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PHARMACEUTICAL & TOXICOLOGICAL INVESTIGATIONS OF METALS: INVESTIGATIONS OF SUPPLEMENTAL CHROMIUM(III) AND IRON OXIDE NANOPARTICLES IN RODENT MODELS by KRISTIN ROGERS DI BONA JANE F. RASCO, COMMITTEE CHAIR RYAN L. EARLEY JANIS M. O’DONNELL KATRINA M. RAMONELL JOHN B. VINCENT A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences in the Graduate School of The University of Alabama TUSCALOOSA, ALABAMA 2014

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Page 1: PHARMACEUTICAL & TOXICOLOGICAL INVESTIGATIONS OF METALSacumen.lib.ua.edu/content/u0015/0000001/0001804/u0015_0000001_0001804.…pharmaceutical & toxicological investigations of metals:

PHARMACEUTICAL & TOXICOLOGICAL INVESTIGATIONS OF METALS:

INVESTIGATIONS OF SUPPLEMENTAL CHROMIUM(III) AND IRON OXIDE

NANOPARTICLES IN RODENT MODELS

by

KRISTIN ROGERS DI BONA

JANE F. RASCO, COMMITTEE CHAIR

RYAN L. EARLEY

JANIS M. O’DONNELL

KATRINA M. RAMONELL

JOHN B. VINCENT

A DISSERTATION

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

in the Department of Biological Sciences

in the Graduate School of

The University of Alabama

TUSCALOOSA, ALABAMA

2014

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Copyright Kristin R. Di Bona 2014

ALL RIGHTS RESERVED

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ABSTRACT

Trivalent chromium (Cr3+) has widely been accepted as a nutritional element necessary for

proper carbohydrate and lipid metabolism in mammals. Upon closer examination, beneficial

effects resulting from Cr supplementation in many rodent studies are actually pharmaceutical in

nature due in part to additional stressors and supranutritional doses. Zucker lean, obese (ZOB),

and diabetic fatty (ZDF) rats were used to examine the effects of Cr supplementation on healthy

and insulin-resistant models of type 2 diabetes and pre-diabetes, respectively. Increased insulin

sensitivity was observed in Zucker lean rats receiving a highly Cr-supplemented diet (+1,000 μg

Cr/kg diet), although urinary Cr levels did not correlate with supplementation. ZDF rats displayed

both increased absorption and increased urinary excretion of 51Cr when given a single 51CrCl3 dose.

With extended Cr supplementation, elevated kidney Cu levels in the ZDF rats decreased in the

highest CrCl3 and Cr3 treatments (1,000 μg Cr/kg body mass).

Nanoparticles (NPs) are widely being explored for use in biomedicine. One concern with the

increased prevalence and availability of pharmaceutical NPs is the potential developmental

toxicity that may result from exposure in utero. Due in part to their small size, NPs may have the

ability to cross the placenta and accumulate in the fetus. Iron oxide NPs are currently being used

as supplements for patients with Fe deficiencies as well as contrast agents for magnetic resonance

imaging. In order to aid in a more “intelligent design” of NPs to which pregnant women may be

exposed, the developmental toxicity of surface-charged iron oxide NPs was investigated in CD-1

mice in order to determine whether iron oxide NPs cross the placenta and accumulate in the fetus

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and whether the surface-charge influences toxicity. Pregnant CD-1 mice were exposed to 1 or 8

doses of 10 mg NPs/kg body mass, the equivalent of one MRI exposure. Exposure to positively-

charged polyethylenimine-coated NPs resulted in greater toxicity compared to controls or

negatively-charged poly(acrylic acid)-coated NPs, exhibiting increased fetal resorptions,

decreased maternal weight gain, and increased Fe accumulation in the fetal liver.

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DEDICATION

This manuscript and the years of work that went into producing it would not have been possible

without the support of my family. My husband Caleb and our son Ronin are the most important

people in my life, and are the driving force for all of my pursuits. Ronin and Caleb are constant

sources of love, support, silliness, and encouragement and I can never thank them enough. I also

want to thank my parents, Robin and Lil, for everything they have done and continue to do for me.

I am sure I would not be the person I am today if it were not for them. For these reasons and many,

many more I dedicate this manuscript to my family (related or not) and thank them as best I can

for continuing to love and support me, as I hope to do for them.

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LIST OF ABBREVIATIONS AND SYMBOLS

± Plus or minus

AAALAC Association for the advancement and accreditation of laboratory animal care

ANOVA Analysis of variance

AUC Area under the curve

cm Centimeter

CO2 Carbon dioxide

Cr Chromium(III)

Cr3 [Cr3O(propionate)6(H2O)3]+

CrCl3 Chromium chloride

51CrCl3 Chromium-51 labelled chromium chloride

g Gram

h Hour(s)

H2O2 Hydrogen peroxide

125I RIA Iodine-125 labelled radio immunoassay

kg Kg

L Liter

LMWCr Low molecular weight chromium binding substance

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LSD Least significant difference

mg Milligram

min Minute(s)

mL Milliliter

mm Millimeter

mmol Millimolar

MRI Magnetic resonance imaging

NPs Nanoparticles

nm Nanometer

PAA Poly(acrylic acid)

PEI Polyethylenimine

pic Picolinate

ppm Parts per million

r2 Coefficient of determination

s Second

SEM Standard error of the mean

STZ Streptozotocin

TPN Total parenteral nutrition

ZDF Zucker diabetic fatty

ZOB Zucker obese

µg Microgram

µL Microliter

µM Micromolar

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ACKNOWLEDGMENTS

I would like to acknowledge and thank everyone who aided in the completion of the work

described herein. First and foremost I would like to acknowledge my advisor Dr. Jane Rasco for

all of her guidance and mentorship throughout my graduate studies. I would also like to thank the

other members of my committee: Dr. John Vincent, Dr. Janis O’Donnell, Dr. Ryan Earley, Dr.

Katrina Ramonell, and previously Dr. Perry Churchill who provided invaluable insight and

guidance throughout my graduate studies.

The chromium research would not have been possible without the contributions of the Vincent

group, especially Drs. John Vincent and Sharifa Love-Ruteledge, as well as DeAna McAdory, Ge

Deng, and Drs. Nick Rhodes, Sharmistha Sinha, and Yuan Chen. I would especially like to thank

Sharifa for measuring the chromium content in the urine samples, and being an excellent office

mate for the past couple of years. I would like to acknowledge the Krejpcio lab in Poland for

measuring tissue metal concentrations.

I would like to acknowledge and thank Drs. Yuping Bao and Yaolin Xu for the work and support

they provided in the nanoparticle research. I would especially like to thank Yaolin for synthesizing

iron oxide nanoparticles for this research.

I would also like to thank all of the undergraduates in the Rasco group throughout the past few

years for their help with the studies. I would like to thank Julia Kent specifically for helping

perform glucose and insulin challenges on the chromium research and Paul and Javeia for the work

they provided on the nanoparticle research.

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CONTENTS

ABSTRACT ............................................................................................................ ii

DEDICATION ....................................................................................................... iv

LIST OF ABBREVIATIONS AND SYMBOLS ....................................................v

ACKNOWLEDGMENTS .................................................................................... vii

LIST OF TABLES ................................................................................................ xii

LIST OF FIGURES ............................................................................................. xiii

1. INTRODUCTION: PHYSIOLOGICAL INVESTIGATIONS INTO THE

USE OF CHROMIUM(III) AS A PHARMACEUTICAL AND THE

DEVELOPMENTAL TOXICITY OF SURFACE-CHARGED IRON

OXIDE NANOPARTICLES .......................................................................1

1.1 Pharmaceutical versus Nutritional Studies of Chromium ............................1

1.2 Chromium, Diabetes, and Zucker Rats ........................................................4

1.3 Exposure to Pharmaceutical Metal NPs In Utero: Considerations to

Reduce the Developmental Toxicity of Engineered Metal Oxide NPs by

More Intelligent Design ...............................................................................7

1.4 References ..................................................................................................10

2. INVESTIGATIONS INTO THE EFFECTS OF EXTENED

CHROMIUM(III) SUPPLEMENTATION ON GLUCOSE

METABOLISM AND INSULIN SENSITIVITY AND URINARY

CHROMIUM LOSS AS A BIOMARKER FOR DIETARY CHROMIUM

STATUS IN HEALTHY ZUCKER LEAN RATS ...................................15

2.1 Introduction ................................................................................................15

2.2 Materials and Methods ...............................................................................18

2.2.1 Chemicals, Assays, and Instrumentation ...................................................18

2.2.2 Animals and Husbandry .............................................................................18

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2.2.3 Treatments..................................................................................................19

2.2.4 Metal-Free Housing ...................................................................................19

2.2.5 Food and Water Containers.......................................................................20

2.2.6 Data Collection ..........................................................................................21

2.2.7 Cr Concentration in Diets ..........................................................................21

2.2.8 Cr Concentration in Urine .........................................................................22

2.2.9 Statistical Analyses ....................................................................................23

2.3 Results and Discussion ..............................................................................24

2.3.1 Carefully Controlled Access to Cr: Metal-Free Caging............................24

2.3.2 Carefully Controlled Access to Cr: Analysis of the Diets..........................27

2.3.3 Effects of Cr Supplementation on Physiological Factors ..........................29

2.3.4 Effects of Cr Supplementation on Response to Glucose and Insulin

Challenges..................................................................................................32

2.3.4.1 Glucose Levels in Response to Challenges ................................................33

2.3.4.2 Insulin Levels in Response to Challenges ..................................................39

2.3.4.3 Urinary Cr Loss as a Biomarker for Cr Administration Status ................42

2.4 Conclusions ................................................................................................50

2.5 References ..................................................................................................52

3. PHARMACOKINETICS OF A SINGLE ORALLY ADMINISTERED

DOSE OF 51CrCl3 IN ZUCKER LEAN, TYPE 2 DIABETIC (ZUCKER

DIABETIC FATTY), AND PRE-DIABETIC (ZUCKER OBESE)

RATS .........................................................................................................56

3.1 Introduction ................................................................................................56

3.2 Materials and Methods ...............................................................................58

3.2.1 Materials and Instrumentation ..................................................................58

3.2.2 Animals and Husbandry .............................................................................58

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3.2.3 Sample Collection .....................................................................................59

3.2.4 Statistical Analyses ....................................................................................60

3.3 Results and Discussion ..............................................................................60

3.3.1 Cr Supplementation ...................................................................................60

3.3.2 51Cr Pharmacokinetics ...............................................................................61

3.3.3 51Cr Absorption ..........................................................................................72

3.4 Conclusions ................................................................................................77

3.5 References ..................................................................................................78

4. THE EFFECTS OF DIABETES AND EXTENDED CHROMIUM

SUPPLEMENTATION ON THE TISSUE METAL

CONCENTRATIONS OF ZUCKER LEAN, ZUCKER OBESE, AND

ZUCKER DIABETIC FATTY RATS .......................................................80

4.1 Introduction ................................................................................................80

4.2 Materials and Methods ...............................................................................84

4.2.1 Animals and Husbandry .............................................................................84

4.2.2 Treatments..................................................................................................85

4.2.3 Surgeries and Organ Collection ................................................................85

4.2.4 Atomic Absorption Spectrometry for Metal Analyses ................................86

4.2.5 Chromium Compounds ..............................................................................86

4.2.6 Statistical Analyses ....................................................................................86

4.3 Results and Discussion ..............................................................................87

4.3.1 Differences Between Strains (Healthy, Obese/Pre-Diabetic, Type 2

Diabetic) ....................................................................................................87

4.3.2 Chromium and Vanadium Supplementation .............................................90

4.3.3 Effects of Supplementation of Cr and Vanadium on Tissue Metal

Concentrations ...........................................................................................92

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4.4 Conclusions ..............................................................................................109

4.5 References ................................................................................................111

5. SURFACE CHARGE AND DOSAGE DEPENDENT

DEVELOPMENTAL TOXICITY AND BIODISTRIBUTION OF IRON

OXIDE NANOPARTICLES IN PREGNANT CD-1 MICE ...................114

5.1. Introduction ..............................................................................................114

5.2. Materials and Methods .............................................................................117

5.2.1 Animals and Husbandry ...........................................................................117

5.2.2 Nanoparticle Preparation and Characterization.....................................117

5.2.3 Treatments................................................................................................118

5.2.4 Data Collection ........................................................................................119

5.2.5 Statistical Analysis ...................................................................................121

5.3 Results and Discussion ............................................................................121

5.3.1 Nanoparticle Synthesis and Characterization .........................................121

5.3.2 Effect of Surface-Charged NPs on Dams.................................................123

5.3.3 Effects of Charged NPs on Litter Values .................................................125

5.3.4 Biodistribution of Surface-Charged NPs in Fetal Tissues .......................128

5.4 Conclusions ..............................................................................................131

5.5 References ................................................................................................133

6. OVERALL CONCLUSIONS ..................................................................137

6.1. References ................................................................................................140

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LIST OF TABLES

2.1 Actual Cr content of purified AIN-93G rodent diets measured by GFAA

....................................................................................................................28

5.1 Treatment groups and number of animals per group (n) .........................119

5.2 Maternal weight gain (g ± SEM) for treatment groups as follows

(1) Controlx8, (2) PEIx1+, (3) PAAx1-, (4) PEIx8+, and (5) PAAx8-,

n = 14-18. *indicates significant differences compared to all other groups

(p < 0.05) ..................................................................................................123

5.3 Litter values for treatment groups as follows (1) Controlx8, (2) PEIx1+,

(3) PAAx1-, (4) PEIx8+, and (5) PAAx8-, n = 14-18, *indicates

significant difference versus control and single dosed treatment groups

(p < 0.05) ..................................................................................................126

5.4 Resorptions and dead fetus distribution. Total resorptions and dead

fetuses are expressed as the average percentage in each litter. Early and

late resorptions are presented as a percentage of the total resorptions.

n = 14-18, *indicates significant difference versus control and single

dosed treatment groups (p < 0.05) ...........................................................128

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LIST OF FIGURES

2.1 Metal-free housing for rodents...................................................................25

2.2 Body mass of Zucker lean rats on the standard and modified AIN-93G

diets: AIN-93G without Cr added to the mineral mixture (low Cr); the

standard AIN-93G diet (Cr sufficient); an additional 200 μg Cr/kg diet

(+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg).

Different letters indicate significant differences between groups

(p ≤ 0.05) ....................................................................................................30

2.3 Non-heme plasma Fe levels for Zucker lean rats on the standard and

modified AIN-93G diets: AIN-93G without Cr added to the mineral

mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an additional

200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet

(+ 1000 μg Cr/kg) ......................................................................................31

2.4 Plasma glucose levels for Zucker lean rats on the standard and modified

AIN-93G diets during glucose tolerance testing: AIN-93G without Cr

added to the mineral mixture (low Cr); the standard AIN-93G diet

(Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an

additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). Different letters indicate

significant differences between groups ......................................................33

2.5 Plasma glucose concentrations during glucose tolerance testing

represented by the area under the curve for Zucker lean rats on the

standard and modified AIN-93G diets: AIN-93G without Cr added to the

mineral mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an

additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg

Cr/kg diet (+ 1000 μg Cr/kg). Different letters indicate significant

differences between groups........................................................................34

2.6 Plasma glucose levels during insulin tolerance testing for Zucker lean rats

on the standard and modified AIN-93G diets: AIN-93G without Cr added

to the mineral mixture (low Cr); the standard AIN-93G diet (Cr

sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an

additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). Different letters indicate

significant differences between groups ......................................................35

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2.7 Plasma glucose concentrations during insulin tolerance testing represented

by the area under the curve for Zucker lean rats on the standard and

modified AIN-93G diets: AIN-93G without Cr added to the mineral

mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an additional

200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet

(+ 1000 μg Cr/kg) ......................................................................................36

2.8 Plasma insulin levels during glucose tolerance testing for Zucker lean rats

on the standard and modified AIN-93G diets: AIN-93G without Cr added

to the mineral mixture (low Cr); the standard AIN-93G diet

(Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an

additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). Different letters indicate

significant differences between groups ......................................................39

2.9 Plasma insulin concentrations during glucose tolerance testing represented

by the area under the curve for Zucker lean rats on the standard and

modified AIN-93G diets: AIN-93G without Cr added to the mineral

mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an additional

200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet

(+ 1000 μg Cr/kg). Different letters indicate significant differences

between groups ..........................................................................................41

2.10 Rate of urinary Cr loss (ng Cr/h) in response to a glucose challenge for

Zucker lean rats on the standard and modified AIN-93G diets: for Zucker

lean rats on the standard and modified AIN-93G diets: AIN-93G without

Cr added to the mineral mixture (low Cr); the standard AIN-93G diet

(Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an

additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). The initial time point is

the rate of Cr loss measured throughout 6 h before a glucose challenge.

Rates were subsequently measured from t = 0 through t = 2, then from

t = 2 to t = 6, and finally from t = 6 through t = 12 h after glucose

injection......................................................................................................44

2.11 Rate of urinary Cr loss (ng Cr/h) in response to an insulin challenge for

Zucker lean rats on the standard and modified AIN-93G diets: for Zucker

lean rats on the standard and modified AIN-93G diets: AIN-93G without

Cr added to the mineral mixture (low Cr); the standard AIN-93G diet

(Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an

additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). The initial time point is

the rate of Cr loss measured throughout 6 h before an insulin challenge.

Rates were subsequently measured from t = 0 through t = 2, then from

t = 2 to t = 6, and finally from t = 6 through t = 12 h after insulin

injection......................................................................................................45

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2.12 Rate of urinary Cr loss in response to a glucose challenge represented by

the area under the curve for Zucker lean rats on standard and modified

AIN-93G diets: AIN-93G without Cr added to the mineral mixture (low

Cr); the standard AIN-93G diet (Cr sufficient); an additional 200 μg Cr/kg

diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg

Cr/kg) .........................................................................................................46

2.13 Rate of urinary Cr loss in response to an insulin challenge represented by

the area under the curve for Zucker lean rats on standard and modified

AIN-93G diets: AIN-93G without Cr added to the mineral mixture (low

Cr); the standard AIN-93G diet (Cr sufficient); an additional 200 μg Cr/kg

diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg

Cr/kg) ....................................................................................................47

2.14 Rate of urinary Cr loss (ng Cr/h) in response to an insulin challenge for

individual Zucker lean rats on the standard and modified AIN-93G diets:

for Zucker lean rats on the standard and modified AIN-93G diets: AIN-

93G without Cr added to the mineral mixture (low Cr); the standard AIN-

93G diet (Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg);

or an additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). The initial time

point is the rate of Cr loss measured throughout 6 h before an insulin

challenge. Rates were subsequently measured from t = 0 through t = 2,

then from t = 2 to t = 6, and finally from t = 6 through t = 12 h after insulin

injection......................................................................................................48

3.1.A Concentration of 51Cr measured in the gastrointestinal tract and feces after

an oral dose of 51CrCl3 in Zucker lean rats. Concentration is represented by

the percentage of the applied dose measured for each sample as a function

of time. Letters indicate the concentration of 51Cr is (b) significantly

different from ZOB rats and (c) significantly different from ZDF rats

(p ≤ 0.05) ....................................................................................................62

3.1.B Concentration of 51Cr measured in the gastrointestinal tract and feces after

an oral dose of 51CrCl3 in ZOB rats. Concentration is represented by the

percentage of the applied dose measured for each sample as a function of

time. Letters indicate the concentration of 51Cr is (a) significantly different

from Zucker lean rats or (c) significantly different from ZDF rats

(p ≤ 0.05). ...................................................................................................63

3.1.C Concentration of 51Cr measured in the gastrointestinal tract and feces after

an oral dose of 51CrCl3 in ZDF rats. Concentration is represented by the

percentage of the applied dose measured for each sample as a function of

time. Letters indicate the concentration of 51Cr is (a) significantly different

from Zucker lean and (b) significantly different from ZOB rats (p ≤ 0.05)

....................................................................................................................64

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3.2.A Concentration of 51Cr measured in the blood and urine after an oral dose

of 51CrCl3 in Zucker lean rats. Concentration is represented by the

percentage of the applied dose measured for each sample as a function of

time. Letters indicate the concentration of 51Cr is (b) significantly different

from ZOB rats and (c) significantly different from ZDF rats (p ≤ 0.05) ...65

3.2.B Concentration of 51Cr measured in the blood and urine after an oral dose

of 51CrCl3 in ZOB rats. Concentration is represented by the percentage of

the applied dose measured for each sample as a function of time. Letters

indicate the concentration of 51Cr is (a) significantly different from Zucker

lean rats and (c) significantly different from ZDF rats (p ≤ 0.05) .............66

3.2.C Concentration of 51Cr measured in the blood and urine after an oral dose

of 51CrCl3 in ZDF rats. Concentration is represented by the percentage of

the applied dose measured for each sample as a function of time. Letters

indicate the concentration of 51Cr is (a) significantly different from Zucker

lean rats and (b) significantly different from ZOB rats (p ≤ 0.05) ............67

3.3.A Concentration of 51Cr measured in the right femur, heart, skeletal muscle,

testes, and epididymal fat after an oral dose of 51CrCl3 in Zucker lean rats.

Concentration is represented by the percentage of the applied dose

measured for each sample as a function of time ........................................68

3.3.B Concentration of 51Cr measured in the right femur, heart, skeletal muscle,

testes, and epididymal fat after an oral dose of 51CrCl3 in ZOB rats.

Concentration is represented by the percentage of the applied dose

measured for each sample as a function of time. Letters indicate the

concentration of 51Cr is (c) significantly different from ZDF rats (p ≤ 0.05)

....................................................................................................................69

3.3.C Concentration of 51Cr measured in the right femur, heart, skeletal muscle,

testes, and epididymal fat after an oral dose of 51CrCl3 in ZDF rats.

Concentration is represented by the percentage of the applied dose

measured for each sample as a function of time. Letters indicate the

concentration of 51Cr is (b) significantly different from ZOB rats

(p ≤ 0.05) ....................................................................................................70

3.4.A Concentration of 51Cr measured in the pancreas, spleen, liver and kidney

after an oral dose of 51CrCl3 in Zucker lean rats. Concentration is

represented by the percentage of the applied dose measured for each

sample as a function of time ......................................................................71

3.4.B Concentration of 51Cr measured in the pancreas, spleen, liver and kidney

after an oral dose of 51CrCl3 in ZOB rats. Concentration is represented by

the percentage of the applied dose measured for each sample as a function

of time ........................................................................................................72

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3.4.C Concentration of 51Cr measured in the pancreas, spleen, liver and kidney

after an oral dose of 51CrCl3 in ZDF rats. Concentration is represented by

the percentage of the applied dose measured for each sample as a function

of time ........................................................................................................73

4.1.A Body masses of Zucker lean rats supplemented daily with Cr or vanadyl

sulfate. No significant differences were observed .....................................89

4.1.B Body masses of ZOB rats supplemented daily with Cr or vanadyl sulfate.

No significant differences were observed ..................................................89

4.1.C Body masses of ZDF rats supplemented daily with Cr or vanadyl sulfate.

