

The rapid turnover of monocytes or an unrecognized aspect of monocyte physiology may

explain some of the within subject variation observed during zinc supplementation.

The hMT monocyte mRNA values within the control group were fairly consistent

from day to day, but these baseline values were low. It is difficult to predict whether the

competitive RT-PCR assay will be sensitive enough to detect differences between zinc

deficient MT mRNA levels in monocytes and control values. Additional longitudinal

studies involving graded differences in total zinc intake including a markedly reduced
intake will be necessary to determine if the monocyte hMT mRNA response is the result of

an initial response to dietary zinc, or a measurement of total body zinc status. However, it
is reasonable to predict that monocyte hMT mRNA has potential as an indicator of zinc

status, because MT protein and mRNA levels have been shown to reflect overall zinc

status in animals (Bremner and Marshall, 1974; Richards and Cousins, 1976; McCormick

et al.,1981; Blalock et al., 1988). The current competitive RT-PCR assay could be of

practical value in studies where supplementation efficacy must be monitored, for example,

zinc prophylaxis in treatment of persistent diarrhea (Sazawal et al., 1996). It could also be
useful for the detection of compliance in epidemiological studies of the effects of zinc
supplementation when studying free-living subjects. The bioavailability of different forms

of supplemental zinc (i.e. zinc gluconate vs. zinc carbonate) could also be evaluated using

the competitive RT-PCR assay.









Comparison of Monocyte MT mRNA and Erythrocyte
MT Protein Response to Zinc Supplementation


0 Erythrocyte Metallothionein
U Monocyte Metallothionein mRNA


U Z 4 b $ 1U 10 1 :
A Zinc Supplementation A
Days of Study


Figure 5-1. Comparison of monocyte metallothionein mRNA and erythrocyte metallothionein protein levels during the 36 day
longitudinal study. Metallothionein protein and mRNA are expressed as relative ratio to day zero.









Compared to erythrocyte hMT (Figure 5-1), monocyte hMT mRNA levels

responded more rapidly to the initiation and discontinuation of supplemental dietary zinc.

These data probably reflect the turnover properties of monocytes and erythrocytes. The

turnover rate for erythrocytes is 120 days (Ganong, 1987), while the turnover rate for

monocytes is approximately one day (Johnston, 1988). Thus, monocyte hMT mRNA may

provide a better reflection of acute changes in zinc status.

To minimize blood loss, plasma zinc and copper were measured weekly in subjects

(on 6 occasions instead of 12) because an additional 10 mL of blood was required to assay

these minerals. The increased plasma zinc values by day 6 reflected the excellent

compliance with consumption of supplemental zinc. Although still elevated by day 15,

plasma zinc levels had declined which reflects the homeostatic regulation of body zinc and

redistribution of zinc from the plasma to other body compartments.

Plasma copper levels were within normal ranges (10-22 WM) throughout the study

period. They suggest that under the conditions of this study, copper status was not

altered. These results reflect those of other studies that have examined the effects of zinc

supplementation on copper status (Taper et al., 1980; Fischer et al., 1984). The

mechanism of the inhibition of copper absorption by high zinc intake is believed to involve

the increased synthesis of MT (Cousins, 1985). However, balance studies have reported

that even moderate supplementation of zinc (15 mg/day) may increase the requirements

for copper (Sandstead, 1982). Severe copper deficiency has been documented when zinc

intake exceeds 100 mg/day (Prasad et al., 1978; Porter et al., 1977). The trend toward

increased serum copper concentrations during the study may also be a reflection of a

response to multiple blood draws. Induction of ceruloplasmin, the major copper protein in

plasma, may respond to factors (cytokines) released following multiple blood draws.

The results presented in this study have shown for the first time that cellular MT

mRNA levels change in vivo in human subjects in response to dietary zinc. Two decades









of data with animals and isolated cells support the deduction that zinc is the direct inducer

of increased MT mRNA and protein expression in humans.
When compared to the former competitive ELISA, the sandwich ELISA

developed in this study provides a more convenient method for the measurement of

erythrocyte hMT during zinc supplementation. The response of monocyte MT mRNA

and erythrocyte hMT protein together could serve as an index of zinc supplementation and

that could be of value in studies where supplementation efficacy must be monitored.

These new methods may also have value in future experiments for assessment of zinc

deficiency if the responsiveness ofmonocyte MT mRNA and erythrocyte MT protein to

depletion and varying zinc intake can be demonstrated using these newly developed

methods. In addition, the RT-PCR approach has wide applicability for nutrient assessment

in those situations where genes are differentially expressed, either directly or indirectly

through mediators, by changes in dietary intake of a specific nutrient.