No significant differences were observed ..................................................90

4.2.A Liver Cr concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. Dagger represents significant difference from

Zucker lean rats (p ≤ 0.05) .........................................................................93

4.2.B Kidney Cr concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. Dagger represents significant

difference from Zucker lean rats (p ≤ 0.05) ...............................................93

4.3.A Liver Cu concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. Double dagger represents significant difference

from ZDF rats (p ≤ 0.05). Single asterisk indicates significant difference

from the other two rat strains (p ≤ 0.05) ....................................................94

4.3.B Spleen Cu concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains .....................................................94

4.3.C Spleen Cu concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains .....................................................95

4.3.D Heart Cu concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. No significant differences were observed

between treatments or strains .....................................................................95

4.4.A Liver Zn concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. Double dagger represents significant difference

from ZDF rats (p ≤ 0.05). Single asterisk indicates significant difference

from the other two rat strains (p ≤ 0.05) ....................................................96

4.4.B Kidney Zn concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains .....................................................96

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4.4.C Spleen Zn concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains .....................................................97

4.4.D Heart Zn concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. No significant differences were observed

between treatments or strains .....................................................................97

4.5.A Liver Fe concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. Single asterisk indicates significant difference

from the other strains (p ≤ 0.05). Double asterisk indicates all strains are

significantly different from each other (p ≤ 0.05) ......................................98

4.5.B Kidney Fe concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains .....................................................98

4.5.C Spleen Fe concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. Double dagger represents

significant difference from ZDF rats (p ≤ 0.05) ........................................99

4.5.D Heart Fe concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. No significant differences were observed

between treatments or strains .....................................................................99

4.6.A Liver Mg concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. Single asterisk indicates significant difference

from the other two rat strains (p ≤ 0.05). Dagger represents significant

difference from Zucker lean rats (p ≤ 0.05). Double dagger represents

significant difference from ZDF rats (p ≤ 0.05) ......................................100

4.6.B Kidney Mg concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains ...................................................100

4.6.C Spleen Mg concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains ...................................................101

4.6.D Heart Mg concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains ...................................................101

4.7.A Liver Ca concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. No significant differences were observed

between treatments or strains ...................................................................102

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4.7.B Kidney Ca in Zucker lean, ZOB, and ZDF rats supplemented with Cr or

vanadyl sulfate. Single asterisk indicates significant difference from the

other rat strains (p ≤ 0.05) ........................................................................102

4.7.C Spleen Ca concentrations in Zucker lean, ZOB, and ZDF rats

supplemented with Cr or vanadyl sulfate. No significant differences were

observed between treatments or strains ...................................................103

4.7.D Heart Ca concentrations in Zucker lean, ZOB, and ZDF rats supplemented

with Cr or vanadyl sulfate. No significant differences were observed

between treatments or strains ...................................................................103

4.8.A Kidney Cr concentrations of Zucker lean rats supplemented with Cr or

vanadyl sulfate. Different letters indicate significant difference between

treatment groups (p ≤ 0.05) ......................................................................104

4.8.B Kidney Cr concentrations of ZOB rats supplemented with Cr or vanadyl

sulfate. Different letters indicate significant difference between treatment

groups (p ≤ 0.05). .....................................................................................104

4.8.C Liver Ca concentrations of ZOB rats supplemented with Cr or vanadyl

sulfate. Different letters indicate significant difference between treatment

groups (p ≤ 0.05) ......................................................................................105

4.8.D Kidney Cu concentrations of ZDF rats supplemented with Cr or vanadyl

sulfate. Different letters indicate significant difference between treatment

groups (p ≤ 0.05) ......................................................................................105

5.1 TEM images of (A) PEI-NPs and (B) PAA-NPs in H2O.........................122

5.2 Zeta potentials of (A) PEI-NPs and (B) PAA-NPs in H2O ......................122

5.3 Maternal weight gain assessed by subtracting the female body mass

measured on GD 0 from the final body mass minus gravid uteri on GD 17,

n = 14-18, *indicates significant differences compared to all other groups

(p < 0.05) ..................................................................................................124

5.4 Percent resorbed fetuses, n = 14-18, *indicates significant difference

versus control and single dosed treatment groups (p < 0.05) ..................127

5.5 Fetal livers stained for iron content using Prussian Blue (blue indicates

presence of iron) in (A) Control (H2O treated) (1), (B) 1 dose of PEI NPs

(2), and (C) 8 doses of PEI coated NPs (4) ..............................................129

5.6 Fetal liver iron content, n = 9, *indicates significant differences compared

to all other groups (p < 0.05) ...................................................................130

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CHAPTER 1

INTRODUCTION: PHYSIOLOGICAL INVESTIGATIONS INTO THE USE OF

CHROMIUM(III) AS A PHARMACEUTICAL AND THE DEVELOPMENTAL TOXICITY

OF SURFACE-CHARGED IRON OXIDE NANOPARTICLES

1.1: Pharmaceutical versus Nutritional Studies of Chromium

Trivalent Cr is currently considered an essential nutrient (trace element) in mammals, necessary

for proper glucose and lipid metabolism. Chemical elements required by the body in order to

function properly, such as Ca, K, Mg, and Na, are considered dietary elements. Trace elements are

dietary elements that are required at much lower concentrations. Examples of trace elements

include Fe, Zn, and Se.1 Cr has long been investigated as a potential trace element responsible for

maintaining normal glucose metabolism since it was suggested in the 1950’s that Cr is the active

part of a biological complex designated “glucose tolerance factor.”2 Whether Cr is actually

essential to maintain homeostasis of these essential functions in the human body is a topic of debate

with compelling arguments both pro- and anti-essentiality.3-9 Regardless of status, most agree that

the amount that would be required in the diet daily is so small (25 or 35 μg Cr/d for women and

men respectively)10 that greater than 98 % of Americans would receive this amount in their self-

selected diets without additional supplementation.5, 6

The scope of the research presented throughout this dissertation is primarily focused on the

pharmaceutical effects of large doses of orally administered Cr. Many studies that have sought to

examine the essentiality of Cr by inducing a Cr deficient model and supplementing their diet with

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Cr have actually been investigating the pharmaceutical benefits of Cr supplementation due in part

to the small amount of Cr considered adequate and the utilization of large or unregulated doses of

Cr. Additionally, extraneous sources of Cr were often not properly controlled, such as continuous

access to wire cages which contain appreciable levels of Cr. Rats often bite or chew on the wire

caging which allows access to Cr, a large component of stainless steel. In general, supplementation

of Cr to rodents with impaired glucose tolerance results in beneficial effects to carbohydrate and/or

lipid metabolism though many also report no or minimal beneficial effects. Large variability exists

in the results of Cr supplementation in humans and rodent models due to many factors including

the variety of Cr compounds utilized (CrCl3, Cr(pic)3, etc.)11 and the variability of the dosage

parameters (different dose levels, frequency of administration, and length of supplementation to

name a few). Different strains may also respond differently to supplementation, such as Wistar

rats versus Zucker lean rats (see discussion in Chapter 4). Direct comparison may be drawn in

Chapter 2 to studies investigating the nutritional benefit of Cr supplementation, but the doses

required to observe any effect on glucose or insulin were far higher (~140 times higher) than a

recommended Cr adequate daily intake (recommended for humans), indicating pharmaceutical

activity.

Several lines of evidence suggest Cr should be considered essential but upon further

investigation seem to indicate a pharmaceutical role of Cr supplementation. In rodent studies,

apparent Cr deficiency induced by a low Cr diet resulted in insulin insensitivity in glucose

tolerance when compared to Cr supplemented rats.12-14 The improved insulin sensitivity in

response to glucose tolerance tests in the supplemented rats should be evaluated more as a dose-

response relationship than a Cr deficiency versus Cr sufficiency. The “Cr deficient” diets

employed either the lowest reported value (~33 μg Cr/kg diet or higher) or did not report the level

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of Cr contained in the diet.12-15 This “Cr deficient” diet already contains more than enough Cr to

be considered sufficient relative to the recommended adequate daily intake in humans.10, 12-15 These

rats were also given additional stressors such as high-fat or high-fructose diets and increased Cu

in their diets to induce compromised glucose and lipid metabolism as well as decrease pancreatic

function, further confounding the results.12, 13 Cr supplementation in drinking water (5 ppm CrCl3)

resulted in decreased fasting insulin and slightly faster glucose clearance compared to non-

supplemented.12-14 The effects of Cr supplementation in healthy rats residing in controlled metal-

free environments without additional dietary stressors and with the lowest level of Cr possible in

the diet have yet to be examined.

Another line of research used as an argument for the essentiality of Cr involves studies in human

subjects on total parenteral nutrition (TPN). TPN provides a way for people who cannot eat to

maintain adequate nutrition through intravenous administration. Five patients receiving TPN as

their only source of nutrition developed symptoms similar to type 2 diabetes such as glucose

intolerance, weight loss, neuropathy, encephalopathy, and glycosuria.15-18 Addition of Cr to the

TPN resulted in reversal of glucose intolerance and resolution of neurological changes in these

five patients.17 Orally administered Cr is only absorbed with approximately 0.5-2 % efficiency,

dependent partially on the compound administered leading to approximately 0.15 μg Cr present in

the bloodstream for the adequate daily intake (~30 μg Cr/d). 7, 19 The intravenous administration

of Cr received by the patients on TPN results in Cr administered directly into the bloodstream. For

patients receiving ~125-250 μg Cr/d added to the TPN resulted in ~1,000 times higher Cr levels

than would be nutritionally relevant.7, 17 These studies seem to indicate potential beneficial effects

of supplemental Cr in a pharmaceutical nature.

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1.2: Chromium, Diabetes, and Zucker Rats

As described above, some evidence exists to suggest Cr supplementation may improve

carbohydrate and lipid parameters in rodents and possibly in humans in extreme situations

throughout prolonged dietary supplementation as well as intravenous administration.3, 13, 14, 20-25

These effects appear to be pharmaceutical in nature due to the large doses required to observe

changes. The next logical question to ask is “Who benefits from pharmaceutical Cr

supplementation?” As Cr supplementation may result in increased insulin sensitivity, it would

follow that insulin-resistant disorders such as type 2 diabetes or pre-diabetic obesity would benefit

most from this result. A large amount of research has gone into investigating the relationship

between Cr supplementation and diabetes.

Diabetes mellitus is a condition that results in impaired glucose tolerance among other

symptoms. Type 1 diabetes results from the inability to produce insulin in the pancreas often due

to autoimmune destruction of insulin-producing β-cells. The absence of insulin leads to the

inability to properly metabolize glucose and the necessity of insulin treatment. Type 2 diabetes is

a disorder resulting in severe insulin resistance and hyperglycemia. Though insulin is not absent

as in type 1 diabetes, the body has lost the ability to properly respond to it, and the condition is

referred to as “insulin-resistant” or “insulin insensitive.” Since Cr supplementation may lead to

increased insulin sensitivity and/or lipid metabolism, type 2 diabetics and those pre-disposed to

diabetes through moderate insulin resistance brought on by factors such as obesity (pre-diabetics)

may benefit from Cr supplementation and should be further investigated.6

Few studies in diabetic human models have demonstrated potential beneficial effects of Cr

supplementation, though the results are extremely variable in nature; and it has been postulated

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that the observed beneficial effects are perhaps a side effect of Cr toxicity.3, 26-29 For example,

Anderson, et al. supplemented diabetic patients with various levels of Cr and observed lowered

levels of fasting plasma insulin, glucose, and total cholesterol as well as decreased insulin and

glucose levels in response to a glucose challenge.28 Most human studies, however, fail to correlate

beneficial effects with Cr supplementation. Previous meta-analyses of human studies in diabetic

models have indicated improved glucose and lipid parameters in Cr-supplemented diabetics,

thought the methodology of the analyses have since been refuted.30, 31 A recent meta-analysis of

human studies indicates that Cr supplementation had no effect on glycated hemoglobin, HDL

cholesterol, or triglycerides, but does significantly reduce fasting plasma glucose in type 2

diabetics.32 While yet another, more thorough meta-analyses observed no beneficial effects of Cr

supplementation in humans regardless of disease state (both diabetics and non-diabetics), not

including the often-cited Anderson study described above due to lack of sufficient data for

analysis.33

In order to investigate the relationship between Cr supplementation and diabetes, a proper model

of the disease must be utilized. Zucker obese (ZOB) and Zucker diabetic fatty (ZDF) rats represent

models of pre-diabetes (obese, slightly insulin-resistant) and type 2 diabetes, respectively.34 To

model responses to Cr in pre-diabetic, slightly insulin-resistant rats, the ZOB rat model may be

utilized. The ZOB rats originated from a healthy Zucker lean model. ZOB rats have a single

missense mutation in the gene encoding for the leptin receptor. The hormone leptin is primarily

secreted by adipocytes and is partially responsible for food intake regulation, energy homeostasis,

appetite behaviors, as well as energy expenditure regulation.35 This mutation results in a glycine

to proline change in the leptin receptor and the production of non-functional mRNA.35 Thus, the

rats are not able to recognize leptin and respond appropriately. Due to this mutation, ZOB rats

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become obese due mostly to hyperphagia and display insulin resistance, high cholesterol, and mild

hyperglycemia and are considered “pre-diabetic” models. ZDF rats are an inbred strain derived

from the ZOB rats. ZDF rats have the same mutation of the gene encoding the leptin receptor plus

an additional, undescribed mutation not related to the leptin receptor. This additional mutation

results in hyperglycemia as well as other symptoms of type 2 diabetes. Male ZDF rats initially

become obese, like the ZOB rats from which they are derived, but lose the weight as they age,

becoming smaller than the healthy Zucker lean rats over time. ZDF rats are commonly used as a

model for type 2 diabetes as they display hyperglycemia, β-cell dysfunction, insulin resistance,

and high cholesterol similar to that of the disease state.

ZOB and ZDF rats are excellent models for insulin resistance as “pre-diabetic” and type 2

diabetic models. Research has been conducted into how Cr supplementation may influence insulin

and glucose levels in diabetic models, but further information is needed to determine whether the

pharmacokinetics of healthy Zucker lean rats varies from that of the insulin-resistant ZOB or ZDF

models. To understand how insulin resistance may alter Cr transport, healthy Zucker lean rats must

first be examined for comparison. Many studies have observed increased urinary Cr loss in both

diabetic humans and diabetic rats.36-40 This increased urinary Cr loss indicates either an alteration

in the absorption of Cr in diabetic models compared to healthy models, or is simply an alteration

in excretion due to the increase of urinary output that is a symptom of diabetes. Further study is

needed into the absorption, biodistribution, and excretion of Cr in Zucker lean, ZOB, and ZDF rats

in order to determine if the absorption of Cr by insulin-resistant models is altered compared to the

healthy control.

If the pharmacokinetics (absorption, biodistribution, metabolism, excretion) of Cr are altered in

diabetic rats compared to healthy controls, it may be possible for the altered levels of Cr to

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influence dietary minerals present in the body. This alteration could be possible due to many

factors including shared transport mechanisms of metals. For example, the Fe-transport protein,

transferrin, has been shown to competitively bind Cr in the bloodstream.41 Increased levels of Cr

in the bloodstream may result in altered Fe transport and decrease the levels of Fe in tissues. Type

2 diabetes or obesity results in altered levels of tissue metals even without Cr supplementation. A

few studies have analyzed the tissue metal concentrations of ZOB and Zucker lean, but not ZDF,

rats in the past as well as Wistar rats and models of type 1 diabetes. It is necessary to analyze the

basal levels of Zucker lean, ZOB, and ZDF rats tissue metal concentrations, as well as post-Cr

supplementation, in order to evaluate strain differences in tissue metals and if there are any

beneficial or detrimental effects of extended Cr-supplementation on tissue metal homeostasis.

1.3: Exposure to Pharmaceutical Metal NPs In Utero: Considerations to Reduce the

Developmental Toxicity of Engineered Metal Oxide NPs by More Intelligent Design

Nanoparticles (NPs) are small particles (1-100 nm in diameter). NP’s small size and large surface

area to size ratio have generated ample interest in NP technology and increased development of

NPs for in vivo uses, such as biomedicine. By 2015, an estimated 240 nano-enabled products are

estimated to enter the pharmaceutical pipeline.42 Concern remains about the safety of engineered

NPs and a deeper understanding of interactions of NPs in vivo would facilitate the ability to design

safer NPs initially, before discovering a potential health hazard. The small size of the NPs allow

for potentially increased biodistribution, as size exclusion is one of the body’s main lines of

defense. Many types of NPs (gold, silver, platinum, iron, titanium dioxide, etc.) have been

investigated for many biomedical uses such as carriers in drug delivery systems, imaging contrast

agents, cancer treatments, contraceptives, and diagnostics.43, 44

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The risk of NP exposure to pregnant women and the developing fetus is of great concern and is

often not investigated.45, 46 Several studies using perfused human placenta to examine the ability

of NPs to cross the placenta have produced various results. Gold NPs47 were able to perfuse into

the placental tissue but were not found in fetal circulation; however, various quantum dot NPs

perfused into the placental tissue and entered the fetal circulation.48, 49 NP size and length of

perfusion time were examined as factors for placental transfer efficiency.48 Animal studies have

indicated exposure to NPs can affect pregnant mice and their offspring. For example, titanium

dioxide NPs can not only cross the placenta and transfer to the fetus, but exposure results in brain

damage, nerve system damage and reduced sperm production in male offspring.50, 51 Platinum NPs

have been shown to increase in pup mortality and a decrease in growth rate of offspring exposed

to NPs in utero.52

Very little information exists on the potential developmental toxicity of exposure to iron oxide

NPs in utero, as well as the effect that surface charge may have on the ability of the NPs to induce

toxicity or cross the placenta and accumulate in the fetus. Iron oxide NPs have been widely

explored in drug delivery,53, 54 as contrast agents in magnetic resonance imaging (MRI),55 for soil

and groundwater remediation,56 and as photocatalysts.57, 58 In addition, iron oxide is a major

potential product of zero-valence iron NPs, the most popular metallic NPs in environmental

remediation applications.59-64 Pregnant women may be at risk for multiple exposures to iron oxide

NPs through biomedical uses (MRIs, drug delivery, etc.) as well as through environmental

applications. The influence of surface charge on developmental toxicity was evaluated and can be

compared to other similar NPs. The risk of exposure of pregnant women to other NPs, such as

TiO2, Au, and Ag NPs, is also concerning due to the ubiquitous nature of these products in

consumer products such as sunscreens and food additives. Surface charge should be considered

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when designing NPs to which a pregnant women may come in contact. Only one study has to this

point examined the in vivo developmental toxicity of iron oxide NPs.65 Using a 50 mg NPs/ kg

body mass intraperitoneal dose decreased infant growth, as well as an alteration in testicular

morphology in offspring that had been exposed to NPs in utero, was observed.65, 66 The present

study seeks to correlate the developmental toxicity and fetal biodistribution of iron oxide NPs with

surface charge and dosage.

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1.4: References

1. Goldhaber, S. B., Trace element risk assessment: essentiality vs. toxicity. Regulatory

Toxicology and Pharmacology 2003, 38, 232-242.

2. Schwarz, K.; Mertz, W., Chromium(III) and the glucose tolerance factor. Archives of

Biochemistry and Biophysics 1959, 85, 292-295.

3. Anderson, R. A., Chromium, glucose intolerance and diabetes. Journal of the American

College of Nutrition 1998, 17, 548-55.

4. Anderson, R. A., Chromium as an essential nutrient for humans. Regulatory Toxicology and

Pharmacology 1997, 26, S35-41.

5. Vincent, J. B.; Love, S. T., The need for combined inorganic, biochemical, and nutritional

studies of chromium(III). Chemistry & Biodiversity 2012, 9, 1923-1941.

6. Di Bona, K. R.; Love, S.; Rhodes, N. R.; McAdory, D.; Sinha, S. H.; Kern, N.; Kent, J.;

Strickland, J.; Wilson, A.; Beaird, J.; Ramage, J.; Rasco, J. F.; Vincent, J. B., Chromium is

not an essential trace element for mammals: effects of a "low-chromium" diet. Journal of

Biological Inorganic Chemistry 2011, 16, 381-390.

7. Vincent, J. B., Chromium: celebrating 50 years as an essential element? Dalton Transactions

2010, 39, 3787-3794.

8. Lay, P. A.; Levina, A., Chromium: biological relevance. Encyclopedia of Inorganic and

Bioinorganic Chemistry 2012.

9. Stearns, D. M., Is chromium a trace essential metal? Biofactors 2000, 11, 149-162.

10. National Research Council, Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic,

Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon,

Vanadium, and Zinc. A Report of the Panel on Micronutrients, Subcommittee on Upper

Reference Levels of Nutrients and of Interpretations and Uses of Dietary Reference Intakes,

and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes.

National Academy of Sciences: Washington, D. C., 2002.

11. Olin, K. L.; Stearns, D. M.; Armstrong, W. H.; Keen, C. L., Comparative retention absorption

of chromium-51 (Cr-51) from Cr-51 chloride, Cr-51 nicotinate and Cr-51 picolinate in a rat

model. Trace Elements and Electrolytes 1994, 11, 182-186.

12. Striffler, J. S.; Polansky, M. M.; Anderson, R. A., Overproduction of insulin in the chromium-

deficient rat. Metabolism 1999, 48, 1063-8.

13. Striffler, J. S.; Polansky, M. M.; Anderson, R. A., Dietary chromium decreases insulin

resistance in rats fed a high-fat, mineral-imbalanced diet. Metabolism 1998, 47, 396-400.

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14. Striffler, J. S.; Law J. S.; Polansky M. M.; Bhathena S. J.; Anderson R. A., Chromium

improves insulin response to glucose in rats. Metabolism 1995, 44, 1314-20.

15. Jeejeebhoy, K. N.; Chu, R.; Marliss, E. B.; Greenberg, G. R.; Brucerobertson, A., Chromium

deficiency, diabetes and neuropathy, reversed by chromium infusion in a patient on total

parenteral nutrition (TPN) for 3-1/2 years. Clinical Research 1975, 23, A636-A636.

16. Anderson, R. A., Chromium and parenteral nutrition. Nutrition 1995, 11, 83-6.

17. Jeejeebhoy, K. N., Chromium and parenteral nutrition. Journal of Trace Elements in

Experimental Medicine 1999, 12, 85-89.

18. Jeejeebhoy, K. N.; Chu, R. C.; Marliss, E. B.; Greenberg, G. R.; Brucerobertson, A.,

Chromium Deficiency, Glucose-Intolerance, And Neuropathy Reversed By Chromium

Supplementation, In A Patient Receiving Long-Germ Total Parenteral Nutrition. American

Journal of Clinical Nutrition 1977, 30, 531-538.

19. Laschinsky, N.; Knottwitz K.; Freund B.; Dresow B.; Fischer R.; Nielsen P., Bioavailability

of chromium(III)-supplements in rats and humans. BioMetals 2012, 25, 1051-60.

20. Lukaski, H. C., Chromium as a supplement. Annual Review of Nutrition 1999, 19, 279-302.

21. Mertz, W., Chromium in human nutrition: a review. Journal of Nutrition 1993, 123, 626-633.

22. Anderson, R. A.; Polansky M. M.; Bryden N. A.; Canary J. J., Supplemental chromium effects

on glucose, insulin, glucagon, and urinary chromium losses in subjects consuming controlled

low-chromium diets. American Journal of Clinical Nutrition 1991, 54, 909-16.

23. Vincent, J. B., The bioinorganic chemistry of chromium(III). Polyhedron 2001, 20, 1-26.

24. Sun, Y.; Clodfelder, B. J.; Shute, A. A.; Irvin, T.; Vincent, J. B., The biomimetic

[Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma insulin, cholesterol, and triglycerides in

healthy and type 2 diabetic rats but not type 1 diabetic rats. Journal of Biological Inorganic

Chemistry 2002, 7, 852-62.

25. Clodfelder, B.; Gullick, B.; Lukaski, H.; Neggers, Y.; Vincent, J., Oral administration of the

biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ increases insulin sensitivity and improves blood

plasma variables in healthy and type 2 diabetic rats. Journal of Biological Inorganic

Chemistry 2005, 10, 119-130.

26. Levina, A.; Lay P., Chemical properties and toxicity of chromium(III) nutritional

supplements. Chemical Research In Toxicology 2008, 21, 563-71.

27. Anderson, R. A., Chromium in the prevention and control of diabetes. Diabetes & Metabolism

2000, 26, 22-7.

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28. Anderson, R. A.; Cheng, N. Z.; Bryden, N. A.; Polansky, M. M.; Cheng, N. P.; Chi, J. M.;

Feng, J. G., Elevated intakes of supplemental chromium improve glucose and insulin

variables in individuals with type 2 diabetes. Diabetes 1997, 46, 1786-1791.

29. Vincent, J. B., Beneficial effects of chromium(III) and vanadium supplements in diabetes.

Nutritional and Therapeutic Interventions for Diabetes and Metabolic Syndrome 2012, 381-

391.

30. Suksomboon, N.; Poolsup, N.; Yuwanakorn, A., Systematic review and meta-analysis of the

efficacy and safety of chromium supplementation in diabetes. Journal of Clinical Pharmacy

& Therapeutics 2014, 1-15.

31. Balk, E. M.; Tatsioni, A.; Lichtenstein, A. H.; Lau, J.; Pittas, A. G., Effect of chromium

supplementation on glucose metabolism and lipids. Diabetes Care 2007, 30, 2154-2163.

32. Abdollahi, M.; Farshchi, A.; Nikfar, S.; Seyedifar, M. J., Effect of chromium on glucose and

lipid profiles in patients with type 2 diabetes; a meta-analysis review of randomized trials.

Journal of Pharmacy & Pharmaceutical Sciences 2013, 16, 99-114.

33. Bailey, C. H., Improved meta-analytic methods show no effect of chromium supplements on

fasting glucose. Biological Trace Element Research 2014, 157, 1-8.

34. Etgen, G. J.; Oldham, B. A., Profiling of Zucker diabetic fatty rats in their progression to the

overt diabetic state. Metabolism 2000, 49, 684-8.

35. Wang, B.; Chandrasekera, P. C.; Pippin, J. J., Leptin- and leptin receptor-deficient rodent

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39. Morris, B. W.; MacNeil, S.; Hardisty, C. A.; Heller, S.; Burgin, C.; Gray, T. A., Chromium

homeostasis in patients with type II (NIDDM) diabetes. Journal of Trace Elements in

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40. Rhodes, N. R.; McAdory, D.; Love, S.; Di Bona, K. R.; Chen, Y.; Ansorge, K.; Hira, J.; Kern,

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41. Clodfelder, B. J.; Emamaullee, J.; Hepburn, D. D. D.; Chakov, N. E.; Nettles, H. S.; Vincent,

J. B., The trail of chromium(III) in vivo from the blood to the urine: the roles of transferrin

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42. Powers, M., Nanomedicine and nano device pipeline surges 68%. NanoBiotech News 2006,

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48. Chu, M. Q.; Wu, Q.; Yang, H.; Yuan, R. Q.; Hou, S. K.; Yang, Y. F.; Zou, Y. J.; Xu, S.; Xu,

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54. Namdeo, M.; Saxena, S.; Tankhiwale, R.; Bajpai, M.; Mohan, Y. M.; Bajpai, S. K., Magnetic

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Particles. Elements 2010, 6, 395-400.

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CHAPTER 2

INVESTIGATIONS INTO THE EFFECTS OF EXTENED CHROMIUM(III)

SUPPLEMENTATION ON GLUCOSE METABOLISM AND INSULIN SENSITIVITY AND

URINARY CHROMIUM LOSS AS A BIOMARKER FOR DIETARY CHROMIUM STATUS

IN HEALTHY ZUCKER LEAN RATS

2.1: Introduction

There are two primary forms of Cr that arise in both pharmaceutical and toxicological studies,

Cr(III) and Cr(VI). Cr(VI) is highly toxic and can lead to toxicity of the kidney, liver,

gastrointestinal system, and immune system with chronic oral exposure.1 Cr(III) on the other hand

is the bioactive and pharmaceutically relevant form of Cr, influencing glucose, insulin, and lipid

metabolism, though it’s specific roles are not clearly understood. The effects of Cr(III)

supplementation have been widely studied in both humans and rats over several decades, though

it should be noted that the results of these studies vary widely. Currently Cr is considered an

essential trace element responsible for maintenance of glucose and lipid metabolism, meaning Cr

is considered necessary for normal function and absence of the recommended daily amount could

lead to deficiency, resulting in negative effects. The essentiality of Cr is under debate as conflicting

evidence of its necessity exists. A recent meta-analysis of Cr supplementation of human subjects

found no significant effect of Cr supplementation indicating supplementations to alleviate “Cr

deficiency” are unnecessary and will not prove to be beneficial.2 Though its essentiality remains

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uncertain, clearly Cr plays a role in enhancing glucose/insulin interactions in rodents, though again

the mechanisms are unknown.3-11

Since the 1950s, Cr has been implicated as a potential aide for impaired glucose tolerance.12 Rats

fed a Torula yeast-based diet developed an impaired glucose tolerance in response to an

intravenous glucose challenge when compared to rats on a standard chow.12 This yeast-based diet

was found to be deficient in selenium, which led to liver disease. Supplementation with selenium

led to improved liver function but did not relieve the impaired glucose metabolism. These

researchers identified what they believed to be an active, Cr-containing, compound that could

improve the glucose intolerance, which was previously impaired by the yeast-based diet.12

Supplementation of rats given this glucose-intolerance inducing yeast-based diet with inorganic

Cr-containing compounds (200 μg Cr/kg body mass) improved their glucose clearance rates.

Though the interpretation of this work has been questioned, these studies lead to increased interest

in Cr and how supplementation could affect glucose and insulin metabolic pathways, and posed

the question of Cr’s essentiality in nutrition.4, 12, 13

Many studies have been performed in order to determine the effects of Cr supplementation on

glucose and insulin, as well as to elucidate a biomarker for Cr status. In order to elucidate the

effects of Cr supplementation, an as low Cr as reasonably possible diet, must be established.

Previous attempts to observe the effects of a low Cr diet in rats have been reported, with the lowest

previously reported dietary Cr concentration at 33 ± 14 μg Cr/kg diet.6, 7, 14 Lower fasting plasma

insulin levels as well as insulin levels in response to a glucose challenge were observed in Cr

supplemented rats (5 ppm CrCl3 in water) versus rats on the low Cr diet.6, 15 No differences were

observed in glucose clearance rates or fasting glucose levels between treatments.6, 15 Many of these

investigations into Cr involved inducing a state of impaired glucose metabolism by the use of

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additional stressors such as high fat, high iron, high carbohydrates to induce increased urinary Cr

loss or copper to decrease pancreatic function.6, 7, 14 The presence of these additional stressors does

not allow for objective evaluation of the physiological actions of the Cr itself but does allow for

investigation into alleviation of stress-induced increased fasting insulin levels as a possible

pharmaceutical use.

The research presented in this chapter seeks to improve upon this previous research and remove

unnecessary variables such as extraneous sources of Cr and unnecessary stressors. Zucker lean rats

will be supplemented with diets varying in Cr concentration based on the current standard rodent

diet, the AIN-93G diet. The diets examined herein will consist of the standard AIN-93G as well

as the AIN-93G supplemented with additional Cr (+ 200 μg Cr/kg diet and + 1000 μg Cr/kg diet)

and the AIN-93G diet without any Cr added into the mineral mix (low Cr) to observe the effects

of increased dietary Cr on glucose metabolism and insulin sensitivity.

Another goal of this research is an investigation into the use of urinary Cr excretion in response

to a glucose or insulin challenge as a biomarker for Cr status. Cr can be mobilized in the body in

response to insulin (or the increase in insulin as a response to a plasma glucose increase). This

movement of Cr leads to an increase in Cr excretion in the urine in response to a glucose or insulin

challenge. The rate of Cr excretion in response to insulin may allow for determination of dietary

Cr intake. Previous studies to elucidate a biomarker for Cr dietary status have been unsuccessful.

Plasma Cr or urinary Cr levels do not correlate with tissue Cr, plasma glucose, plasma insulin, or

lipid levels.3 Instead of examining the overall loss of urinary Cr as previous studies have, this study

seeks to examine the rate of urinary Cr loss as a response to a glucose or insulin challenge. Cr

binds to the protein transferrin in the bloodstream. A current model of Cr transport proposed by

Vincent et al., indicates that after a glucose or insulin challenge, transferrin-bound Cr travels to

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the tissues and provides some function in glucose metabolism, after which it is ultimately lost in

the urine.16-19 Measuring urinary Cr response instead of total Cr could provide a better indication

of dietary Cr intake. This line of inquiry may also aid in determining whether dietary Cr intake

effects the rates of Cr mobilization in response to a glucose or insulin challenge.

2.2 Materials and Methods

2.2.1 Chemicals, Assays, and Instrumentation

Glucose and insulin (bovine, zinc) were obtained from Sigma-Aldrich. The final concentrations

of glucose and insulin were prepared using doubly deionized water. Plasma insulin was measured

using an 125I RIA kit from MP Biomedicals. Gamma counting was performed using a Packard

Cobra II Auto-Gamma counter. Blood glucose levels were measured using a OneTouch glucose

meter. Fe content was determined using a modified colorimetric method for determining non-heme

Fe concentration in biological samples.20

2.2.2 Animals and Husbandry

Thirty-two male Zucker lean rats were obtained from Charles River Laboratories International

at 6 weeks of age and acclimated for 2 weeks prior to treatment. Male rats were chosen for

consistency with previous studies, while the use of Zucker lean rats would allow for the effects of

health condition to subsequently be examined by comparison of results with those of Zucker obese

and Zucker diabetic fatty rats to determine if urinary Cr loss in response to an insulin or glucose

challenge would prove to be a potential biomarker for Cr status. Rats were maintained in an

AAALAC-approved animal care facility in rooms at 22 ± 2 ºC and 40-60 % humidity with a 12 h

photoperiod. The animals were housed individually in specially constructed metal-free housing

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(vide infra) to prevent the introduction of additional Cr into their diets. Rats were provided specific

diets and distilled water ad libitum for a 23 week period prior to glucose and insulin challenges.

Rats were weighed and food consumption was measured twice weekly. All procedures involving

these animals were reviewed and approved by The University of Alabama’s Institutional Animal

Care and Use Committee.

2.2.3 Treatments

Following a 2 week acclimation period, male Zucker lean rats were randomly separated into four

treatment groups, each containing eight rats as follows: (1) rats on a purified AIN-93G Cr-

sufficient diet, (2) rats on the AIN-93G diet without Cr included in the mineral mix, (3) rats on the

AIN-93G Cr-sufficient diet with an additional 200 μg Cr/kg diet, and (4) rats on the AIN-93G Cr-

sufficient diet with an additional 1,000 μg Cr/kg diet. Purified AIN-93G rodent diets and modified

AIN-93G diets were obtained from Dyets (Bethlehem, PA, USA). Diets were received in powder

form.

2.2.4 Metal-Free Housing

Iris Buckle Up boxes were obtained from Target; the boxes were approximately 18 cm high, 45

cm wide, and 28 cm deep. These boxes are made of clear plastic with a removable lid that attaches

with latches on both 28 cm sides of the boxes. Holes (4 mm in diameter) were drilled with an

electric hand drill in all five sides of the box and in the lid using a square grid pattern with

approximately 5 cm between holes. Holes (4 mm in diameter) were also drilled in the corners of

the bottom of each box to facilitate urine drainage. Shavings of plastic were removed from the

holes, and any rough spots were smoothed using fine sandpaper. An additional hole was drilled in

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the lid with an appropriate diameter to accommodate the tube of the water bottles, and another hole

was drilled in the lip of the box to accommodate a hanging cage card holder. Tube tread no. 116

wet area anti-fatigue mats were purchased from General Mat Company. The matting is made of

vinyl with a tensile strength of 139 kg/cm and is flexible from -10 to 100 ºC. The matting was cut

with a knife to fit inside the base of the boxes. Both the boxes and the matting could pass through

multiple cycles of a cage washing machine without noticeable damage. As the boxes are similar

in size to shoebox-type housing, they were kept on standard racks for animal cages. The cages

were placed on absorbent bench paper or newspaper. The rear of the cage was elevated

approximately 1 cm using scrap pieces of the matting material placed under the rear of the cage to

ensure drainage of urine.

2.2.5 Food and Water Containers

Wheaton clear straight-sided, wide-mouth glass jars (about 9 cm in diameter, 9.5 cm in height,

473 mL) and plastic lids (89-400 mm screw cap size) were obtained from Fisher Scientific and

were used to hold food. A 5 cm-diameter circular opening was cut in the polyvinyl-lined plastic

lids to allow the animals access to food. To prevent the rats from dumping the powdered food from

the jars, a 2 cm-thick Plexiglas disk (about 7 cm in diameter) was placed on the food. The disk had

a 14 mm-diameter circle cut out in the center, with six other 14 mm-diameter circles cut in a

hexagonal pattern around the center circle; the disks were prepared by The University of Alabama

College of Arts and Sciences machine shop. To provide water, the stainless steel tubes were

removed from the water bottles and replaced with glass tubes. The University of Alabama glass

shop cut and bent glass tubing of the appropriate diameter to match the length and shape of the

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stainless steel tubes. To prevent potential injury, the end of the tubing exposed to the rats was fire-

polished.

2.2.6 Data Collection

Rats were weighed, and food consumption was measured twice weekly. At 23 and 25 weeks,

respectively, rats were fasted for between 10 and 12 h then given an intravenous glucose challenge

(1.25 mg glucose/kg body mass) or an intravenous insulin challenge (5 insulin units/kg body

mass). Blood was collected in EDTA-lined capillary tubes by a tail vein prick. Blood was collected

before intravenous challenges and 30, 60, 90, and 120 min after the challenge injections.

After 23 weeks on the diets, the rats were placed in metabolic cages for 6 h prior to and removed

12 h after an intravenous glucose challenge (1.25 mg glucose/kg body mass). Urine was collected

prior to injection and 2, 6, and 12 h post injection. The first 8 h of the urine collection occurred

during the dark period with the remainder occurring during the photoperiod. The urine was

transferred to pre-weighed disposable centrifuge tubes and stored at -20 ° C. After continuing on

the diet another 2 weeks, the rats were placed in metabolic cages for 6 h prior to and removed 12

h after an intravenous insulin (five insulin units (bovine, zinc) per kg of body mass)21 challenge.

Urine was collected as described for the glucose challenge. Rats had unrestricted access to food

and water during the urine collection period.

2.2.7 Cr Concentration in Diets

Samples of each powdered diet (200 mg) were digested with a 30:1 mixture of ultra-high-purity

concentrated HNO3 (99.99 % trace-element free) and ultra-high-purity concentrated H2SO4

(99.99 % trace-element free). The digestion was continued with controlled heating (sub-boiling)

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until the samples had been heated to dryness. Then, the residue was diluted to 10 mL with doubly

deionized water (Milli-Q, Millipore). All glassware was acid-washed. Blank digestions were

carried out in the same fashion. Cr concentrations were determined utilizing a PerkinElmer Analyst

400 atomic absorption spectrometer equipped with an HGA-900 graphite furnace and an AS-800

autosampler using a Cr hollow cathode lamp operating at 10 mA; a spectral bandwidth of 0.8 nm

was selected to isolate the light at 353.7 nm. The operating conditions were as follows

(temperature, ramp time, hold time): drying 1 (100 ºC, 5 s, 20 s), drying 2 (140 ºC, 15 s, 15 s),

ashing (1600 ºC, 10 s, 20 s), atomization (2500 ºC, 0 s, 5 s), and cleaning (2600 ºC, 1 s, 3 s). Other

instrumental parameters included the following: pyrolytic cuvette, argon carrier gas (flow rate 250

mL/min), 20 μL sample volume, and peak area measurement mode. The digestion and atomic

absorption methods were verified by analysis of a certified reference material, 1573a Tomato

Leaves (NIST).

2.2.8 Cr Concentration in Urine

Each urine sample was digested with a mixture of ultra-high purity concentrated HNO3 (99.99 %

trace element free) and 30 % H2O2. The digestion was continued with controlled heating (sub-

boiling) for 15 h. All glassware was acid washed. Blank digestions were carried out in the same

fashion. Cr concentrations were determined utilizing a PerkinElmer Analyst 400 atomic absorption

spectrometer equipped with HGA-900 graphite furnace and an AS-800 autosampler using a Cr

hollow cathode lamp operating at 8 mA; a spectral bandwidth of 0.8 nm was selected to isolate the

light at 353.7 nm, with operating conditions (temperature (ºC), ramp time (s), hold time (s)): drying

1 (90, 45, 20), drying 2 (140, 20, 20), ashing (800, 15, 15), atomization (2500, 0, 5), and cleaning

(2700, 1, 5). Other instrumental parameters included the following: pyrolytic cuvette, argon carrier

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gas (flow rate 250 mL/min), 20 μL sample volume, and peak area measurement mode. Urine Cr

concentrations were calculated using the method of standard additions with samples spiked with

10, 20, 30, and 50 μg/L of PerkinElmer Pure Atomic Spectroscopy Standard 1,000 μg Cr/mL in

HNO3. Fits of the standard addition lines had r2 values > 0.98, while each triplicate point generally

had standard deviations less than 2 %. To test whether urine could be contaminated by feces in the

metabolic cages, 51Cr-containing rat feces (available from previous work)22 were used to line the

urine and feces collection component of the metabolic cage; a rat was housed in the cage (with

food and water) and urine was collected. 51Cr content of the urine was then determined by gamma

counting. Contamination of the urine with Cr from the feces was insignificant.

2.2.9 Statistical Analyses

Statistical analyses were performed using SPSS (SPSS, Chicago, IL, USA). Data are represented

graphically as average values with standard error of mean (SEM) bars. Data were calculated

independently, tested for homogeneity of variance with Levene’s test, and analyzed using

univariate analysis of variance and descriptive statistics. For eight animals per group, an expected

difference between two means would be significant at the α = 0.05 level and 1 - β = 0.01 if the

difference between the means is twice the standard deviation; these values are reasonable based

on the effects of insulin on urinary Cr in Sprague–Dawley and Zucker obese rats.18 Blood insulin

and blood glucose tolerance tests were further analyzed for the area under each curve. Post hoc

least significant difference analyses were used to indicate significant differences at a 95 %

confidence level (p ≤ 0.05). Area under the curve was calculated using the trapezoid rule.

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2.3: Results and Discussion

2.3.1 Carefully Controlled Access to Cr: Metal-Free Caging

In order to properly assess the effects of Cr supplementation, Cr exposure must be carefully

controlled. The typical environment of an experimental rodent allows for Cr exposure through

multiple avenues. The diets used throughout this study were carefully controlled for Cr content as

described in the materials and methods. Typical caging involves the use of a plastic “shoe-box”

style base with a metal grating placed over the top with an inverted water bottle sipper tube inserted

to allow access to food and water. This metal grating and sipper tube are composed of stainless

steel, which consists of > 10 % Cr. Rodents actively gnaw on the metal grating and sipper tube,

which allow for additional Cr than presented in the diets. In addition, the hardwood bedding

present in most rodent housing habitats also contains numerous metal ions, including Cr. In order

to examine the effects Cr supplementation and ensure that rats on the low Cr diet received as little

Cr as possible, metal-free caging free caging was a necessity.

A sub-goal of this project was to design and implement inexpensive and easily constructed

metal-free housing for the rodents in this study. Previous plastic caging set-ups have been

described in order to attempt to remove access to metal caging components.23-25 These previous

metal-free constructs involved more arduous construction specifications than are described herein

such as bonding specifically measured sheets of plastic and plastic grating to construct boxes.23-25

Due to the current ubiquitous nature of inexpensive plastic snap-top boxes, construction of the

metal-free caging described herein was greatly simplified. Metal-free caging was designed in order

to provide a non-hazardous environment for the rats. The components of the caging also were

designed to be resistant to damage from the animals, such as gnawing or scratching, as well as

impervious to experimental stresses such as the high temperatures used in cage washing.

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Figure 2.1: Metal-free housing for rodents.

Caging components were designed to mimic the ambient conditions present in classic, shoe-box

housing. Air-flow occurs in the traditional shoe-box housing through the lid to allow for proper

ventilation, therefore ventilation concerns must be addressed when replacing this metal grating.

Simply replacing the metal grating with a plastic grating or mesh would not be feasible, due to the

ability of the rats to chew through plastic evenly spaced enough to provide adequate air circulation

over time. Snap-top Iris Buckle Up plastic boxes were chosen for the rodent enclosure as an

alternative to construction due in part to their similarity in size (22 L), as shown in Figure 2.1.

Holes drilled into the boxes allowed for proper ventilation and did not compromise the structural

integrity of the boxes.

Rats were prevented from standing on the floor of the box for additional comfort as well as to

prevent walking in undrained urine using vinyl anti-fatigue mats, cut to the size of the cages and

placed inside. The anti-fatigue matting in the cages held up well throughout the study, including

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through the cage wash, and was not frequently chewed by the rats. The design of the metal-free

caging also allows for use as metabolic caging. Urine drains through the drilled holes and can

readily be collected by placing a receptacle under the cages. Feces can also be collected as it falls

between the treads of the anti-fatigue mats placed inside the cages, but does not drain through the

holes in the cages themselves.

In order to maintain a clean, non-hazardous environment, cages were cleaned and absorbent

matting was replaced every 2 days. This cleaning prevented the accumulation of feces and hair

and sterilized the cages by running the cages through a high-temperature and high-pressure

mechanical cage washer. During the initial period of the greater than 6 month study, some of the

cages were accumulating moisture in the upper portion of the boxes, observed as condensation, in

certain areas of the animal care facility. In order to improve circulation, a household fan was placed

in each room, which eliminated the excess moisture and allowed for sufficient ventilation in the

cages. The temperature inside of the metal-free cages measured about 1-2 ºC lower than traditional

shoe-box housing, perhaps due to the lack of bedding and lack of side and bottom ventilation in

traditional caging.

An important consideration during this study was the effect that different caging conditions

could have on the health of the rats. In order to determine if there were any detrimental effects on

the rats due to caging, body mass as well as clinical signs of toxicity (such as soft stool, abnormal

activity levels, chromodoacryorrhea (red tears), and abnormal postures)26 were observed for rats

in typical shoe-box housing with bedding and the described metal-free housing. Zucker lean rats

with the same birthdate and shipping date were monitored over a three month period. No

differences in body mass or clinical signs of toxicity, including illness and behavioral

abnormalities, were observed between rats maintained in traditional shoe-box housing versus the

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metal-free housing (data not shown). These results indicate the constructed metal-free caging

provides a suitable environment for experimental rodents, free of extraneous chromium present in

stainless steel caging components.