Areas that would be of interest for future study include immunohistochemistry for

the detection of hMT protein levels in monocytes. The assessment of acute phase proteins

and FACS analysis ofmonocyte production may help to factor out the effects of illness on

monocyte hMT mRNA if future experiments show that illness will influence these levels.

In addition evaluation of MT protein and mRNA levels during dietary zinc restriction, and

comparison studies in female subjects would provide further insight on the usefulness of

MT as an index of human zinc status. Eventually, definitive methods for the measurement

of dietary zinc status in humans could lead to a better understanding of the level of dietary

zinc that is essential for optimal health.














APPENDIX A
QUESTIONNAIRE FOR ZINC SUPPLEMENTATION STUDY'


Date_______


Nami


e_______________


Last


First


Ss#
Phone


Age__


Day


Middle



Evening


Date of Birth______________


Adress


Sex______
Race Caucasian
______Black
______Asian

Education Completed


______Hispanic

______Other


_____ High School Graduate
_____ Bachelor's Degree
____ Master's Degree
_____Doctoral Degree


Present Work/Student Status (check all that apply):
_____ Working Full-time ____ Full-time Student
____ Working Part time ____ Part-time Student
____Not Employed

Medical History


:Adopted from a questionnaire developed by Dr. Gail P.A. Kauwell.





74


Indicate if you have had or currently have any of the following medical problems (check
all that apply):

Past Now
__ Alcholism
____ Allergies
__ Anemia
Arthritis
__Asthma
__ Blood Clots
__ Bronchitis
__ Cardiovascular Disease
(Atherosclerosis/ Heart Disease)
__ Claudication
S__ Cystic Fibrosis
__ Dermatitis
__ Emotional Disorder
__ Eating Disorder
__ Gall Bladder Disease
__ Glaucoma
__Gout
__ Hair Loss (Excessive)
__ Headaches
__ Hemorrhoids
____ Hepatitis
__ Hernia
___ Hypercholesterolemia/hyperlipidemia
__ Intestinal Disorders
__- Kidney Disease
Liver Disease
___ Lung Disease
__ Mental Illness
S __ Neurologic Disorder
___ Obesity/Overweight
Prostate Trouble
__ Rheumatic Fever
Seizure Disorders
__ Stomach Disease
__ Stroke
___ Thyroid Disease
Tumors/Cancer List type:
__ Ulcers
___ Other Please Specify: ___________

Have you ever had any type of surgery? If so please indicate:_______













Indicate any prescription /non-prescription medicines you currently use on a regular basis
(check all that apply):
___ Allergy Medicine/Antihistamines ___ Diuretics
___ Antacids ___ Heart Medicines
___ Antibiotics ___ Hormones
___ Anti-arrhythmics ___ Laxatives
___ Aspirin ___ Nitroglycerin
___ Asthma Medicines ___ Pain Medicines
___ Beta Blockers ___ Psychiatric Medicines/
___ Blood Pressure Medicines Antidepressants
___ Blood Thinners (i.e. anticoagulants) ___ Sedatives/Sleeping Pills
___ Cortisone ___ Seizure Medicines
___ Decongestants Thyroid Medicines
___ Diabetes Medicines/Insulin ___ Tranquilizers

____ Other Specify_______________________________

Do you currently take any type of nutritional supplement (i.e. vitamin/mineral pills; protein
supplements; health food store supplements; Slimfast, Dexatrim, or any other diet
products, etc?
___ yes ___ No

If you answered "yes" to the last question, indicate the brand(s), type of supplements(s),
frequency, and amountss.

Do you use any tobacco products? If so please specify.

Indicate your usual exercise activities, and frequency below. Please circle any activities
below that you participate in more than once per month:

Badminton Baseball/Softball Basketball Boating Bowling Cycling (motor)

Cycling (road) Cycling (stationery) Climbing (scaling) Dancing (aerobic) *

Dancing (social) *Golf(with cart) Golf(walking) Gymnastics Hiking

Horseback Riding Hunting Jogging/Running Martial Arts *
Racquetball/Handball

Rope Jumping Rowing/Canoeing Skating/Roller blading Skiing








Soccer/football Stairmaster Swimming Table Tennis Tennis Treadmill

Triathlons Volleyball Walking Weight Training Wind surfing

Yard Work/Gardening Other -
Specifiy_____________





For all activities that were circled above, indicate the following:
Activity Frequency/month Minutes/Session