2.3.2 Carefully Controlled Access to Cr: Analysis of the Diets

Diets were selected which varied only in Cr content in order to examine the effects of Cr

supplementation in rats as well as to assess the nutritional relevance of Cr. This varying level of

Cr in the diets should also allow for elucidation of whether urinary Cr levels in response to an

insulin or glucose challenge could be identified as a biomarker of Cr supplementation. A standard

rodent diet recommended by the American Institute of Nutrition for both short-term and long-term

rodent studies is the AIN-93 purified diet.27, 28 Of the two formulations of the AIN-93 diet

described by the American Institute of Nutrition, the AIN-93G diet, designed for young animals

during periods of rapid growth, was chosen for this study due to the young age of the rats. The

alternatively available formulation of the AIN-93 diet is AIN-93M which is designed for adult

maintenance.27, 28 Cr was not included in the mineral mix for the lowest Cr treatment in an attempt

to induce an extremely low Cr exposure and if Cr is essential, a Cr deficiency. This lowest Cr diet

is referred to throughout the text at “low Cr” due to the omission of Cr from the mineral mix.

Graphite furnace atomic absorption spectroscopy (GFAA) was utilized to determine the actual

amount of Cr in the diets, reported as μg Cr/kg diet (Table 2.1).

The measured Cr concentration of the low Cr diet (Cr omitted from mix) indicates that the diet

was much lower in Cr than standard rodent diets. GFAA measurements indicate the presence of

16 μg Cr/kg diet, over 1,000 μg Cr lower per kg diet than the AIN-93G. Studies performed in

rodents which observed an apparent Cr deficiency were used a purified “Cr deficient” diet

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containing approximately twice the amount of Cr than the current low Cr diet (33 μg Cr/kg diet),

though they are within error of each other.6, 7, 15 It should be noted that the “Cr deficient” diet used

by Anderson and coworkers is in excess of the recommended adequate daily intake for human

nutritional consumption and would be considered Cr sufficient.29 The Cr content of the low Cr diet

examined herein is very close to the recommended daily intake for humans when converted. Cr

content of the other diets (Cr sufficient, +200 μg Cr/kg diet, and +1000 μg Cr/kg diet) were close

to anticipated values as seen in Table 2.1. Cr in the AIN-93G mix and supplemental Cr was in the

form of CrK(SO4)2 · 12 H2O.

Treatment Groups Measured Cr content

(1) Low Cr 16 μg Cr/kg diet

(2) Cr Sufficient (AIN-93G) 1,135 μg Cr/kg diet

(3) AIN-93G +200 μg Cr/kg diet 1,331 μg Cr/kg diet

(4) AIN-93G +1000 μg Cr/kg diet 2,080 μg Cr/kg diet

Table 2.1: Actual Cr content of purified AIN-93G rodent diets measured by GFAA

A few additional calculations must be performed in order to relate the studies described in this

chapter with studies in humans, as the overall goal is to better understand the role of Cr in human

health, using rats as a model organism. Food consumption was measured throughout the study and

found to be constant at ~15 g of food per day for Zucker lean rats. As seen in Figure 2.2, the body

masses of the Zucker lean rats in this study were around 450 g. The lowest amount of Cr measured

in the diets (16 μg Cr/kg diet) would equate to approximately 0.53 μg Cr/kg body mass/d (for a

450 g rat consuming 15 g of food per day). The recommended adequate daily intake for humans

is approximately 30 μg Cr/d (25 and 35 μg Cr/d for females and males, respectively).30 Assuming

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an average body mass of 62 kg,31 30 μg Cr/d would equate to 0.48 μg Cr/kg body mass/d in

humans. This would mean the current study’s lowest possible Cr diet would be Cr sufficient by

human standards as it would roughly equal the recommended daily intake. Previous low Cr diets

of 33 μg Cr/kg diet for a 100 g rat would equate to over ten times this recommended intake (~3.3 μg

Cr/kg body mass/d).6, 7, 14 The 33 μg Cr/kg diet adjusted for the difference in metabolic rate

between rats and humans (multiplied by ~5) would still be greater than the adequate intake for

humans. The previous “Cr deficient” diet would be considered comparably Cr sufficient in μg

Cr/kg body mass/d when compared to human recommended intake. The effects observed from

diets receiving supplemental Cr should be considered pharmacological as they are considerably

higher than the recommended daily nutritional intake.

2.3.3 Effects of Cr Supplementation on Physiological Factors

Body mass and food intake were measured throughout the study in order to help elucidate the

effects of Cr removal and supplementation in the Zucker lean rats. Cr has been touted as a weight

loss aid, by increasing lean muscle mass and decreasing fat. Literature investigating these claims

are generally not consistent, claiming both body mass gain and body mass loss as well as no

changes in body mass at all.3, 32-37 A recent meta-analyses of articles analyzing how Cr

supplementation effects body composition and obesity in humans indicates a very small decrease

in body mass (~0.06 %) and percentage body fat compared to placebos, but stress that these results

should be “interpreted with caution” due to significant heterogeneity.38 Many studies in rats have

indicated that there is no change in body mass due to Cr supplementation.36, 39

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Time (day)

0 20 40 60 80 100 120 140

Bo

dy m

ass (

g)

200

250

300

350

400

450

500

550

Low Cr

Cr Sufficient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

a

abab

bb

a

abab

a

b

ab

b

Figure 2.2: Body mass of Zucker lean rats on the standard and modified AIN-93G diets: AIN-93G

without Cr added to the mineral mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an

additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg

Cr/kg). Different letters indicate significant differences between groups (p ≤ 0.05).

In the current study, no differences were observed in food intake (data not shown) and body

masses were not affected by removal or supplementation of Cr as shown in Figure 2.2. The body

masses of the “Cr sufficient” and the diet supplemented with 200 μg Cr/kg diet of the young rats

were statistically different for a few days at the beginning of the study, but these differences

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Low Cr

Cr Suffic

ient

+200 ug Cr/kg

+1000 ug Cr/kg

Non-H

em

e F

e (

ug/1

00m

L p

lasm

a)

0

50

100

150

200

250

300

350

Figure 2.3: Non-heme plasma Fe levels for Zucker lean rats on the standard and modified AIN-

93G diets: AIN-93G without Cr added to the mineral mixture (low Cr); the standard AIN-93G diet

(Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg

diet (+ 1000 μg Cr/kg).

disappeared within the first month. No differences were observed in the rate or type of health issues

or demeanor between any of the treatment groups.

The effects of Cr supplementation on non-heme plasma Fe content were also measured. The

proposed mechanisms of Cr transport indicate systemic transport through blood plasma. Cr can

tightly bind the glycoprotein transferrin, as shown previously.16, 40 Transferrin tightly, yet

reversibly, binds Fe in the blood plasma to create a utilizable pool of mobilized Fe. When needed,

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transferrin-bound Fe binds to transferrin-Fe receptors at the cell surface and undergoes endocytosis

to raise intracellular Fe levels. Previous studies have indicated that Cr can compete with ferric ions

in the bloodstream to bind the metal-binding sites of transferrin.16 The concentration of non-heme

Fe was analyzed in order to determine if Cr supplementation negatively affected the mobilizable

pool of Fe in the plasma due to competitive binding. The non-heme Fe concentrations were not

significantly affected by the removal or addition of Cr to diets and did not vary among treatment

groups (Figure 2.3). These results were as expected as the mineral mix used (AIN-93 G) provides

far more Fe than Cr even with the additional amounts. AIN-93 G provides 35 mg Fe/kg diet while

the treatments contained between 16 μg Cr and ~2 mg Cr per kg of diet. A significant change in

plasma Fe content was not expected and not observed.

2.3.4 Effects of Cr Supplementation on Response to Glucose and Insulin Challenges

Alterations in Cr intake have been shown to lead to abnormal glucose metabolism and insulin

sensitivity.7, 32 In mammalian studies, when given “low Cr” diets, animals display an altered

response to an insulin or a glucose challenge to those Cr supplemented as described previously.

Low Cr diets resulted in decreased response efficiency in returning to resting (“normal”) blood

plasma glucose levels and blood plasma insulin levels after an insulin or glucose challenge. After

being on the controlled diets for 23 weeks, rats in this study were given an intravenous glucose

challenge (1.25 mg glucose/kg body mass) to monitor their glucose and insulin response. After 25

weeks on the diets, rats received an insulin challenge (5 units insulin (bovine, zinc) per kg body

mass). Prior to challenges, rats were fasted 10-12 h to negate the impact of food on glucose and

insulin levels. Blood samples were collected prior to the challenges as well as 30, 60, 90, and 120

minutes after injection or glucose or insulin.

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Time after glucose injection (min)

0 20 40 60 80 100 120

Glu

co

se

(m

g/d

L)

50

100

150

200

250

300

350

400

Low Cr

Cr Sufficient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

a

ab

b

b

Figure 2.4: Plasma glucose levels for Zucker lean rats on the standard and modified AIN-93G diets

during glucose tolerance testing: AIN-93G without Cr added to the mineral mixture (low Cr); the

standard AIN-93G diet (Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an

additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). Different letters indicate significant differences

between groups.

2.3.4.1 Glucose Levels in Response to Challenges

Plasma glucose levels of rats on the standard and modified AIN-93G diets in response to a

glucose challenge (glucose tolerance tests) are displayed in Figure 2.4. As expected, plasma

glucose levels elevated in response to intravenous glucose injection. Plasma glucose levels should

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Low Cr Cr Sufficient + 200 ug Cr/kg + 1000 ug Cr/kg

Glu

co

se

are

a u

nd

er

cu

rve

0

10000

20000

30000

40000

ab

a

ab

b

Figure 2.5: Plasma glucose concentrations during glucose tolerance testing represented by the area

under the curve for Zucker lean rats on the standard and modified AIN-93G diets: AIN-93G

without Cr added to the mineral mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an

additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg

Cr/kg). Different letters indicate significant differences between groups.

spike and be at their highest soon after injection, then gradually decrease back to the resting

glucose level through the action of insulin. Figure 2.4 shows the initial increase in plasma glucose

levels, with a gradual decrease (glucose clearance) back to near baseline levels 2 h after glucose

injection (120 m). Most of the diets with various levels of Cr followed the same trajectory with the

exception of the timepoint 60 min after injection. At 60 min after introduction of the glucose

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Time after insulin injection (min)

0 20 40 60 80 100 120

Glu

cose (

mg/d

L)

0

100

200

300

400

Low Cr

Cr Sufficient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

a

ab

b

ab

Figure 2.6: Plasma glucose levels during insulin tolerance testing for Zucker lean rats on the

standard and modified AIN-93G diets: AIN-93G without Cr added to the mineral mixture (low

Cr); the standard AIN-93G diet (Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg);

or an additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). Different letters indicate significant

differences between groups.

challenge, the Cr sufficient diet (unmodified AIN-93G) remained elevated while the low-Cr, + 200

μg Cr/kg, and + 1000 μg Cr/kg diets had already begun to return to pre-challenge levels (baseline).

All other timepoints were statistically equivalent.

The results of the glucose challenge on plasma glucose levels indicate that only the rats given

the Cr sufficient (unmodified AIN-93G) diet displayed elevated glucose levels, even then only at

the 1 h (60 min) timepoint. Another way to visualize the blood plasma glucose levels after a

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Low Cr Cr Sufficient + 200 ug Cr/kg + 1000 ug Cr/kg

Glu

co

se

are

a u

nd

er

cu

rve

0

10000

20000

30000

40000

Figure 2.7: Plasma glucose concentrations during insulin tolerance testing represented by the area

under the curve for Zucker lean rats on the standard and modified AIN-93G diets: AIN-93G

without Cr added to the mineral mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an

additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg

Cr/kg).

glucose challenge are by calculating and visualizing the areas under the curves (AUCs). The AUCs

are given in Figure 2.5. The AUCs are consistent with the glucose levels over time and show a

slightly elevated glucose levels in the Cr sufficient diet. This elevated glucose level is only

statistically different from the group of rats given the + 1000 μg Cr/kg diet. There are no

differences in plasma glucose levels in response to a glucose challenge in the low Cr diet versus

any other diet.

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Glucose clearance rates (KG) can be used as an indicator of glucose metabolism, e.g. as the

efficiency of the animals to remove excess systemic glucose (above baseline) from the blood after

a glucose challenge. Based on the results from the glucose tolerance testing (see Figure 2.4), KG

can be calculated for the rats on the various diets. It is expected that the glucose clearance rates

will be equivalent or very similar for the low Cr, +200 μg Cr/kg, and + 1000 μg Cr/kg diets due to

the observation that all measured points in the glucose challenges were very similar and not

statistically different. KG was calculated for the rats on each diet as described previously, see the

equation below.7, 12, 41, 42

𝐾𝐺 = 𝑚𝑔 × 100

The variable mg represents the slope of the best fit line for the plot of the natural log of the “% of

baseline” glucose measurement over time. Percent of baseline is described in the equation below.

(% of baseline) =𝐺𝑙𝑢𝑐𝑜𝑠𝑒𝑡

𝐺𝑙𝑢𝑐𝑜𝑠𝑒0× 100

Use of this strategy instead of excess glucose measurements allowed for measurement of the

clearance rates of total glucose in calculating KG.

Glucose clearance was calculated for the rats receiving each diet. KG was calculated to be 1.1 %,

0.6 %, 0.5 %, and 0.7 % per minute for the rats given the low Cr, Cr sufficient AIN-93G, + 200

μg Cr/kg, and + 1000 μg Cr/kg diets, respectively. Due in part to the wide variability between rats

on the same treatment, these rates of excess glucose clearance are not statistically different. It is

interesting to note that the highest rate of glucose clearance (therefore fastest to metabolize glucose

and remove it from systemic circulation) was observed in the rats receiving the lowest Cr diet (no

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Cr in the mineral mix), though these observations are not statistically significant. Glucose tolerance

tests indicate that diets with variable amounts of Cr including as low as possible (low Cr) have the

ability to metabolize Cr equally efficiently.

Insulin tolerance tests were performed in addition to glucose tolerance testing. Figure 2.6 shows

the plasma glucose levels of rats on each diet in response to an intravenous insulin challenge.

Insulin functions to promote storage of glucose and removal from the bloodstream. In response to

an insulin challenge, the plasma glucose levels are expected to decrease dramatically due to this

rise in plasma insulin levels. All timepoints after the introduction of insulin were statistically

equivalent and as expected. Fasting plasma glucose levels in the low Cr diet (no Cr added to the

mineral mix) are significantly higher than the diet supplemented with + 200 μg Cr/kg. This

difference is not observed 2 weeks earlier during the glucose tolerance testing, though in general

it appears slightly higher. The Cr sufficient (AIN-93G) as well as the + 1000 μg Cr/kg diets were

not statistically different from any other diets.

AUCs for the insulin challenges are displayed in Figure 2.7. No differences were observed

between diets. The results of the glucose and insulin challenges on the plasma glucose levels in

rats given diets varying in Cr content for 23 and 25 weeks indicate that the level of Cr in the diet

does not influence glucose management after a glucose or an insulin challenge. An interesting

effect was observed in the fasting glucose levels of Cr present versus very low Cr after 25 weeks

which was not observed at 23 weeks with Cr supplemented fasting plasma levels appearing

lowered in comparison to rats fed a very low Cr diet.

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Time after glucose injection (min)

0 20 40 60 80 100 120

Insu

lin (

mic

ro insu

lin u

nits/m

L)

0

20

40

60

80

100

120

Low Cr

Cr Sufficient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

a

ab

ab

b

a

b

b

b

a

ab

ab

b

Figure 2.8: Plasma insulin levels during glucose tolerance testing for Zucker lean rats on the

standard and modified AIN-93G diets: AIN-93G without Cr added to the mineral mixture (low

Cr); the standard AIN-93G diet (Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg);

or an additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). Different letters indicate significant

differences between groups.

2.3.4.2 Insulin Levels in Response to Challenges

In addition to the effects on plasma glucose, plasma insulin levels were measured in response to

a glucose and insulin challenge to measure the amount of insulin required to overcome the

challenges. Insulin levels after an insulin challenge were statistically equivalent at all timepoints

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for all diets (data not shown). Figure 2.8 displays plasma insulin concentrations measured in

response to a glucose challenge. In response to a glucose challenge, circulating insulin levels

should raise initially and then lower as they will first be released by the pancreatic β-cells then

used to promote glucose metabolism/storage. No increase was observed in insulin concentration,

as Zucker lean rats have been shown to return to baseline prior to the first timepoint (30 m), as

early as 5 min after a bolus intravenous injection of glucose.43 Prior to glucose challenges the

resting levels of insulin were markedly different between diets of various Cr content. The diet

containing no Cr added to the mineral mix (low Cr) displayed the highest resting insulin levels,

while the diet containing + 1000 μg Cr/kg displayed the lowest. Previously, daily Cr

supplementation for 24 weeks resulted in lower fasting plasma insulin concentrations as well as

lower insulin plasma levels after a glucose challenge.11 Due at least in part to this differential in

starting levels, the plasma insulin levels of the low Cr diet remained highest and the + 1000 μg

Cr/kg diet remained lowest through 60 min post glucose challenge.

These results indicate that it takes a substantial amount of additional Cr in the diet in order to

see a marked reduction of plasma insulin levels (~2080 μg versus ~16 μg per kg diet). After 60 m,

plasma insulin levels between groups became statistically similar and overlapped. These results

indicate that Cr supplementation could affect resting (baseline) plasma insulin concentrations and

that supplementation of large amounts (over 100 times the low Cr diet and ~140 times higher than

the recommended daily intake) of Cr could lead to lower resting plasma insulin.

The difference in plasma insulin levels in response to a glucose challenge is represented by the

AUCs of the glucose tolerance testing in Figure 2.9. Here the relationship between Cr in the diet

and insulin levels in response to glucose are more easily observed. The plasma insulin

concentrations do not differ significantly between the Cr sufficient (unmodified AIN-93G) and the

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Low Cr Cr Sufficient + 200 ug Cr/kg + 1000 ug Cr/kg

Insu

lin a

rea u

nd

er

cu

rve

0

2000

4000

6000

8000

10000

12000

a

ab

bc

c

Figure 2.9: Plasma insulin concentrations during glucose tolerance testing represented by the area

under the curve for Zucker lean rats on the standard and modified AIN-93G diets: AIN-93G

without Cr added to the mineral mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an

additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg

Cr/kg). Different letters indicate significant differences between groups.

low Cr diet. Though the AIN-93G contains ~70 times the amount of Cr as the lower diet (which is

about half of previously reported “deficient” diets), the glucose and insulin responses are nearly

identical.

Interestingly, when the Cr content is additionally supplemented with + 200 μg Cr/kg and + 1000

μg Cr/kg, the resulting insulin levels in response to a glucose challenge are significantly decreased

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from the low Cr diet. Additionally, the insulin levels measured for the + 1000 μg Cr/kg diet is

significantly lower than the Cr sufficient AIN-93G. As the glucose levels in response to a glucose

challenge were equivalent between all Cr diets, the differences in plasma insulin levels indicate

that less insulin was necessary in order to respond to a glucose challenge. This difference as well

as the difference in resting insulin levels indicates that a supranutritional dose of Cr (+ 1000 μg

Cr/kg diet, ~2080 μg Cr/kg) can result in increased insulin sensitivity in healthy Zucker lean rats.

2.3.4.3 Urinary Cr Loss as a Biomarker for Cr Administration Status

Another goal of this study was to elucidate whether urinary Cr loss in response to a glucose or

an insulin challenge could be used as a biomarker of Cr dietary (or supplementary) status. As

shown above, supplementation of Cr at very high levels leads to an increase in insulin sensitivity

in response to a glucose challenge. Previous studies have also observed altered glucose or insulin

levels when supplementing Cr, as described in the introduction. Since Cr seems to have a role in

the interactions between insulin and glucose signaling, Cr should be mobilized or utilized in

response to insulin or glucose challenges. If Cr were to be considered essential, as evidence has

pointed away from in this chapter, and urinary Cr loss is a biomarker of Cr status, the Cr sufficient

(AIN-93G), + 200 μg Cr/kg, and + 1000 μg Cr/kg diets should have comparable levels of urinary

Cr loss, saturable in response to a challenge, while the low Cr diet should have significantly

different levels. This would lead to decreased release of Cr in the urine in response to a glucose or

insulin challenge. This could also be the case if Cr is not essential for proper glucose metabolism

and has a pharmacological role. It is possible that increased Cr in the diets could lead to increased

Cr utilization overall in response to a challenge. This would result in an increase in urinary Cr loss

proportional to the additional Cr present in the diets.

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Urine samples were collected prior to and during glucose and insulin challenges given after 23

and 25 weeks on the carefully controlled diets, respectively as described in the above subsections.

The amount of Cr in the urine was measured and the rate of urinary Cr loss (ng Cr/h) was calculated

for pre-injection as well as post-challenge for 2 h (120 m). The resulting rates of urinary Cr loss

were plotted over time in Figures 2.10 and 2.11 for glucose and insulin challenges, respectively.

Though the differences are modest, a trend of increased Cr in the diets leading to increased rates

of Cr loss in the urine can be observed at the initial time point on Figures 2.10 and 2.11. This time

point represents the rates of urinary Cr loss pre-challenge and indicate that as dietary Cr increases,

urinary Cr loss increases. This observation seems logical and indicates that without a glucose or

insulin challenge, the amount of Cr ingested in the diet is proportional to the amount lost in the

urine. Previous studies in both rats and humans have observed a similar effect.14, 44 In the initial

2 h after a glucose challenge (Figure 2.10), urinary Cr loss increased dramatically in all groups

except in the case of the AIN-93G diet with an additional + 1000 μg Cr/kg diet after 2 h. The rate

of urinary Cr loss decreased for rats given the low Cr, Cr sufficient, and +200 μg Cr/kg diets after

2 h and had returned to baseline levels by 12 h post-challenge.

The rate of Cr loss in the rats given a + 1000 μg Cr/kg diet did not resemble the lower Cr diets.

The + 1000 μg Cr/kg diet remained stable over the course of the glucose challenges, indicating the

rate of urinary Cr loss was unaffected by a glucose challenge. This could be due to the high level

of Cr in the diet. This high Cr level could be saturating whichever pathways utilize Cr during

glucose metabolism, leading to a decreased need for Cr mobilization in response to a challenge. A

study in humans observed a similar result noting increased urinary Cr loss in response to a glucose

challenge with non-supplemented individuals, while Cr supplemented individuals’ (+ 200 μg Cr/d)

urinary Cr loss was unaffected by a glucose challenge.45 These results seem to indicate that an

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Time (h)

0 2 4 6 8 10 12

ng C

r/h

0

20

40

60

80

100

120

140

Low Cr

Cr Sufficient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

Figure 2.10: Rate of urinary Cr loss (ng Cr/h) in response to a glucose challenge for Zucker lean

rats on the standard and modified AIN-93G diets: for Zucker lean rats on the standard and modified

AIN-93G diets: AIN-93G without Cr added to the mineral mixture (low Cr); the standard AIN-

93G diet (Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000

μg Cr/kg diet (+ 1000 μg Cr/kg). The initial time point is the rate of Cr loss measured throughout

6 h before a glucose challenge. Rates were subsequently measured from t = 0 through t = 2, then

from t = 2 to t = 6, and finally from t = 6 through t = 12 h after glucose injection.

increase in daily Cr intake could saturate or overwhelm the mechanism of Cr transport in response

to a glucose challenge. In this case it is expected that with even high Cr levels in the diet, the

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Time (h)

0 2 4 6 8 10 12

ng C

r/h

0

20

40

60

80

100

120

140

Low Cr

Cr Sufficient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

Figure 2.11: Rate of urinary Cr loss (ng Cr/h) in response to an insulin challenge for Zucker lean

rats on the standard and modified AIN-93G diets: for Zucker lean rats on the standard and modified

AIN-93G diets: AIN-93G without Cr added to the mineral mixture (low Cr); the standard AIN-

93G diet (Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000

μg Cr/kg diet (+ 1000 μg Cr/kg). The initial time point is the rate of Cr loss measured throughout

6 h before an insulin challenge. Rates were subsequently measured from t = 0 through t = 2, then

from t = 2 to t = 6, and finally from t = 6 through t = 12 h after insulin injection.

urinary Cr loss in response to a glucose challenge would remain unchanged. Further studies would

be necessary to test this hypothesis.

The urinary Cr loss in response to an insulin challenge was also measured with similar results.

As seen in Figure 2.11, in response to an insulin challenge, rats given the low Cr, Cr sufficient

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Low Cr

Cr Suffic

ient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

Are

a U

nd

er

the

Cu

rve

0

100

200

300

400

500

Figure 2.12: Rate of urinary Cr loss in response to a glucose challenge represented by the area

under the curve for Zucker lean rats on standard and modified AIN-93G diets: AIN-93G without

Cr added to the mineral mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an additional

200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg).

(unmodified AIN-93G), as well as the +200 μg Cr/kg diets followed a similar trajectory as they

had in the glucose challenge (Figure 2.10) though on smaller scale. The rates of urinary Cr loss in

these three groups raised slightly during the two h after injection of insulin. This result is consistent

with similar studies in humans and rats.14, 44, 45 Over the next four h the rate of Cr loss in the urine

dropped to levels well below the pre-challenge levels. These urinary Cr rates remained low (below

baseline) through 12 h after receiving insulin. While an increase in plasma glucose seems to

mobilize Cr and promote Cr excretion in the lower Cr treatment groups, plasma insulin seems to

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Low Cr

Cr Suffic

ient

+ 200 ug Cr/kg

+ 1000 ug Cr/kg

Are

a U

nde

r th

e C

urv

e

0

100

200

300

400

500

Figure 2.13: Rate of urinary Cr loss in response to an insulin challenge represented by the area

under the curve for Zucker lean rats on standard and modified AIN-93G diets: AIN-93G without

Cr added to the mineral mixture (low Cr); the standard AIN-93G diet (Cr sufficient); an additional

200 μg Cr/kg diet (+ 200 μg Cr/kg); or an additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg).

have an opposing effect over time. The urinary Cr output in the AIN-93G diet supplemented with

+ 1000 μg Cr/kg diet, however, appeared to remain unchanged as in the glucose challenge, though

the differences were not statistically different between groups. Over time, the introduction of

plasma insulin to the rats seems to lower the overall Cr loss while glucose appears to increase Cr

excretion.

The amount of Cr lost through the urine can also be observed by calculating the AUCs. The

AUCs are displayed as the absolute value of the total measured AUC minus the projected AUC

that would result from no change in the rate observed pre-challenge. These AUCs are displayed

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Time (h)

0 2 4 6 8 10 12 14

ng C

r/h

0

50

100

150

200

250

Figure 2.14: Rate of urinary Cr loss (ng Cr/h) in response to an insulin challenge for individual

Zucker lean rats on the standard and modified AIN-93G diets: for Zucker lean rats on the standard

and modified AIN-93G diets: AIN-93G without Cr added to the mineral mixture (low Cr); the

standard AIN-93G diet (Cr sufficient); an additional 200 μg Cr/kg diet (+ 200 μg Cr/kg); or an

additional 1000 μg Cr/kg diet (+ 1000 μg Cr/kg). The initial time point is the rate of Cr loss

measured throughout 6 h before an insulin challenge. Rates were subsequently measured from t = 0

through t = 2, then from t = 2 to t = 6, and finally from t = 6 through t = 12 h after insulin injection.

for urinary Cr loss in response to glucose and insulin in Figures 2.12 and 2.13, respectively. No

differences in the AUCs for glucose or insulin challenge are observed for rats on any of the diets.

It should be noted the areas representing the urinary Cr loss in response to a glucose challenge

are positive values (increased urinary Cr loss) while the values in response to an insulin challenge

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are negative values (decreased urinary Cr loss). The rate of Cr loss represented by the AUCs were

overlapping and statistically the same.

A wide range of individual responses were observed throughout the study to both glucose and

insulin challenges even from rats within the same treatment group (same diets). The standard error

was large throughout the study due to this individualized response and not due to Cr measurement

in the samples. The rates of urinary Cr loss were noticeably more stable pre-challenges, as can be

observed by the standard deviations represented in Figures 2.10 and 2.11. This change in deviation

post-insulin or glucose challenge indicates that individual rats may respond differently and

regulate Cr transport differently even when exposed to the same amount dietary Cr. Individual

responses to an insulin challenge are presented in Figure 2.14 to illustrate this wide variability in

responses for rats given the same diet. Figure 2.14 represents the urinary Cr loss for rats given the

standard AIN-93G (Cr sufficient) diet when given an intravenous insulin challenge. The initial

response observed at the 2 h timepoint varies widely. Three of the rats in this treatment group

appear to respond with increased Cr output, though at various levels, while the other five rats in

this group of eight appear to be non-responders. Wide variability in individual responses has also

been observed in human studies attempting to elucidate the role of Cr in glucose and insulin

metabolism as well as to determine a Cr status biomarker.44, 45 This wide range of individual

responses in both humans and rats leads to the conclusion that urinary Cr loss in response to a

glucose or insulin challenge would not be a viable biomarker for Cr status.

Previous attempts have been made to elucidate a biomarker for Cr status in humans as well as

rats. Correlations between dietary Cr intake and urinary Cr levels3, 44, 46 or serum Cr levels47 have

been observed in human studies. Human urinary Cr concentrations do not, however, correlate with

serum glucose, serum insulin, age, body mass, or lipid parameters.44 Similar results to this chapter

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were observed in human subjects either taking or not taking a Cr supplement (200 μg Cr as CrCl3).

Supplemented subjects did not show an altered urinary Cr response to a glucose challenge while

non-supplemented subjects (self-selected diets) responded to a glucose challenge with an increase

in urinary Cr output.45 Also like this article, the urinary Cr loss in response to a challenge were

found to be unpredictable and unreliable.

2.4: Conclusions

Though the clearance rates (KG) of glucose in the blood plasma in response to a glucose

challenge were identical among rats given purified diets of various levels of Cr (low Cr, Cr

sufficient, +200 μg Cr/kg diet, and +1000 μg Cr/kg diet), the plasma insulin concentration in

response to the same glucose challenge varied between treatments. As the amount of dietary Cr

increased, the amount of insulin necessary to overcome a glucose challenge decreased, indicating

an increased sensitivity to the circulating insulin. Fasting insulin levels were also affected as the

lowest Cr diet had the highest fasting plasma insulin and the highest Cr diet had the lowest fasting

plasma insulin. The results of this chapter indicate that very large increases in dietary Cr (~140

times higher than the dietary recommendations, a pharmaceutical dose) could lead to increased

insulin sensitivity and decreased resting insulin levels in healthy Zucker lean rats. No differences

were observed between any treatment group in body mass, food intake, plasma iron concentration,

or urinary Cr loss in response to an insulin challenge. Very low Cr diets did not affect glucose

metabolism or insulin sensitivity when compared to rats given standard chow. Results of this study

indicate the pharmaceutical nature and lack of apparent essential function of Cr supplementation.

Increased investigation is needed into the use of supranutritional levels of Cr supplementation as

a treatment for conditions involving insulin insensitivity such as type 2 diabetes.

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The rate of urinary Cr loss in response to an insulin or glucose challenge seem to follow certain

trends, but these trends are obscured by the possible appearance of “responders” and “non-

responders.” Further investigation is necessary to confirm the existence of responders versus non-

responders. This rate of urinary Cr loss was fairly consistent between the low Cr, Cr sufficient,

and +200 μg Cr/kg diets with a large increase in the rate of Cr loss when given glucose followed

a decrease in rate until baseline is reached at approximately 12 h after injection. When given insulin

these same three groups follow a similar trend with a slight increase in urinary Cr loss followed

by a sharp rate decrease, dipping to levels of Cr loss below the starting urinary Cr loss rate which

is not fully recovered by 12 h after insulin introduction. The treatment group with the largest

amount of Cr in the diet (AIN-93G + 1000 μg Cr/kg diet) did not display a significant change in

the rate of urinary Cr loss when exposed to an insulin or a glucose challenge. This indicates that

the Cr transport system may be saturated or overwhelmed. The wide variability between

“responders” and “non-responders” as well as the lack of difference observed between treatment

groups, indicates that urinary Cr loss in response to a glucose or insulin challenge would not be a

reliable biomarker of Cr status.

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2.5: References

1. Saha, R.; Rumki, N.; Saha, B., Sources and toxicity of hexavalent chromium. Journal of

Coordination Chemistry 2011, 64, 1782-1806.

2. Bailey, C. H., Improved meta-analytic methods show no effect of chromium supplements on

fasting glucose. Biological Trace Element Research 2014, 157, 1-8.

3. Lukaski, H. C., Chromium as a supplement. Annual Review of Nutrition 1999, 19, 279-302.

4. Mertz, W., Chromium in human nutrition: a review. Journal of Nutrition 1993, 123, 626-633.

5. Anderson, R. A., Chromium, glucose intolerance and diabetes. Journal of the American

College of Nutrition 1998, 17, 548-55.

6. Striffler, J. S.; Polansky, M. M.; Anderson, R. A., Dietary chromium decreases insulin

resistance in rats fed a high-fat, mineral-imbalanced diet. Metabolism 1998, 47, 396-400.

7. Striffler, J. S.; Law J. S.; Polansky, M. M.; Bhathena S. J.; Anderson R. A., Chromium

improves insulin response to glucose in rats. Metabolism 1995, 44, 1314-20.

8. Anderson, R. A.; Polansky M. M.; Bryden N. A.; Canary J. J., Supplemental chromium effects

on glucose, insulin, glucagon, and urinary chromium losses in subjects consuming controlled

low-chromium diets. American Journal of Clinical Nutrition 1991, 54, 909-16.

9. Vincent, J. B., The bioinorganic chemistry of chromium(III). Polyhedron 2001, 20, 1-26.

10. Sun, Y.; Clodfelder, B. J.; Shute, A. A.; Irvin, T.; Vincent, J. B., The biomimetic

[Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma insulin, cholesterol, and triglycerides in

healthy and type 2 diabetic rats but not type 1 diabetic rats. Journal of Biological Inorganic

Chemistry 2002, 7, 852-62.

11. Clodfelder, B.; Gullick, B.; Lukaski, H.; Neggers, Y.; Vincent, J., Oral administration of the

biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ increases insulin sensitivity and improves blood

plasma variables in healthy and type 2 diabetic rats. Journal of Biological Inorganic Chemistry

2005, 10, 119-130.

12. Schwarz, K.; Mertz, W., Chromium(III) and the glucose tolerance factor. Archives of

Biochemistry and Biophysics 1959, 85, 292-295.

13. Mertz, W.; Schwarz, K., Relation of glucose tolerance factor to impaired intravenous glucose

tolerance of rats on stock diets. American Journal of Physiology 1959, 196, 614-8.

14. Anderson, R. A.; Bryden, N. A.; Polansky, M. M.; Reiser, S., Urinary chromium excretion and

insulinogenic properties of carbohydrates. American Journal of Clinical Nutrition 1990, 51,

864-868.

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15. Striffler, J. S.; Polansky, M. M.; Anderson, R. A., Overproduction of insulin in the chromium-

deficient rat. Metabolism 1999, 48, 1063-8.

16. Clodfelder, B. J.; Emamaullee, J.; Hepburn, D. D.; Chakov, N. E.; Nettles, H. S.; Vincent, J.

B., The trail of chromium(III) in vivo from the blood to the urine: the roles of transferrin and

chromodulin. Journal of Biological Inorganic Chemistry 2001, 6, 608-617.

17. Clodfelder, B. J.; Upchurch, R. G.; Vincent, J. B., A comparison of the insulin-sensitive

transport of chromium in healthy and model diabetic rats. Journal of Inorganic Biochemistry

2004, 98, 522-533.

18. Clodfelder, B. J.; Vincent, J. B., The time-dependent transport of chromium in adult rats from

the bloodstream to the urine. Journal of Biological Inorganic Chemistry 2005, 10, 383-393.

19. Vincent, J. B., Elucidating a biological role for chromium at a molecular level. Accounts of

Chemical Research 2000, 33, 503-10.

20. Fish, W. W., Rapid colorimetric micromethod for the quantitation of complexed iron in

biological samples. Methods in Enzymology 1988, 158, 357-364.

21. Cefalu, W. T.; Wang, Z. Q.; Zhang, X. H.; Baldor, L. C.; Russell, J. C., Oral chromium

picolinate improves carbohydrate and lipid metabolism and enhances skeletal muscle glut-4

translocation in obese, hyperinsulinemic (jcr-la corpulent) rats. Journal of Nutrition 2002, 132,

1107-1114.

22. Rhodes, N. R.; McAdory, D.; Love, S.; Di Bona, K. R.; Chen, Y.; Ansorge, K.; Hira, J.; Kern,

N.; Kent, J.; Lara, P.; Rasco, J. F.; Vincent, J. B., Urinary Chromium Loss Associated with

Diabetes is Offset by Increases in Absorption. Journal of Inorganic Biochemistry 2010, 104,

790-797.

23. Nielsen, F. H.; Bailey, B., Fabrication of plastic cages for suspension in mass air-flow racks.

Laboratory Animal Science 1979, 29, 502-506.

24. Polansky, M. M.; Anderson, R. A., Metal-free housing units for trace-element studies in rats.

Laboratory Animal Science 1979, 29, 357-359.

25. Mohr, H. E.; Hopkins, L. L., All plastic system for housing small animals in trace-element

studies. Laboratory Animal Science 1972, 22, 96-&.

26. Van Vleet, T. R.; Rhodes, J. W.; Waites, C. R.; Schilling, B. E.; Nelson, D. R.; Jackson, T. A.,

Comparison of technicians' ability to detect clinical signs in rats housed in wire-bottom versus

solid-bottom cages with bedding. Journal of the American Association for Laboratory Animal

Science 2008, 47, 71-75.

27. Reeves, P. G.; Nielsen, F. H.; Fahey, G. C., AIN-93 purified diets for laboratory rodents: final

report of the american institute of nutrition ad hoc writing committee on the reformulation of

the AIN-76A rodent diet. Journal of Nutrition 1993, 123, 1939-1951.

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54

28. Reeves, P. G., Components of the AIN-93 diets as improvements in the AIN-76A diet. Journal

of Nutrition 1997, 127, 838S-841S.

29. Trumbo, P.; Yates, A. A.; Schlicker, S.; Poos, M., Dietary reference intakes: vitamin A,

vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel,

silicon, vanadium, and zinc. Journal of the American Dietetic Association 2001, 101, 294-301.

30. National Research Council, Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic,

Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon,

Vanadium, and Zinc. A Report of the Panel on Micronutrients, Subcommittee on Upper

Reference Levels of Nutrients and of Interpretations and Uses of Dietary Reference Intakes,

and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes.

National Academy of Sciences: Washington, D. C., 2002.

31. Walpole, S. C.; Prieto-Merino, D.; Edwards, P.; Cleland, J.; Stevens, G.; Roberts, I., The

weight of nations: an estimation of adult human biomass. BMC Public Health 2012, 12.

32. Vincent, J. B., Chromium: celebrating 50 years as an essential element? Dalton Transactions

2010, 39, 3787-3794.

33. Lay, P. A.; Levina, A., Chromium: Biological Relevance. Encyclopedia of Inorganic and

Bioinorganic Chemistry 2012.

34. Cefalu, W. T.; Hu, F. B., Role of chromium in human health and in diabetes. Diabetes Care

2004, 27, 2741-2751.

35. Anderson, R. A., Effects of chromium on body composition and weight loss. Nutrition Reviews

1998, 56, 266-270.

36. Bennett, R.; Adams, B.; French, A.; Neggers, Y.; Vincent, J., High-dose chromium(III)

supplementation has no effects on body mass and composition while altering plasma hormone

and triglycerides concentrations. Biological Trace Element Research 2006, 113, 53-66.

37. Jeejeebhoy, K. N.; Chu, R. C.; Marliss, E. B.; Greenberg, G. R.; Brucerobertson, A., Chromium

deficiency, glucose-intolerance, and neuropathy reversed by chromium supplementation, in a

patient receiving long-germ total parenteral nutrition. American Journal of Clinical Nutrition

1977, 30, 531-538.

38. Onakpoya, I.; Posadzki, P.; Ernst, E., Chromium supplementation in overweight and obesity:

a systematic review and meta-analysis of randomized clinical trials. Obesity Reviews 2013, 14,

496-507.

39. Stout, M. D.; Nyska, A.; Collins, B. J.; Witt, K. L.; Kissling, G. E.; Malarkey, D. E.; Hooth,

M. J., Chronic toxicity and carcinogenicity studies of chromium picolinate monohydrate

administered in feed to F344/N rats and B6C3F1 mice for 2 years. Food and Chemical

Toxicology 2009, 47, 729-733.

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55

40. Sun, Y.; Ramirez, J.; Woski, S. A.; Vincent, J. B., The binding of trivalent chromium to low-

molecular-weight chromium-binding substance (LMWCr) and the transfer of chromium from

transferrin and chromium picolinate to LMWCr. Journal of Biological Inorganic Chemistry

2000, 5, 129-36.

41. Woolliscroft, J.; Barbosa, J., Analysis of chromium induced carbohydrate intolerance in rat.

Journal of Nutrition 1977, 107, 1702-1706.

42. Di Bona, K. R.; Love, S.; Rhodes, N. R.; McAdory, D.; Sinha, S. H.; Kern, N.; Kent, J.;

Strickland, J.; Wilson, A.; Beaird, J.; Ramage, J.; Rasco, J. F.; Vincent, J. B., Chromium is not

an essential trace element for mammals: effects of a "low-chromium" diet. Journal of

Biological Inorganic Chemistry 2011, 16, 381-390.

43. Zanchi, A.; Perregaux, C.; Maillard, M.; Cefai, D.; Nussberger, J.; Burnier, M., The PPAR

gamma agonist pioglitazone modifies the vascular sodium-angiotensin II relationship in

insulin-resistant rats. American Journal of Physiology, Endocrinology and Metabolism 2006,

291, E1228-E1234.

44. Anderson, R. A.; Polansky, M. M.; Bryden, N. A.; Patterson, K. Y.; Veillon, C.; Glinsmann,

W. H., Effects of chromium supplementation on urinary Cr excretion of human-subjects and

correlation of cr excretion with selected clinical-parameters. Journal of Nutrition 1983, 113,

276-281.

45. Anderson, R. A.; Polansky, M. M.; Bryden, N. A.; Roginski, E. E.; Patterson, K. Y.; Veillon,

C.; Glinsmann, W., Urinary chromium excretion of human subjects: effects of chromium

supplementation and glucose loading. American Journal of Clinical Nutrition 1982, 36, 1184-

1193.

46. Anderson, R. A., Chromium as an essential nutrient for humans. Regulatory Toxicology and

Pharmacology 1997, 26, S35-41.

47. Anderson, R. A.; Bryden N. A.; Polansky M. M., Serum chromium of human subjects: effects

of chromium supplementation and glucose. American Journal of Clinical Nutrition 1985, 41,

571-7.

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CHAPTER 3

PHARMACOKINETICS OF A SINGLE ORALLY ADMINISTERED DOSE OF 51CrCl3 IN

ZUCKER LEAN, TYPE 2 DIABETIC (ZUCKER DIABETIC FATTY), AND PRE-DIABETIC

(ZUCKER OBESE) RATS

3.1: Introduction

Chapter 2 investigated the effects of dietary Cr(III) supplementation on glucose metabolism and

insulin sensitivity as well as the urinary Cr loss in response to a glucose or insulin challenge in

Zucker lean rats. Increased insulin sensitivity and decreased fasting insulin levels were observed

in rats fed the highest Cr-containing diet (AIN-93G + 1000 μg Cr/kg diet). Urinary Cr output in

response to a glucose or insulin challenge did not differ significantly except again in this highest

Cr level diet. The AIN-93G + 1000 μg Cr/kg diet remained unresponsive in glucose and insulin

levels in response to a challenge with a consistent rate of Cr release, possible due to saturation of

Cr transport pathways. Zucker lean rats represent a healthy model with normal functioning glucose

metabolism, so the observed increased insulin sensitivity is not necessary for the Zucker lean

model. Conditions in which this result of Cr supplementation would be beneficial include models

of improper glucose metabolism and/or insulin sensitivity such as type 2 diabetes. Zucker Diabetic

Fatty (ZDF) rats and the Zucker obese (ZOB) rats represent models of type 2 diabetes and pre-

diabetic obesity, respectively.

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A major focus of Cr research has been to investigate whether Cr supplementation at a nutritional

level, may provide beneficial responses in individuals with conditions that include improper

carbohydrate metabolism such as glucose intolerance or improper lipid metabolism, such as type

2 diabetes. Studies in humans indicate that Cr supplementation may benefit subjects with observed

glucose intolerance.1, 2 A study by Anderson, et al. observed a dose responsive lowering of fasting

levels of plasma insulin, glucose, and total cholesterol in human diabetic subjects supplemented

with various levels of Cr.3 The same subjects also displayed decreased insulin and glucose levels

in response to glucose challenges at pharmacological levels of Cr.3 It should be noted that most

studies involving human subjects have failed to observe effects of chromium supplementation.

This could be due to the variability inherent with clinical studies as well as the low number of

subjects generally involved in studies involving humans. Studies using rodent models of diabetes

on the other hand have consistently observed beneficial effects in plasma glucose, plasma insulin,

total cholesterol, and/or levels of triglycerides when supplementing with pharmacological levels

of Cr.4

It has been observed that conditions such as type 2 diabetes, glucose loading, obesity, acute

exercise, or physical trauma can lead to increased urinary Cr loss.5, 6 Whether these Cr losses are

influenced by or influence changes in the absorption of Cr, have not been investigated. For

example, type 2 diabetic patients exhibited ~33 % lower plasma Cr in addition to ~100 % higher

Cr in the urine than healthy individuals throughout their first 6 years of onset.6 Urinary Cr loss of

diabetic patients diagnosed with diabetes for several years (> 8) decreased to about 44 % higher

and plasma Cr lowered to ~60 % lower than control, healthy individuals.6 These differences in

plasma and urinary Cr levels indicate a difference in how Cr is transported and utilized in healthy

versus insulin resistant subjects. If Cr were to be used as an aide to reverse or alleviate insulin

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resistance, it is important to understand and measure the changes in Cr biodistribution, absorption,

and excretion induced by these stressors (diabetes, obesity).