Does your usual job/schoolwork require sustained physical activity ?
_yes ___no

If yes please specify



Diet History

Height (without shoes) _______ Feet & inches
Weight(dressed) Pounds
Is your weight fairly stable? yes ___no, If no please specify_______
Are you satisfied with your current weight? If no please explain_______

Have you lost or gained more than 10 pounds in the past year? ___ yes ___ no
If yes explain
How many times (meals & snacks) do you eat each day? ________
Are you allergic to any foods? ___ Yes ___ No
If yes, please list all foods: ________________








Are there any foods you cannot or will not eat? yes ___ no
If yes please list these foods:______________

How would you describe your appetite (check only one)?
___Small ___Medium ___Large



Do you follow any of the modified diets listed below?
___ Diabetic ___ Weight Reduction
____ Low Sodium ____ Weight Gain
___ Renal ___ Gastric Banding
___ Ulcer/Bland ___ Kosher
___ Vegetarian ___ Macrobiotic
___ Low Cholesterol/Low Fat
___ Other; Please specify______________

How often do you drink the following beverages?
PER DAY PER WEEK
Coffee
Tea
Soda (Specify Type)

Could you limit your intake of these beverages to 1 cup/day if necessary without any
difficulty? ___Yes ___No

Do you consume alcoholic beverages (i.e. beer, wine, wine coolers, hard liquor, etc.)?
___Yes ___No

If yes, could you discontinue your intake of these for 5 weeks? ___ Yes ___ No


Food Frequency List

Indicate the amount and frequency with which you consume the following:
Amt. Never/ Occas. Feql. Always
Rarely (per/day)
Beef ___ ___ ___ ___ ___

Beer(regular) ___ ___ ___ ___ ___

Beer (light) ___ ___ ___ ___ ___

Bread





78


Cake ___ ___ ___ ___ ___

Candy ___ ___ ___ ___ ___

Cereal ___ ___ ___ ___ ___

Chicken ___ ___ ___ ___ ___

Coffee (regular)___ ___ ___ ___ ___

Cola (regular) ___ ___ ___ ___

Cola (diet) ___ ___ ___ ___

Cheese ___ ___ ___ ___ ___

Cookies ___ ___ ___ ___

Crackers ___ ___ ___

Eggs ___ ___

Fish ___ ___ ___ ___ ___

Fruit ___ ___ ___ ___

Fruit Juice ___ ___ ___ ___

Hard Liquor ___ ___ ___

Legumes ___ ___ ___ ___
(beans)

Milk ___ ___ ___ ___ ___

Pork ___ ___ ___ ___ ___

Shellfish ___ ___ ___ ___ ___

Tea ___ ___ ___ ___ ___

Turkey ___ ___ ___ ___ ___

Vegetables ___ ___ ___ ___ _____



78








Wine ___ ___ ___ ___ ___

Wine Coolers ___ ___ ___ ___


Indicate if you disagree (DA), moderately agree (MA) or strongly agree (SA) with each of
the following statements:

DA MA SA
I am very conscious of what I eat. ___ ___ ___

I consider myself to have a lot of
will power. ___ ___

I don't usually pay much attention to
what I eat. ___ ___














APPENDIX B


INFORMATION SHEET FOR ZINC STUDY



Thank you for participating in this study. You have been given a schedule that

includes all of the days that you are required to come in to have blood drawn and/or take

an oral supplement. To be eligible for a $200 honorarium, you must complete the entire

36 days of the study and fulfil the following requirements:

1. Report to the FSHN building (2nd floor) between 7:00 7:45 a.m. on the days
listed on your schedule. Our phlebotomist will only be available during these
hours, so please do not be late.

2. On the days that you will have blood drawn, do not eat anything after 10:00
p.m. the night before, only water until after the blood draws.

3. Do not drink alcohol during the 36 days of the study.

4. Limit your intake of caffinated drinks (i.e. coffee, tea, soda, diet soda) to three
cups per day.

5. Do not donate blood or plasma at anytime during the study. If you have any
type of a medical situation come up for which you must take medication or
have medical attention during the 36 days, please make us aware of this as
soon as possible. Even if its just a common cold, just let me know so I can
make a note of it.

6. Please avoid the following zinc rich foods: oysters, shrimp, and any type of
organ meats.

7. If you normally take any type of a supplement (vitamins, diet products,
proteins shakes etc.) please discontinue them during the study.













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Apgar, J. (1985) Zinc and reproduction. Annu. Rev. Nutr. 5: 43-68.

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Burke, J.P. & Fenton, M.R. (1985) Effect of a zinc-deficiency diet on lipid peroxidation in
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