In the previous chapter supplemented dietary Cr (upwards of 2000 μg Cr/kg diet) improved

insulin sensitivity in Zucker lean rats (representing normal glucose metabolism and insulin

sensitivity). Studies herein were performed in Zucker lean rats in order to further examine the

pharmacokinetics of orally administered Cr, including absorption, biodistribution, and excretion.

To address the need for evaluation of the effects of stressors, such as diabetes, and evaluate the

potential of Cr as a pharmaceutical, orally administered Cr was also examined in ZOB and ZDF

rats to examine if the insulin-resistant rats would exhibit altered Cr pharmacokinetics.

3.2 Materials and Methods

3.2.1 Materials and Instrumentation

51CrCl3 was obtained from MP Biomedicals, Inc. and diluted with 100 mL of doubly-deionized

water. Gamma-counting was performed on a Packard Cobra II auto-gamma counter.

3.2.2 Animals and Husbandry

The University of Alabama Institutional Animal Care and Use Committee approved all

procedures involving the use of rats. Twenty-one male rats of each Zucker lean, Zucker obese

(ZOB, an insulin-resistant model of obesity and early stage type 2 diabetes), and Zucker diabetic

fatty, ZDF, (a type 2 diabetes model) were obtained from Charles River Breeding Laboratories

(six-weeks of age) and acclimated to their cages for two weeks (2 rats per cage). Zucker obese rats

have a mutation in the gene for a receptor for the hormone leptin; as a result, the rats become obese,

have elevated plasma cholesterol and triglycerides levels, and have high normal plasma glucose

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and insulin concentrations; the ZDF rats have an additional (yet to be identified) mutation which

results in their developing the full range of symptoms associated with type 2 diabetes. The rats

were maintained on a 12 h light/dark cycle. The rats were housed for 16 weeks, to ensure the ZDF

rats developed diabetes, prior to the absorption experiments. The Zucker lean and obese rats were

fed a standard commercial chow diet (Harlan Teklad LM-485 Mouse/ Rat Sterilizable Diet). The

ZDF rats were fed a commercial high fat chow diet (Formulab Diet 5008), previously shown to

assist in the development of diabetic symptoms. Both diets have previously been shown to be

chromium sufficient.7, 8 The rats were allowed to access to food and water ad libitum. After the 16

week period, experiments were initiated by gavaging the rats with an appropriate volume of an

aqueous 51CrCl3 solution (3 μg Cr/kg body mass), after which they were placed in metabolic cages

for selected time intervals for the purpose of feces and urine collection. The time intervals were

30, 60, 120, 360, 720, 1440, and 2880 min. The rats were allowed access to food and water ad

libitum after Cr administration. Before administration of Cr, the blood glucose levels of the ZDF

rats were tested to ascertain and confirm the development diabetes; blood glucose measurements

were made using a OneTouch meter collected from tail slits.

3.2.3 Sample Collection

At the end of each time interval, the rats were sacrificed by CO2 asphyxiation. Blood and tissue

samples were harvested and placed into pre-weighed 50 mL disposable centrifuge tubes. The

stomach, small intestine, large intestine, heart, liver, spleen, testes, kidneys, epididymal fat, right

femur, pancreas, and muscle (musculus triceps surae) from right hind leg were collected and

weighed; urine and feces were also collected and weighed. Blood and muscle were assumed to

comprise 6 % and 30 % of the total body mass, respectively, for calculations.9 For the Zucker

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obese rats with their large fat content, muscle was assumed to compromise 16 % of the total body

mass. Studies of the muscle content of the hind legs10 and carcass (body minus tail, internal organs,

and gastrointestinal tract)11 both reveal about a 53 % reduction in the percent muscle composition

of ZOB rats versus their normal counterparts.

3.2.4 Statistical Analyses

Each data point in the figures represents the average value for three rats, except for the 30 min

ZDF rats as one rat died of apparent heart complications (common for the disease model) shortly

before the Cr administration was to begin. Error bars in the figures denote standard deviation. The

data from each replicate was calculated independently, tested for homogeneity of variance by the

Levine statistic using SPSS (SPSS Inc. Chicago, IL), and pooled and analyzed to give the reported

results. Data was analyzed by repeated measures ANOVA and MANOVA. Specific differences at

95 % confidence (p ≤ 0.05) were determined by a Bonferroni post-hoc test.

3.3: Results and Discussion

3.3.1 Cr Supplementation

The purpose of the experiments described throughout this chapter was to examine the

pharmacokinetics of Cr supplementation in vivo both in healthy rats (Zucker lean) as well as

models with impaired glucose tolerance brought on by obesity (pre-diabetic, ZOB) or type 2

diabetic rat models (ZDF). Though the suggested daily intake of Cr in humans is 25-35 μg for

females and males, respectively, commercially available Cr supplements contain approximately

~200-800 μg of Cr, indicating the results of these Cr supplements are pharmaceutical in nature. In

order to assess the relevancy of these studies to currently available Cr supplements, relevant dose

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and route of administration must be utilized. Cr was given orally in the form of 51CrCl3 at 3 μg

Cr/kg body mass. Assuming an average body mass of 62 kg,12 the selected dosage would equate

to approximately 200 μg in humans, relevant to the currently available Cr supplements.

3.3.2 51Cr Pharmacokinetics

As this was an oral dose, 51Cr distribution can be followed throughout the gastrointestinal tract

over time (Figure 3.1). The oral dose of 51Cr passes from the stomach to small intestine, peaking

at 60 min post 51CrCl3 administration for Zucker lean and ZOB, and 120 min post-administration

for ZDF rats. After 2 h, at least 90 % of the administered dose clears the stomach for the Zucker

lean and ZDF rats, while only ~80 % of the dose clears the stomach for the ZOB rats. In previous

studies into the fate of 51CrCl3 in rats, ~90 % of the Cr had passed through the stomach and first

15 cm of small intestine within 30 min.13 After an hour, almost 100 % of the Cr was in the small

and large intestine.13 This amount was reduced to 55 % after 24 h, though the quantity of Cr in the

feces was not measured.13 In the current study, the quantity of Cr in the small intestine reached

near 100 % of the applied dose after 1 h for all groups. The Zucker lean and ZOB rats’ small

intestinal Cr levels decreased at the 2 h timepoint, while the ZDF rats Cr level remained near

100 %. Cr was no longer detectable in the small intestine 6 h after treatment.

Levels of radiolabeled Cr were highest in the large intestines at 6 h for all groups. At 12 h post-

dose, the Cr levels in the large intestine remained high (~80 %) for the Zucker lean rats, while

ZOB and ZDF rats large intestine Cr levels dropped to ~25 %. ZOB and ZDF rats’ large intestinal

Cr levels reached near-baseline levels by 24 h while the Zucker lean rat retained ~35 % of the

applied dose until 48 h. Interestingly, at 12 h after Cr administration, 50-70 % of the administered

dose was lost in the feces for ZOB and ZDF rats, while only about 10 % of the dose was present

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Time (Minutes)

0 500 1000 1500 2000 2500 3000

% o

f A

pp

lied

Dose

0

20

40

60

80

100

120

Stomach

Small Intestine

Large Intestine

Feces

Figure 3.1.A: Concentration of 51Cr measured in the gastrointestinal tract and feces after an oral

dose of 51CrCl3 in Zucker lean rats. Concentration is represented by the percentage of the applied

dose measured for each sample as a function of time. Letters indicate the concentration of 51Cr is

(b) significantly different from ZOB rats and (c) significantly different from ZDF rats (p ≤ 0.05).

in feces of Zucker lean rats. This appears to be due to retention of the radiolabeled Cr in the large

intestine of Zucker lean rats. The passage through the gastrointestinal tract is followed ~80-100 %

of the administered Cr present in the feces after 48 h for all groups of rats.

b,c

b

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Time (Minutes)

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Dose

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20

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120

Stomach

Small Intestine

Large Intestine

Feces

Figure 3.1.B: Concentration of 51Cr measured in the gastrointestinal tract and feces after an oral

dose of 51CrCl3 in ZOB rats. Concentration is represented by the percentage of the applied dose

measured for each sample as a function of time. Letters indicate the concentration of 51Cr is (a)

significantly different from Zucker lean rats or (c) significantly different from ZDF rats (p ≤ 0.05).

In the bloodstream, levels of 51Cr rose rapidly with peaks of 30-60 minutes for all groups of rats

(Figure 3.2). Though the levels of Cr present in the blood peaked for all groups at this time, the

magnitude of this peak differed between groups. Zucker lean and ZOB rats shared similar plasma

Cr levels or ~0.2 % and ~0.25 %, respectively while the peak Cr plasma concentration for ZDF

was ~0.4 % of the applied dose. This level of Cr in the blood remained elevated for ZDF until 2 h,

a,c a

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Time (Minutes)

0 500 1000 1500 2000 2500 3000

% o

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lied

Do

se

0

20

40

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80

100

120

Stomach

Small Intestine

Large Intestine

Feces

Figure 3.1.C: Concentration of 51Cr measured in the gastrointestinal tract and feces after an oral

dose of 51CrCl3 in ZDF rats. Concentration is represented by the percentage of the applied dose

measured for each sample as a function of time. Letters indicate the concentration of 51Cr is (a)

significantly different from Zucker lean and (b) significantly different from ZOB rats (p ≤ 0.05).

when the level of Cr in the blood returned to near-baseline levels. Zucker lean rats also had returned

to near-baseline levels by 2 h, but ZOB rats’ plasma remained slightly elevated 6 h post-

administration. Blood plasma levels of 51Cr remained close to zero percent of the applied dose

until 48 h when the plasma levels of radiolabeled Cr elevated slightly for all groups of rats,

indicating movement or utilization of 51Cr that had been stored in the tissues.

a

b

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Time (Minutes)

0 500 1000 1500 2000 2500 3000

% o

f A

pp

lied

Dose

0.0

0.5

1.0

1.5

2.0

Blood

Urine

Figure 3.2.A: Concentration of 51Cr measured in the blood and urine after an oral dose of 51CrCl3

in Zucker lean rats. Concentration is represented by the percentage of the applied dose measured

for each sample as a function of time. Letters indicate the concentration of 51Cr is (b) significantly

different from ZOB rats and (c) significantly different from ZDF rats (p ≤ 0.05).

The movement of 51Cr into and out of the bloodstream corresponds to tissue 51Cr concentrations

over time. Cr reaches a maximum concentration in the tissues 30-60 min after administration, then

rapidly decreases (Figures 3.3, and 3.4). As near 100 % of the dose of Cr is present in the stomach

and small intestines during this time (Figure 3.2), Cr must be rapidly absorbed from the stomach

and/or small intestine into the bloodstream where it is either absorbed by the tissues or eliminated.

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Time(Minutes)

0 500 1000 1500 2000 2500 3000

% o

f A

pp

lied

Dose

0.0

0.5

1.0

1.5

2.0

Blood

Urine

Figure 3.2.B: Concentration of 51Cr measured in the blood and urine after an oral dose of 51CrCl3

in ZOB rats. Concentration is represented by the percentage of the applied dose measured for each

sample as a function of time. Letters indicate the concentration of 51Cr is (a) significantly different

from Zucker lean rats and (c) significantly different from ZDF rats (p ≤ 0.05).

Previous 51Cr biodistribution investigations have observed Cr accumulation in the bone, kidney,

spleen, and liver over time.9, 14

Tissue Cr levels were highest in the skeletal muscle. The maximum Cr levels recorded for skeletal

muscle samples were ~0.8 %, ~0.4 %, and ~0.6 % for Zucker lean, ZOB and ZDF rats,

respectively. This high level of Cr was reached at 30 min for Zucker lean rats, then decreased back

c

c

c

a,c

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Time (Minutes)

0 500 1000 1500 2000 2500 3000

% o

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Do

se

0.0

0.5

1.0

1.5

2.0

Blood

Urine

Figure 3.2.C: Concentration of 51Cr measured in the blood and urine after an oral dose of 51CrCl3

in ZDF rats. Concentration is represented by the percentage of the applied dose measured for each

sample as a function of time. Letters indicate the concentration of 51Cr is (a) significantly different

from Zucker lean rats and (b) significantly different from ZOB rats (p ≤ 0.05).

to ~0 % of the applied dose by 2 h post-dose while the levels of Cr in the skeletal muscle remained

elevated during 30-60 min, then dropped back to near 0 % by 6 h for the ZOB and ZDF rats. All

groups of rats displayed an increase to ~0.5-0.6 % applied dose at 48 h from a near zero percent

level in the previous 18 h. This is concurrent with an increase in blood and liver Cr levels observed

at 48 h (Figures 3.2, 3.3, and 3.4). The next highest Cr content in the tissues is observed in the

a,b

a,b

a,b

a,b

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Time (Minutes)

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% o

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d D

ose

0.0

0.2

0.4

0.6

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1.0

1.2

Right Femur

Heart

Muscle

Testes

Epid. Fat

Figure 3.3.A: Concentration of 51Cr measured in the right femur, heart, skeletal muscle, testes, and

epididymal fat after an oral dose of 51CrCl3 in Zucker lean rats. Concentration is represented by

the percentage of the applied dose measured for each sample as a function of time.

liver. The liver Cr values peak 30-60 min post-dose with the highest concentration observed in

ZDF rats (~0.17 %) followed by Zucker lean (~0.04 %) and ZOB (~0.06 %). A previous study

examining the biodistribution of Cr following an oral dose of 51CrCl3 and 51Cr nicotinate observed

similar results with muscle containing the greatest Cr concentration at all timepoints up to 24 h.9

The administration of 51CrCl3 followed the results presented herein with liver containing the

second highest Cr concentrations.9 Unlike this study, the Cr nicotinate resulted in higher kidney

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Time (Minutes)

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% o

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Do

se

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Right Femur

Heart

Muscle

Testes

Epid. Fat

Figure 3.3.B: Concentration of 51Cr measured in the right femur, heart, skeletal muscle, testes, and

epididymal fat after an oral dose of 51CrCl3 in ZOB rats. Concentration is represented by the

percentage of the applied dose measured for each sample as a function of time. Letters indicate the

concentration of 51Cr is (c) significantly different from ZDF rats (p ≤ 0.05).

Cr concentrations than the liver for the first three h after administration.9 In this study the kidneys

were the only other tissue besides the skeletal muscle and the liver to register a notable amount of

Cr. Approximately 0.01-0.02 % of the applied dose was present in the kidneys of Zucker lean and

ZOB rats at their highest concentrations. The ZDF rats however reached kidney Cr concentrations

c

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Time (Minutes)

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% o

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se

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Right Femur

Heart

Muscle

Testes

Epid. Fat

Figure 3.3.C: Concentration of 51Cr measured in the right femur, heart, skeletal muscle, testes, and

epididymal fat after an oral dose of 51CrCl3 in ZDF rats. Concentration is represented by the

percentage of the applied dose measured for each sample as a function of time. Letters indicate the

concentration of 51Cr is (b) significantly different from ZOB rats (p ≤ 0.05).

of ~0.04 % of applied dose. This is likely due to increased metabolism and excretion of Cr in the

urine.

The disappearance of Cr from the bloodstream within approximately the first 1-2 h post-

administration coincides with an increase in Cr in the urine and to a lesser extent the liver and

kidney (Figures 3.2 and 3.4). For the Zucker lean and ZOB rats, the amount of Cr present in the

b

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Time (Minutes)

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0.00

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Pancreas

Spleen

Liver

Kidney

Figure 3.4.A: Concentration of 51Cr measured in the pancreas, spleen, liver and kidney after an

oral dose of 51CrCl3 in Zucker lean rats. Concentration is represented by the percentage of the

applied dose measured for each sample as a function of time.

urine rises to 0.4-0.6 % of the applied dose, while the amount of Cr present in the ZDF rats rises

to twice that amount at ~1.2 % of the applied dose. The Cr content of the urine from ZDF rats is

significantly higher than the Zucker lean and ZOB rats and remains elevated from 2 h to at least

48 h post-administration of 51CrCl3. Results are consistent with previous research into Cr loss in

models of diabetes, as described in further detail below.16-19

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Time (Minutes)

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Pancreas

Spleen

Liver

Kidney

Figure 3.4.B: Concentration of 51Cr measured in the pancreas, spleen, liver and kidney after an

oral dose of 51CrCl3 in ZOB rats. Concentration is represented by the percentage of the applied

dose measured for each sample as a function of time.

3.3.3 51Cr Absorption

Total absorption of Cr was estimated by addition of the Cr content measured in the blood, urine,

kidney, muscle, and liver. Other tissues measured were excluded from this calculation due to the

negligible measurements of Cr. Zucker lean rats retained ~1.1 % of the applied dose at both 30 min

and 1 h. ZOB rats retained ~0.7 % and ~1.2 % of the applied dose at 30 min and 1 h, respectively.

This indicates a slower absorption in ZOB rats in comparison to the healthy Zucker lean rats,

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Time (Minutes)

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% o

f A

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d D

ose

0.00

0.05

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0.15

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Pancreas

Spleen

Liver

Kidney

Figure 3.4.C: Concentration of 51Cr measured in the pancreas, spleen, liver and kidney after an

oral dose of 51CrCl3 in ZDF rats. Concentration is represented by the percentage of the applied

dose measured for each sample as a function of time.

though the level of Cr retention is similar. Unlike Zucker lean and ZOB rats, the level of Cr

absorption in the ZDF rats is elevated for both 30 min and 1 h. ZDF rats retained ~1.3 % of the

applied dose at 30 min and ~1.9 % of the applied dose at 1 h. This high retention of Cr in the

tissues and the bloodstream is followed by an elevated level of Cr excretion in the urine. Based on

these results ZDF rats appear to absorb at least ~50 % more Cr than the Zucker lean and ZOB rats

during the first hour after administration. The amount of absorbed Cr in Zucker lean rats is similar

to what has been observed in other studies. In humans, dietary Cr absorption (as CrCl3) was

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estimated to be between 0.5 and 2 %.15 Absorption has also been studied in rat models which have

observed similarly poor absorption rates (~0.5-1.3 %).9, 16

Similar studies have been performed in rats to examine the distribution of Cr, though the

relevancy to this study is not ideal due to the method of administration being through intravenous

injection instead of oral gavage. A study by Kraszeski, et al. examined serum and tissue Cr

concentrations in both healthy and streptozotocin (STZ)-induced diabetic rats 1 and 3 days after

intravenous injection of 51CrCl3.17 STZ induces type 1 diabetes by damaging insulin-producing

pancreatic beta cells. Serum Cr levels were higher in the diabetic rats compared to the controls,

which was also observed herein in type 2 diabetic rats.17 Interestingly, after diabetic rats received

daily insulin injections, the serum levels of diabetic rats lowered to resemble those of the healthy

rats.17 STZ-treated rats also had elevated Cr levels in the liver after 2 h, which was also reversed

by insulin treatment.18 These results indicate the difference in serum and liver Cr levels is insulin-

dependent and insulin plays an important role in Cr transport.

Cr has been shown to be stored and transported in the blood as a complex with the glycoprotein

transferrin. Increased urinary Cr loss and increased tissue Cr was observed in STZ rats versus

healthy rats given an intravenous injection of Cr2-transferrin.18 After treatment with insulin the

observed increase in urinary Cr loss and tissue Cr was lowered to comparable levels with the

controls.18 This study also compared the serum and tissue Cr levels of ZOB rats to healthy, Sprague

Dawley rats. The ZOB rats also displayed greater urinary Cr loss than the healthy rats, though this

loss was reduced by insulin treatment.18 Plasma Cr levels were similar in the ZOB and healthy rats

and liver Cr levels were only slightly higher in ZOB. Skeletal muscle samples, however, were

greater in Cr content than the healthy rats both before and after insulin treatment. Other labs,

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however have reported no effects from insulin supplementation on Cr retention in both healthy

and STZ-induced diabetic rats, although no data was presented.13

A similar study by Feng, et al. examined radiolabelled Cr biodistribution in healthy and alloxan-

induced diabetic rats.19 Alloxan induces a type 1 diabetic model by selectively destroying insulin-

producing pancreatic beta cells. Diabetic and healthy rats were intragastrically administered

radiolabeled Cr (unspecified form) to fasted rats.19 This differed from the current study in which

rats were allowed to feed ad libitum. Cr content was measured in the stomach, small intestine,

large intestine, feces, urine, blood, liver, kidneys, skeletal muscle, femur, testes, heart, spleen, lung,

pancreas, and brain 1, 2, 4, 8, 24, 48, 96, and 168 h post Cr administration.19 Cr distribution in the

alloxan-induced diabetic rats compared to controls followed with the results of the three groups of

rats presented in the current study as follows. Administered Cr passed through the stomach and

into the small intestine in < 1 h at which time over 80 % of the administered dose was present in

the small intestine in both the healthy and alloxan-induced diabetic rats.19 As seen in the current

study, Cr remained in the small intestine longer for diabetic rats. At 2 h post-dose the alloxan-

induced diabetic rats maintained ~40 % of the administered dose in the small intestine, while only

~8 % of the administered dose is present in the small intestine of the controls, as ~87 % of the

administered dose is present in the large intestine after 2 h.19 Cr was then elevated in the large

intestine peaking at 2 to 8 h for the healthy rats and 4 h for the diabetic rats. Cr levels were higher

in the type 1 diabetic models versus the controls’ skeletal muscle and reached their highest Cr level

at 4 h in contrast to 1 h for the controls. Like the present study, the blood and tissue content rose

initially then began to drop as Cr concentration in the urine increased.19 Cr in the kidneys, liver,

and femur rose from 8-24 h after administration in both groups of rats, indicating movement of

absorbed Cr. Urinary Cr content was again raised in diabetic versus controls with a two-fold higher

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urinary Cr content measured in alloxan-induced diabetic rats at 48 h and over four-fold higher after

168 h versus healthy controls. In the study described herein, the type 2 diabetic model ZDF rats

displayed elevated urinary Cr levels throughout. These results as well as the results of the current

study indicate that type 1 and type 2 diabetic rats absorb greater levels of orally introduced Cr and

in turn excrete greater Cr levels in the urine than healthy rats.19

These results are not entirely unexpected as similar results have been observed with Fe and type

2 diabetes. The iron transport protein transferrin is capable of binding Cr and aiding its transport,

absorption and distribution.20, 21 Subjects with type 2 diabetes displayed increased urinary Fe loss,

which seems to be offset by the presence of increased Fe reserves in these subjects with type 2

diabetes.22, 23 This indicates a possible similarity between the transport and absorption of Fe and

Cr in association with insulin resistance which can be further examined, though Fe is transported

through active transport mechanisms, while Cr is transported primarily through passive diffusion.

This increase in urinary Cr loss may be a result of the increased urinary output observed in both

type 1 and type 2 diabetics and be a result of the diabetes, or it may be a consequence of the

increased urinary Cr absorption observed in these models. This can be determined as Cr content

in urine, blood, and tissues is directly proportional to input. In either case, Cr is increasingly

absorbed and excreted in diabetic models versus healthy models. Few alterations of Cr transport

and distribution were observed in the insulin-resistant ZOB (pre-diabetic) model when compared

to Zucker lean rats. Alterations in urinary Cr loss and Cr absorption were not observed between

ZOB and Zucker lean rats, though ZOB rats appears to absorb Cr slightly slower. Further research

is required to elucidate the benefits or lack thereof of pharmacologically relevant Cr

supplementation on models of diabetes (type 1 or type 2). It is not expected that supplementation

of human diets with nutritional quantities (~30 μg Cr/d) of Cr will provide beneficial effects such

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as increased insulin sensitivity or effects on triglyceride levels and there is no apparent “Cr

deficiency” due to the offsetting of the Cr absorption and Cr excretion in the diabetic models.

These observed beneficial effects are likely due to pharmacological effects of Cr given at high

doses.

3.4: Conclusions

The results presented herein indicate that type 2 diabetic rats (ZDF) absorb increased amounts

of Cr from the gastrointestinal tract in addition to losing increased amounts of urinary Cr when

compared to healthy Zucker lean rats. Zucker lean rats absorb the orally administered Cr at

approximately 1 % efficiency (observed at 30 min and 1 h). ZOB rats have a similar ~1 %

absorption which is reached more slowly than the Zucker lean rats at 1 h. Alternatively, ZDF rats

appear to have approximately twice the Cr absorption observed in the ZOB or Zucker lean rats at

~2 % of the administered dose. These results are similar to those observed by Feng, et al. who

observed increased Cr absorption and increased urinary Cr loss in alloxan-induced type 1 diabetic

rats when compared to healthy control rats.19 It appears any additional urinary Cr loss in diabetic

models is offset by increases in Cr absorption. Large supplementary doses of Cr have been shown

to increase insulin sensitivity in response to a glucose challenge in Chapter 2 in Zucker lean rats.

This result would be beneficial to altered states of glucose metabolism and insulin-resistant

diseases, such as diabetes, though more research is needed as Cr transport and absorption is clearly

influenced by type 1 and type 2 diabetes.

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3.5: References

1. Anderson, R. A., Chromium, glucose intolerance and diabetes. Journal of the American

College of Nutrition 1998, 17, 548-55.

2. Anderson, R. A.; Polansky, M. M.; Bryden, N. A.; Roginski, E. E.; Mertz, W.; Glinsmann, W.,

Chromium supplementation of human subjects: effects on glucose, insulin, and lipid variables.

Metabolism 1983, 32, 894-9.

3. Anderson, R. A.; Cheng, N. Z.; Bryden, N. A.; Polansky, M. M.; Cheng, N. P.; Chi, J. M.;

Feng, J. G., Elevated intakes of supplemental chromium improve glucose and insulin variables

in individuals with type 2 diabetes. Diabetes 1997, 46, 1786-1791.

4. Balk, E. M.; Tatsioni, A.; Lichtenstein, A. H.; Lau, J.; Pittas, A. G., Effect of chromium

supplementation on glucose metabolism and lipids: A systematic review of randomized

controlled trials. Diabetes Care 2007, 30, 2154-2163.

5. Anderson, R. A., Chromium as an essential nutrient for humans. Regulatory Toxicology and

Pharmacology 1997, 26, S35-41.

6. Morris, B. W.; MacNeil, S.; Hardisty, C. A.; Heller, S.; Burgin, C.; Gray, T. A., Chromium

Homeostasis in Patients with Type 2 (NIDDM) Diabetes. Journal of Trace Elements in

Medicine and Biology 1999, 13, 57-61.

7. Clodfelder, B.; Gullick, B.; Lukaski, H.; Neggers, Y.; Vincent, J., Oral administration of the

biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ increases insulin sensitivity and improves blood

plasma variables in healthy and type 2 diabetic rats. Journal of Biological Inorganic Chemistry

2005, 10, 119-130.

8. Sun, Y.; Ramirez, J.; Woski, S. A.; Vincent, J. B., The binding of trivalent chromium to low-

molecular-weight chromium-binding substance (LMWCr) and the transfer of chromium from

transferrin and chromium picolinate to LMWCr. Journal of Biological Inorganic Chemistry

2000, 5, 129-36.

9. Olin, K. L.; Stearns, D. M.; Armstrong, W. H.; Keen, C. L., Comparative retention absorption

of chromium-51 (Cr-51) from Cr-51 chloride, Cr-51 nicotinate and Cr-51 picolinate in a rat

model. Trace Elements and Electrolytes 1994, 11, 182-186.

10. Ardevol, A.; Adan, C.; Remesar, X.; Fernandez-Lopez, J. A.; Alemany, M., Hind leg heat

balance in obese Zucker rats during exercise. Pflugers Arch 1998, 435, 454-64.

11. Rolland, V.; Roseau, S.; Fromentin, G.; Nicolaidis, S. V.; Tome, D.; Even, P. C., Body weight,

body composition, and energy metabolism in lean and obese Zucker rats fed soybean oil or

butter. American Journal of Clinical Nutrition 2002, 75, 21-30.

12. Walpole, S. C.; Prieto-Merino, D.; Edwards, P.; Cleland, J.; Stevens, G.; Roberts, I., The

weight of nations: an estimation of adult human biomass. BMC Public Health 2012, 12.

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13. Anderson, R. A.; Polansky, M. M., Dietary and metabolite effects on trivalent chromium

retention and distribution in rats. Biological Trace Element Research 1995, 50, 97-108.

14. Kottwitz, K.; Laschinsky, N.; Fischer, R.; Nielsen, P., Absorption, excretion and retention of

Cr-51 from labelled Cr-(III)-picolinate in rats. BioMetals 2009, 22, 289-295.

15. Anderson, R. A.; Kozlovsky, A. S., Chromium intake, absorption and excretion of subjects

consuming self-selected diets. American Journal of Clinical Nutrition 1985, 41, 1177-83.

16. Anderson, R. A.; Bryden, N. A.; Polansky, M. M.; Gautschi, K., Dietary chromium effects on

tissue chromium concentrations and chromium absorption in rats. Journal of Trace Elements

in Experimental Medicine 1996, 9, 11-25.

17. Kraszeski, J. L.; Wallach, S.; Verch, R. L., Effect of insulin on radiochromium distribution in

diabetic rats. Endocrinology 1979, 104, 881-5.

18. Clodfelder, B. J.; Upchurch, R. G.; Vincent, J. B., A comparison of the insulin-sensitive

transport of chromium in healthy and model diabetic rats. Journal of Inorganic Biochemistry

2004, 98, 522-533.

19. Feng, W. Y.; Ding, W. J.; Qian, Q. F.; Chai, Z. F., Study on the metabolism of physiological

amounts of Cr(III) intragastrical administration in normal rats using activable enriched stable

isotope Cr-50 compound as a tracer. Journal of Radioanalytical and Nuclear Chemistry 1998,

237, 15-19.

20. Clodfelder, B. J.; Emamaullee, J.; Hepburn, D. D. D.; Chakov, N. E.; Nettles, H. S.; Vincent,

J. B., The trail of chromium(III) in vivo from the blood to the urine: the roles of transferrin and

chromodulin. Journal of Biological Inorganic Chemistry 2001, 6, 608-617.

21. Clodfelder, B. J.; Vincent, J. B., The time-dependent transport of chromium in adult rats from

the bloodstream to the urine. Journal of Biological Inorganic Chemistry 2005, 10, 383-393.

22. Bao, W.; Rong, Y.; Rong, S.; Liu, L., Dietary iron intake, body iron stores, and the risk of type

2 diabetes: a systematic review and meta-analysis. BMC Medicine 2012, 10.

23. Rajpathak, S. N.; Crandall, J. P.; Wylie-Rosett, J.; Kabat, G. C.; Rohan, T. E.; Hu, F. B., The

role of iron in type 2 diabetes in humans. Biochimica et Biophysica Acta-General Subjects

2009, 1790, 671-681.

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CHAPTER 4

THE EFFECTS OF DIABETES AND EXTENDED CHROMIUM SUPPLEMENTATION ON

THE TISSUE METAL CONCENTRATIONS OF ZUCKER LEAN, ZUCKER OBESE, AND

ZUCKER DIABETIC FATTY RATS

4.1: Introduction

As seen in the literature and Chapter 2, extended Cr supplementation was shown to have an

influence on glucose metabolism by increasing the insulin sensitivity in healthy rats.1 In the

previous chapter, the absorption, excretion, and biodistribution of a single orally-administered

dose of radiolabeled 51Cr was examined. Differences were observed in the transport, absorption,

and excretion of a single dose of 51CrCl3 given to healthy Zucker lean rats compared to those in

models of insulin resistance (Zucker Obese (ZOB), and Zucker Diabetic Fatty (ZDF)). These

results raise the question: how does the extended oral administration of Cr influence the transport,

storage, and absorption of other metals and do these tissue metal concentrations differ between

healthy rats versus models of insulin resistance and diabetes?

To begin, the tissue metal distribution for healthy (Zucker lean), pre-diabetic (Zucker Obese,

ZOB), and type 2 diabetic (Zucker Diabetic Fatty, ZDF) should be examined to compare with other

models of diabetes and insulin resistance as well as with the healthy rats. Tissue metal ion

concentrations have been examined in rat models of diabetes or insulin resistance in the past. Some

of the previous studies of this nature involve examining rats on high-fructose diets,2,3

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streptozotocin (STZ)-induced diabetic rats,4 as well as high-fat STZ-induced diabetic rats.5, 6 STZ

targets insulin-producing beta-cells in the pancreas inducing a model of type 1 diabetes at a large

dose and type 2 diabetes at smaller doses. These studies utilized different models of diabetes as

well as different stressors such as high-fat diets resulting in varying levels of tissue metal

concentrations compared to healthy controls. One aspect that has become clear is that a difference

in the metabolism, bioavailability as well as excretion of metals exists between healthy rats versus

the varying models of insulin resistance or diabetes. The underlying mechanisms of these

differences are not well understood.

Dietary changes can affect the tissue metal concentrations as seen in several studies which

examine tissue metal concentrations in rats given a diet high in fructose compared to a standard

balanced rat chow.2, 5 The high-fructose diet utilized in these studies lowered the rats’ liver Cu

concentrations, but not kidney Cu, with little to no effect on kidney or liver Fe, Zn, or Cr.2, 5 In

addition to dietary changes, disease states can also affect tissue metal concentration. Studies

examining STZ-induced diabetic rats observed increased levels of liver Fe and Cu as well as

decreased levels of Zn and Mg versus healthy control rats.4 When combined with a high-fat diet,

the STZ-induced diabetic rats displayed increases in Fe in both the liver and kidney, as well as

increased liver Cu concentrations as seen in the STZ-induced diabetic rats not given a high-fat

diet.4-6 Other effects observed in STZ-induced diabetic rats were not observed when given a high-

fat diet as the results are inconsistent between studies.

ZDF and ZOB rats are common models of type 2 diabetes and insulin resistance (pre-diabetic),

respectively.7 ZOB rats originate from the Zucker lean lineage with a single missense mutation

expressed in the gene encoding for the leptin receptor, producing nonfunctional mRNA and

inducing a glycine to proline change in all isoforms of the leptin receptor.8 Leptin is a hormone

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secreted primarily by mature adipocytes and is partially responsible for food intake regulation,

energy homeostasis, appetite behaviors, as well as energy expenditure regulation.8 In addition to

these roles, leptin has been implicated in other processes involving energy homeostasis and

regulation such as thermoregulation, reproduction, oncogenesis, and skeletal growth.8-10 Due to

mutation in the leptin receptor, ZOB rats display hyperphagia, obesity, insulin resistance, high

cholesterol, and mild hyperglycemia and are considered a “pre-diabetic” model. ZDF rats are an

inbred strain derived from the ZOB rats which have another, undescribed mutation, unrelated to

the leptin receptor, which induces hyperglycemia, among other changes. ZDF rats initially become

obese, much like the ZOB rats, but as they age, male ZDF rats (around 10-12 weeks of age) begin

to display other signs of diabetes such as hyperglycemia, beta-cell dysfunction, insulin resistance,

and high cholesterol. ZDF rats are commonly used as a model for type 2 diabetes.

A few studies have been conducted to determine the tissue metal concentrations in ZOB rats

though no systemic studies in ZDF rats have been reported.11-13 Compared to Zucker lean rats,

ZOB rats are reported to have lower whole body Cu concentrations which disappeared when given

an energy dense cafeteria diet consisting of unhealthy human food ad libitum.11 Another lab

reported ZOB rats have higher Cu kidney concentrations per g of protein at 5 weeks and higher

Cu in the kidney and the liver by 12 weeks compared to their healthy Zucker lean counterparts

while no differences were observed in Zn or Fe concentrations in the liver or the kidney.12

Comparison between studies is difficult due to the use of several different units of measure. Several

labs such as those mentioned above report the tissue metal concentrations in mg of metal per g of

protein or mg metal per entire organ while the studies herein report mg metal per g dry tissue, not

allowing for direct comparison as the size of the organs between ZOB and Zucker lean vary (e. g.

the liver of the ZOB is much larger than the Zucker lean). When comparing mg metal per g dried

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tissue, lower levels of Zn and Cu in the ZOB versus Zucker lean rats were observed.13 These

changes were attributed to the increased adipose tissue in the organs of the ZOB rats. When the

samples were corrected for neutral fat content, the liver Zn and Cu content was equivalent for the

ZOB and Zucker lean rats. When ZOB and Zucker lean rats were treated with STZ, increased

kidney and liver Cu and Zn levels were observed, again per g of dried tissue.13 Due to these

variabilities in ZOB and lack of ZDF tissue metal data, a comparison between Zucker lean, ZOB,

and ZDF is needed. This study tested the tissue metal concentrations of Zucker lean, ZOB and

ZDF rats in order to examine the effects of a pre-diabetic and type 2 diabetic state. Tissue metal

concentrations measured in ZOB and ZDF rats are expected to differ when compared to the healthy

Zucker lean rats.

In addition to the measurement of basal tissue metal concentrations between the different

diabetic states, the rats were supplemented with various Cr compounds in order to measure the

variability in tissue metal concentrations that are induced by sustained Cr supplementation. As

seen in previous chapters, Cr has the ability to increase insulin sensitivity in healthy rats and

displays altered absorption, biodistribution, and excretion in insulin-resistant models. Cr has been

shown multiple times to improve insulin sensitivity in models of diabetes and insulin resistance.1,

14, 15 In addition, some Cr complexes given at high doses have been shown to improve insulin

sensitivity and lipid parameters in healthy rats.1, 16

The addition of supplementary metals, such as Cr, could result in alterations of the absorption,

metabolism, transport, and/or excretion of other metals present in the organism possibly due to

interactions with macro- or microelements. Metals can often utilize the same transport proteins

resulting in imbalances with an excess or deficient of a certain metal in the system. The

glycoprotein transferrin, for example, is responsible for Fe sequestration and transport throughout

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the body, but may also be responsible for the in vivo transport of other metals including Ti4+, VO2+,

and Cr3+ to name a few.17 Cr and Fe competitively bind to sites on transferrin in the bloodstream,

leading to potential ill-effects on Fe biodistribution by Cr supplementation.18 It will be determined

whether large amounts of Cr may result in a Fe imbalance, especially since the positive insulin

sensitizing effects have been observed only at high doses.1, 19 Type 2 diabetes and insulin resistance

result in alterations in tissue metal concentrations that may also be influenced by Cr

supplementation, though whether positively or negatively remains to be seen. This study seeks to

elucidate the effects of supplementation of the Cr picolinate (the most widely used Cr supplement),

CrCl3, Cr3, and vanadyl (as a model of chromate) on the metal tissue concentrations of Zucker

lean, ZOB, and ZDF rats in order to indicate whether extended Cr supplementation can alter tissue

metal concentrations.

4.2 Materials and Methods

4.2.1 Animals and Husbandry

One hundred forty-four male rats, 48 Zucker lean, 48 Zucker obese, and 48 Zucker diabetic fatty

(ZDF), approximately 6 weeks old were obtained from Charles River Breeding Laboratories,

Zucker obese rats are an insulin-resistant model of obesity and early stage type 2 diabetes, while

ZDF rats are a model of type 2 diabetes. Rats were maintained in an AAALAC-approved animal

care facility in rooms with 22 ± 2 °C, 40-60 % humidity, and a 12 h photoperiod. Animals were

housed two rats/cage containing hardwood bedding and were given Harlan Teklad rodent chow

and water ad libitum.

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4.2.2 Treatments

Following a 1 week acclimation period, rats were assigned to the following treatment groups

with treatments administered by gavage daily at circa 9 am for 12 weeks: (groups 1-3) eight Zucker

lean, eight Zucker obese, and eight ZDF as control vehicles; (groups 4-6) eight Zucker lean, eight

Zucker obese, and eight ZDF receiving 1 mg Cr per kg body mass per day as CrCl3; (groups 7-9)

eight Zucker lean, eight Zucker obese, and eight ZDF receiving 33 μg Cr per kg body mass per

day as Cr3; (groups 10-12) eight Zucker lean, eight Zucker obese, and eight ZDF receiving 1 mg

Cr per kg body mass per day as Cr3 ([Cr3O(propionate)6(H2O)3]+); (groups 13-15) eight Zucker

lean, eight Zucker obese, and eight ZDF receiving 1 mg Cr per kg body mass per day as Cr(pic)3;

and (groups 16-18) eight Zucker lean, eight Zucker obese, and eight ZDF receiving 2 mg/kg

vanadyl sulfate (a source of vanadate in vivo) per day. Animals were weighed twice weekly.

4.2.3 Surgeries and Organ Collection

After the 12-week treatment period, rats were anesthetized using isoflurane. A bundle of vastus

lateralis muscle fibers and the end of one segment of epididymal fat were dissected from the right

side of the body for studies beyond the scope of this report. The rats were then treated intravenously

with 5 units of insulin (bovine Zn) per kg body mass; after 30 min, left muscle and fat samples

were collected for studies beyond the scope of this work. The rats were then sacrificed by carbon

dioxide asphyxiation, and the liver, heart, spleen, and kidneys were harvested and weighed.

Tissues were transferred directly to plastic weigh boats for weighing and then to disposable plastic

centrifuge tubes (capable of withstanding at temperature of 105 °C). The heart, spleen, kidneys,

and a weighed aliquot of liver from each rat were then dried to a constant mass in a vacuum oven

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at 105 °C. All procedures with the rats were approved by The University of Alabama Institutional

Animal Use and Care Committee.

4.2.4 Atomic Absorption Spectrometry for Metal Analyses

For metal analyses, samples were digested with concentrated 65 % spectra pure HNO3 (Merck)

in a Microwave Digestion System (MARS-5, CEM). The concentration of Cu, Zn, Fe, Mg, and Ca

was determined by flame atomic absorption spectrometry method F-AAS (Zeiss AA-3, with

background correction). The concentration of Cr was measured using a graphite furnace atomic

absorption spectrometer (AA EA 5 with background correction, Jenoptik). The accuracy of the

determination of Cu and Zn was assured by simultaneous analysis of the certified reference

material bovine liver BCR®-185R (IRMM), while analysis of Fe, Mg, and Ca was controlled using

the certified reference material Virginia tobacco leaves CTA-VTL-2 (Poland). Analysis of Cr was

assured using the certified reference material mussel tissue ERM®-CE278 (ERM). The recovery

for Cu, Zn, Fe, Mg, Ca, and Cr (expressed of the percentage of the mean certified values) were

103 %, 101 %, 97 %, 104 %, 103 %, and 102 %, respectively.

4.2.5 Chromium Compounds

Chromium picolinate and Cr3 were prepared as described previously.20, 21 CrCl3·6H2O (actually

trans-(Cr(H2O)4Cl2)Cl·2H2O and vanadyl sulfate were used as received.

4.2.6 Statistical Analyses

Each data point in the figures represents the average value for eight rats. Error bars in the figures

denote standard deviation. Data were tested for homogeneity of variance by means of the Levine

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statistic and were analyzed by repeated measures ANOVA using SPSS (SPSS, Inc.). Specific

differences (p ≤ 0.05) were determined by LSD and a Bonferroni post hoc test. For eight animals

per group, an expected difference between two means would be significant at the 0.05 level if the

difference between the means is twice the standard deviation.

4.3: Results and Discussion

4.3.1 Differences Between Strains (Healthy, Obese/Pre-Diabetic, Type 2 Diabetic)

Blood samples were not analyzed for metal content as it has been documented that the presence

of insulin can alter blood metal concentrations by moving ions to or from the bloodstream.18, 22

The blood, however, represents a small but rapidly mobilizable pool of certain metal ions and

many metal ions utilize multiple storage pools. Tissues such as the liver on the other hand represent

a large pool of certain metals in the body that allows for a much slower exchange of metals with

the bloodstream. Ingested Cr, for example, is thought to reside in the tissues as a large pool which

slowly exchanges with Cr in the bloodstream (over several months).23-25 As such, the insulin

treatments were followed by approximately an hour (30 min of waiting plus surgeries) and are

anticipated to have little to no effect on the metal concentrations of the tissues selected in this

study. This assumption seems to be confirmed by the results that the Zucker lean rat measurements

were all in the normal, previously examined ranges.

No differences were observed in the body masses of the rats between the various diets for Zucker

lean, ZOB, or ZDF rats, though the strains were in different ranges as expected (Figure 4.1).

Chapter 2 also did not observe a difference in body mass between different Cr concentrations in

the diets.1 By the end of the study, the rats themselves weighed approximately ~450 g, ~650 g, and

~400 g for Zucker lean, ZOB, and ZDF rats, respectively. These differences in body masses are a

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result of the differences in strain. Previous results have indicated an oral dose of 1 mg Cr/kg body

mass as Cr3 may result in an increase of body mass in ZOB rats while an intravenous dose of 20

μg Cr/kg body mass as Cr3 had no effect, though those effects were not observed in this study.16

The tissue levels of Cr, Cu, Zn, Fe, Mg, and Ca were generally similar between the Zucker lean,

ZOB, and ZDF rats except where indicated (Figures 4.2 - 4.7). One of the observed differences

was seen in ZDF rats versus their healthy counterparts. ZDF rats displayed elevated Cu

concentration in the kidneys compared to the Zucker lean and ZOB rats, regardless of treatment

(Figure 4.3.B). No other differences were observed between the ZDF rats and their healthy

counterparts. This result of elevated kidney Cu is similar to results seen in STZ-induced type 1

diabetic rats and high-fat diet induced diabetes.4-6

ZOB rats displayed more variation in tissue metal concentration when reported as metal

concentration in μg per g dried mass. Reduced amounts of many metals in the liver were recorded

for ZOB rats versus both Zucker lean and ZDF rats. ZOB rats had reduced liver Cu and Zn levels

compared to the ZDF rats, reduced liver Fe levels compared to both Zucker lean and ZDF, as well

as reduced liver Mg concentrations compared to Zucker lean rats. Spleen Fe concentrations were

also reduced in the ZOB rat versus the Zucker lean control (Figure 4.5.C). Alternatively, kidney

Ca levels were elevated in the ZOB rats versus the other strains (Figure 4.7.B). The reduced liver

metals (Cu and Zn) observed in ZOB rats have been reported previously.13 These reduced liver

metal concentrations observed in the ZOB rats are likely from the increased adipose tissue present

in the liver of the obese rats compared to both ZDF and Zucker lean rats. Increased kidney Ca

levels have also been observed previously and were attributed to impairment of Ca-ATPase

activity in the ZOB rats.26

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Figure 4.1.A: Body masses of Zucker lean rats supplemented daily with Cr or vanadyl sulfate. No

significant differences were observed.

Figure 4.1.B: Body masses of ZOB rats supplemented daily with Cr or vanadyl sulfate. No

significant differences were observed.

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Figure 4.1.C: Body masses of ZDF rats supplemented daily with Cr or vanadyl sulfate. No

significant differences were observed.

4.3.2 Chromium and Vanadium Supplementation

The current study is designed to examine the effects of extended high doses of various Cr

complexes on tissue metal concentrations of healthy (Zucker lean), insulin-resistant (ZOB), and

type 2 diabetic (ZDF) rats. As described in Chapter 2, extended high doses of Cr were shown to

have the pharmacological effect of increasing insulin sensitivity by reducing the amount of insulin

required to alleviate a glucose challenge in healthy, Zucker lean rats.1 Cr itself has been studied

for over half a century as a potential nutritional supplement as well as therapeutic agent for various

purposes such as weight loss and muscle development agents.19 CrCl3, Cr(pic)3, and Cr3 were

chosen to be compared. CrCl3 and Cr(pic)3 are the most commonly studied forms of Cr for use as

a therapeutic agent or nutritional supplement. Cr3 was chosen due to its observed higher absorption

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efficiency.19 Cr3 has also been studied in Zucker lean, ZOB, and ZDF rats and allows for greater

comparison between studies.16, 27

In addition to the three Cr compounds, vanadyl sulfate was administered to all three rat models

as an analog for chromate (CrO4-). A proposed mechanism of action for the pharmacological effect

of Cr has been proposed by Lay and coworkers stating the observed effects of Cr are actually toxic

effects arising from the generation of chromate from Cr in the body.28, 29 Chromate itself could not

be used in this study as it is reduced to Cr3+ in the gastrointestinal tract and would serve mostly as

another source of Cr. Vanadyl sulfate was utilized in order to mimic the effects that would be

induced by chromate administration. Vanadium containing compounds have previously been

shown to provide beneficial effects in diabetic rodents.30 Vanadyl sulfate was used as the source

of vanadate in this study at 2 mg/kg as it is the most commonly used in studies of vanadyl in rodent

models.31

The doses of the Cr compounds were chosen in order to best compare results between studies as

well as to account for variations in absorption efficiencies. The dose 1 mg Cr/kg body mass was

used for all Cr compounds. This levels of Cr3 has been shown to improve insulin sensitivity and

improve cholesterol levels in Zucker lean, ZOB, and ZDF rats previously.16 Additionally, studies

examining this level of administration of CrCl3, Cr(pic)3, and Cr3 have observed Cr accumulation

in the liver and kidney of rats.16, 32 The lower dose of Cr3 was used to account for the difference

in absorption efficiency from the gastrointestinal tract as CrCl3 and Cr(pic)3 have an absorption of

< 2 %,33-35 while the absorption of Cr3 has been measured at ~40-60 %.36 The two different

concentrations also allow for a dose-response relationship to be investigated.

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4.3.3 Effects of Supplementation of Cr and Vanadium on Tissue Metal Concentrations

Few effects were observed on tissue metal concentrations due to the extended administration of

various Cr compounds and vanadyl sulfate (Figure 4.8). The concentration of Cr in the kidneys

was elevated in Zucker lean and ZOB rats receiving either 1 mg Cr/kg body mass as CrCl3 or Cr3,

but remained unchanged for those receiving Cr(pic)3 or the smaller dose of Cr3 (33 μg Cr/kg body

mass) (Figure 4.2, 4.8). Cr concentration in the ZDF kidneys or liver from Zucker lean, ZOB, and

ZDF rats were not affected by Cr supplementation. The elevated Cu levels observed in the ZDF

rats were significantly reduced by the administration of either 1 mg Cr/kg body mass of CrCl3

orCr3, and appear to be partially reduced for rats given the chromate analog, vanadyl sulfate

(Figure 4.8). This indicates a possible restorative effect of Cr supplementation in ZDF rats with

CrCl3 or Cr3 (but not Cr(pic)3) by reducing kidney Cu levels. One other treatment effect was

observed in Zucker lean rats. Liver Ca concentrations in Zucker lean rats were increased with

administration of 1 mg Cr/kg body mass of Cr3 or Cr(pic)3 (Figure 4.7.A). The significance of this

observation is currently unknown.

Beneficial effects of Cr supplementation on diabetic symptoms have been previously observed

using various Cr supplements, including Cr3 and CrCl3 with disparate results. Several beneficial

effects of Cr3 have been reported using several rat models of diabetes and insulin resistance.

Healthy Sprague-Dawley (SD) rats receiving an intravenous dose of 20 μg Cr/kg body mass daily

for 12 weeks exhibited lower plasma insulin, total cholesterol, LDL cholesterol, HDL cholesterol,

and triglycerides than rats not receiving additional Cr3, though no changes were observed in

glucose levels between the groups.37 Another study similar results when Cr3 was given at the same

dose (20 μg Cr/kg body mass) at 4, 12, 16, 20, and 24 weeks of administration.38

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Figure 4.2.A: Liver Cr concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Dagger represents significant difference from Zucker lean rats (p ≤ 0.05).

Figure 4.2.B: Kidney Cr concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Dagger represents significant difference from Zucker lean rats (p ≤ 0.05).

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Figure 4.3.A: Liver Cu concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Double dagger represents significant difference from ZDF rats (p ≤ 0.05).

Single asterisk indicates significant difference from the other two rat strains (p ≤ 0.05).

Figure 4.3.B: Kidney Cu concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Single asterisk indicates significant difference from the other strains (p ≤ 0.05).

Double asterisk indicates all strains are significantly different from each other (p ≤ 0.05).

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Figure 4.3.C: Spleen Cu concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

Figure 4.3.D: Heart Cu concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.4.A: Liver Zn concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Double dagger represents significant difference from ZDF rats (p ≤ 0.05).

Single asterisk indicates significant difference from the other two rat strains (p ≤ 0.05).

Figure 4.4.B: Kidney Zn concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.4.C: Spleen Zn concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

Figure 4.4.D: Heart Zn concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.5.A: Liver Fe concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Single asterisk indicates significant difference from the other strains (p ≤ 0.05).

Double asterisk indicates all strains are significantly different from each other (p ≤ 0.05).

Figure 4.5.B: Kidney Fe concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.5.C: Spleen Fe concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Double dagger represents significant difference from ZDF rats (p ≤ 0.05).

Figure 4.5.D: Heart Fe concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.6.A: Liver Mg concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. Single asterisk indicates significant difference from the other two rat strains

(p ≤ 0.05). Dagger represents significant difference from Zucker lean rats (p ≤ 0.05). Double

dagger represents significant difference from ZDF rats (p ≤ 0.05).

Figure 4.6.B: Kidney Mg concentrations in Zucker lean, ZOB, and ZDF rats supplemented with

Cr or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.6.C: Spleen Mg concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

Figure 4.6.D: Heart Mg concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.7.A: Liver Ca concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

Figure 4.7.B: Kidney Ca in Zucker lean, ZOB, and ZDF rats supplemented with Cr or vanadyl

sulfate. Single asterisk indicates significant difference from the other rat strains (p ≤ 0.05).

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Figure 4.7.C: Spleen Ca concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

Figure 4.7.D: Heart Ca concentrations in Zucker lean, ZOB, and ZDF rats supplemented with Cr

or vanadyl sulfate. No significant differences were observed between treatments or strains.

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Figure 4.8.A: Kidney Cr concentrations of Zucker lean rats supplemented with Cr or vanadyl

sulfate. Different letters indicate significant difference between treatment groups (p ≤ 0.05).

Figure 4.8.B: Kidney Cr concentrations of ZOB rats supplemented with Cr or vanadyl sulfate.

Different letters indicate significant difference between treatment groups (p ≤ 0.05).

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Figure 4.8.C: Liver Ca concentrations of ZOB rats supplemented with Cr or vanadyl sulfate.

Different letters indicate significant difference between treatment groups (p ≤ 0.05).

Figure 4.8.D: Kidney Cu concentrations of ZDF rats supplemented with Cr or vanadyl sulfate.

Different letters indicate significant difference between treatment groups (p ≤ 0.05).

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Beginning as early as 4 weeks after initiation of Cr3 administration, plasma insulin levels were

decreased in healthy SD rats. Triglyceride levels were decreased in the Cr3 receiving SD rats

beginning at 12 weeks. Total cholesterol was reduced in the Cr3 receiving healthy SD rats

beginning at week 16 while the HDL levels were reduced only at 24 weeks of administration.38 In

addition, the plasma glucose and insulin levels 2 h after a glucose challenge were significantly

decreased in the Cr3 receiving healthy SD rats compared to controls not receiving additional Cr.38

In the same study, STZ-induced diabetic rats did not display any consistent statistically significant

effects though the plasma insulin, total cholesterol, and triglycerides tended to be lower in Cr3

treated rats.38 The STZ treatment seemed to spread the values of the measured variables leading to

a loss of sufficient power to attribute any effects. ZOB rats were also examined in this article and

similar intravenous treatment resulted in lower plasma insulin for those receiving Cr3 beginning

at 4 weeks and persisting throughout the study period (24 weeks).38 Triglyceride concentrations

and total cholesterol were lower beginning at weeks 8, 12, 20, and 24 for ZOB rats receiving Cr3

and HDL levels were lowered on weeks 4, 16, 20, and 24.38 Plasma insulin levels 2 h after a glucose

challenge were lowered in ZOB rats receiving Cr3, while plasma glucose levels remained the

same.38 Zucker lean rats were also examined with disparate results. Zucker lean rats only displayed

lower plasma insulin levels when treated with Cr3 versus non-treated rats, indicating strain

differences between SD and Zucker lean healthy rats though it should be noted total cholesterol

and triglyceride levels tended to be lower in the Zucker lean rats to begin with compared to SD

rats.38

The effects of oral administration of Cr as Cr3 have also been examined.16 Daily oral gavage of

250, 500, or 1,000 μg Cr/kg body mass in healthy SD rats resulted in lowered fasting plasma

insulin, triglycerides, total cholesterol, and LDL cholesterol levels beginning at 4 weeks of

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treatment and persisting throughout the course of study (24 weeks).16 No effects were observed on

plasma glucose or HDL levels for healthy SD rats. The lowered plasma insulin levels with no

change on glucose (maintenance) levels indicates an increased insulin sensitivity in rats receiving

Cr3. Both plasma glucose and insulin were reduced 2 h after a glucose challenge. 16 ZOB rats

receiving 1,000 μg Cr/kg body mass had similar effects to the intravenous administration. In this

study, the effects of Cr3 on ZDF rats were also examined. ZDF rats receiving 1,000 μg Cr/kg body

mass exhibited lowered plasma insulin, total cholesterol, and LDL cholesterol levels while glucose

levels remained consistently lower, yet not statistically different.16 HDL levels were lowered in

ZDF rats receiving Cr3 from a very elevated state and plasma insulin levels 2 h post-glucose

challenge were also lowered.16 Another measurement taken during this study were the levels of

glycated hemoglobin in the plasma in healthy Zucker lean, ZOB and ZDF rats as an indication of

long term blood glucose status after 4, 12, and 24 weeks of treatment. No effects were observed in

healthy rats, though significant effects were observed in diabetic models.16 In ZDF rats, glycated

hemoglobin was lower after 12 and 24 weeks of treatment dropping ~22 % versus the ZDF controls

by week 24. ZOB rats glycated hemoglobin levels were lowered ~27 % by week 24 as well.16

The effects of Cr on healthy and diabetic models were examined by another lab utilizing Wistar

rats as a healthy control and STZ-induced Wistar rats as a model of type 1 diabetes. A diet

containing 5 mg Cr/kg diet as Cr3 was given to male Wistar rats for 10 weeks, resulting in ~15.6 %

reduced plasma insulin levels and ~9.6 % increased glucose transport in erythrocytes when given

the Cr-containing diet versus the control diet.39 STZ-induced diabetic male Wistar rats receiving

Cr in their diet for 5 weeks (also 5 mg Cr/kg diet as Cr3) had ~26 % lower plasma glucose levels

and ~14 % increased HDL levels compared to those not receiving additional Cr in the diet.3 After

8 weeks of supplementation of a high-fructose diet or a control diet (AIN-93M) with Cr3 (0, 1, or

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5 mg Cr/kg body mass daily) resulted in increased insulin sensitivity in male Wistar rats.19 No

changes were observed as an effect of Cr supplementation in the levels of plasma glucose or lipids

as well as body mass (as in this study).19 In another study, male Wistar rats received either a high-

fat diet or a control diet (AIN-93M) with or without additional Cr as Cr3 (0, 1, or 5 mg Cr/kg body

mass daily) for 5 weeks.5 At the completion of 5 weeks of treatment, rats were treated with STZ

then remained on the diets for an additional week.5 The supplementation of Cr3 increased insulin

sensitivity and lowered total cholesterol, LDL cholesterol, and triglyceride levels but had no effect

on blood glucose levels. Rats on the high-fat diet containing the highest amount of Cr3 had lower

body masses on weeks 3 through 5 of treatment compared to those receiving the high-fat diet

without additional Cr.5 This effect disappeared after STZ treatment and no other differences in

body mass were observed.5

The observation of Cr accumulation in the kidney and liver of rats receiving supplemental Cr

has been reported previously for various Cr compounds, indicating the results depend on the

specific Cr compound and dose. Cr accumulation has been investigated in detail for CrCl3 and

Cr(pic)3.32 Kidney and liver Cr content increases linearly with daily doses of CrCl3 or Cr(pic)3 for

24 weeks with doses ranging from 750 μg Cr/kg body mass to 15 mg Cr/kg body mass.32 In contrast

to the study presented here, Cr(pic)3 levels were greater than CrCl3 in the tissues. The results

presented herein are consistent with a recent study on the absorption of CrCl3 and Cr(pic)3

indicating CrCl3 is better absorbed by rats than Cr(pic)3 when tissue concentrations and urinary Cr

output were considered.35

As in the current study, previous studies have indicated no changes observed in the kidney or

liver Cr concentrations in ZOB or ZDF rats with supplementation of Cr3 (1 mg Cr/kg body mass,

orally, daily for 6 months).16 The same study observed a small drop in the kidney Fe concentration

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in the ZOB, but not the ZDF rats, with no changes in liver Fe. In contrast to the current study, SD

rats receiving Cr3 orally, daily for 6 months at doses of 250, 500, or 1,000 μg Cr/kg body mass

did not display accumulation of Cr in the kidney or liver though increases in kidney Cr were

observed for the healthy control (Zucker lean rats) in the current study.16 This difference may be

the result of a strain difference between SD and Zucker lean rats. Wistar rats receiving a high-

fructose diet supplemented daily with Cr3 did not exhibit increased liver or kidney Cr when given

at 1 mg Cr/kg body mass, but did exhibit increased Cr in tissues for rats receiving a higher dosage

of 5 mg Cr/kg body mass.3 A different study performed by the same lab did observe increased

kidney, but not liver, Cr levels when high-fructose fed Wistar rats received 1 mg Cr/kg body mass

as Cr3, similar to the results observed in this study.2 Another study examining the same effects in

STZ-induced Wistar rats on a high-fat diet found increased kidney Cr in a dose dependent manner

when supplemented with Cr3 at 1 or 5 mg Cr/kg diet, similar to the results observed for Zucker

lean rats in this study.5 The ZOB and ZDF rats’ levels of kidney Cr appeared slightly elevated in

the current study at the 1 mg Cr/kg body mass level for Cr3 and CrCl3 though they were not

statistically significant. The result of the current study as well as the studies mentioned above

indicate that 1 mg Cr/kg body mass of Cr3 may be close to the highest level that rats can effectively

remove supplemental Cr from the body and avoid kidney and/or liver accumulation.

4.4: Conclusions

For the first time, the tissue concentrations of the metals Cu, Zn, Fe, Mg, and Ca were compared

and contrasted in the liver, kidney, heart and spleen and Cr levels in the kidney and liver of Zucker

lean, ZOB, and ZDF rats. Reduced liver levels of Cu, Zn, Fe, and Mg were measured per g of

tissue for ZOB rats compared to the Zucker lean and/or ZDF rats, presumably due to the increased

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adipose content in the liver of the obese rats. Splenic Fe concentrations were reduced and kidney

Ca levels were elevated in the ZOB rats compared to Zucker lean rats. ZDF rats displayed ~4 times

higher kidney Cu levels than Zucker lean and ~8 times higher kidney Cu levels than ZOB rats.

Supplementation of Zucker lean, ZOB and ZDF rats with various CrCl3, Cr3, and Cr(pic)3, as

well as the chromate analog vanadyl sulfate resulted in surprisingly few alterations in tissue metal

concentration. Alterations were expected due to previously observed beneficial effects of Cr

supplementation on the symptoms of insulin resistance. Treatment with CrCl3 or Cr3, but not

Cr(pic)s at 1 mg Cr/kg body mass resulted in the accumulation of Cr in the kidneys of Zucker lean,

and ZOB rats, but not ZDF rats. The same level (1 mg Cr/kg body mass) of Cr3 or CrCl3 also

resulted in lowering the elevated levels of kidney Cu observed in the ZDF rats, suggesting a

beneficial effect on this symptom of type 2 diabetes.

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4.5: References

1. Di Bona, K. R.; Love, S.; Rhodes, N. R.; McAdory, D.; Sinha, S. H.; Kern, N.; Kent, J.;

Strickland, J.; Wilson, A.; Beaird, J.; Ramage, J.; Rasco, J. F.; Vincent, J. B., Chromium is not

an essential trace element for mammals: effects of a "low-chromium" diet. Journal of

Biological Inorganic Chemistry 2011, 16, 381-390.

2. Krol, E.; Krejpcio, Z.; Michalak, S.; Wojciak, R. W.; Bogdanski, P., Effects of Combined

Dietary Chromium(III) Propionate Complex and Thiamine Supplementation on Insulin

Sensitivity, Blood Biochemical Indices, and Mineral Levels in High-Fructose-Fed Rats.

Biological Trace Element Research 2012, 150, 350-359.

3. Krol, E.; Krejpcio, Z., Chromium(III) propionate complex supplementation improves

carbohydrate metabolism in insulin-resistance rat model. Food and Chemical Toxicology 2010,

48, 2791-2796.

4. Ozcelik, D.; Tuncdemir, M.; Ozturk, M.; Uzun, H., Evaluation of trace elements and oxidative

stress levels in the liver and kidney of streptozotocin-induced experimental diabetic rat model.

General Physiology and Biophysics 2011, 30, 356-63.

5. Krol, E.; Krejpcio, Z., Evaluation of anti-diabetic potential of chromium(III) propionate

complex in high-fat diet fed and STZ injected rats. Food and Chemical Toxicology 2011, 49,

3217-3223.

6. Dogukan, A.; Sahin, N.; Tuzcu, M.; Juturu, V.; Orhan, C.; Onderci, M.; Komorowski, J.; Sahin,

K., The effects of chromium histidinate on mineral status of serum and tissue in fat-fed and

streptozotocin-treated type 2 diabetic rats. Biological Trace Element Research 2009, 131, 124-

32.

7. Etgen, G. J.; Oldham, B. A., Profiling of Zucker diabetic fatty rats in their progression to the

overt diabetic state. Metabolism 2000, 49, 684-8.

8. Wang, B.; Chandrasekera, P. C.; Pippin, J. J., Leptin- and leptin receptor-deficient rodent

models: relevance for human type 2 diabetes. Current Diabetes Reviews 2014, 10, 131-45.

9. Feve, B.; Bastard, J.P.; Vidal, H., Relationship between obesity, inflammation and insulin

resistance: new concepts. Comptes Rendus Biologies 2006, 329, 587-597.

10. Moran, O.; Phillip, M., Leptin: obesity, diabetes and other peripheral effects-a review.

Pediatric Diabetes 2003, 4, 101-9.

11. Fernandezlopez, J. A.; Esteve, M.; Rafecas, I.; Remesar, X.; Alemany, M., Management of

dietary essential metals (iron, copper, zinc, chromium and manganese) by wistar and zucker

obese rats fed a self-selected high-energy diet. BioMetals 1994, 7, 117-129.

12. Serfass, R. E.; Park, K. E.; Kaplan, M. L., Developmental changes of selected minerals in

Zucker rats. Proceedings of the Society for Experimental Biology 1988, 189, 229-39.

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112

13. Donaldson, D. L.; Smith, C. C.; Koh, E., Effects of obesity and diabetes on tissue zinc and

copper concentrations in the Zucker rat. Nutrition Research 1987, 7, 393-399.

14. Vincent, J. B., Chromium: celebrating 50 years as an essential element? Dalton Transactions

2010, 39, 3787-3794.

15. Vincent, J.B., The Nutritional Biochemistry of Chromium(III). 1st Ed.; Wiley, 2013.

16. Clodfelder, B.; Gullick, B.; Lukaski, H.; Neggers, Y.; Vincent, J., Oral administration of the

biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ increases insulin sensitivity and improves blood

plasma variables in healthy and type 2 diabetic rats. Journal of Biological Inorganic Chemistry

2005, 10, 119-130.

17. Vincent, J. B.; Love, S. T., The need for combined inorganic, biochemical, and nutritional

studies of chromium(III). Chemistry & Biodiversity 2012, 9, 1923-1941.

18. Vincent, J. B.; Love, S., The binding and transport of alternative metals by transferrin.

Biochimica Et Biophysica Acta 2012, 1820, 362-378.

19. Vincent, J. B., The bioinorganic chemistry of chromium(III). Polyhedron 2001, 20, 1-26.

20. Press, R. I.; Geller, J.; Evans, G. W., The effect of chromium picolinate on serum cholesterol

and apolipoprotein fractions in human subjects. Western Journal of Medicine 1990, 152, 41-5.

21. Earnshaw, A.; Figgis, B. N.; Lewis, J., Chemistry of Polynuclear Compounds 6. Magnetic

Properties of Trimeric Chromium and Iron Carboxylates. Journal of the Chemical Society A:

Inorganic Physical Theoretical 1966, 1656-1663.

22. Kandror, K. V., Insulin regulation of protein traffic in rat adipose cells. Journal of Biological

Chemistry 1999, 274, 25210-7.

23. Mertz, W.; Roginski, E. E.; Reba, R. C., Biological activity and fate of trace quantities of

intravenous chromium(3) in rat. American Journal of Physiology 1965, 209, 489-&.

24. Onkelinx, C., Compartment analysis of metabolism of chromium(III) in rats of various ages.

American Journal of Physiology 1977, 232, E478-84.

25. Lim, T. H.; Sargent, T., 3rd; Kusubov, N., Kinetics of trace element chromium(III) in the

human body. American Journal of Physiology 1983, 244, R445-54.

26. Zemel, M. B.; Sowers, J. R.; Shehin, S.; Walsh, M. F.; Levy, J., Impaired calcium metabolism

associated with hypertension in Zucker obese rats. Metabolism 1990, 39, 704-8.

27. Sun, Y.; Clodfelder, B. J.; Shute, A. A.; Irvin, T.; Vincent, J. B., The biomimetic

[Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma insulin, cholesterol, and triglycerides in

healthy and type 2 diabetic rats but not type 1 diabetic rats. Journal of Biological Inorganic

Chemistry 2002, 7, 852-62.

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28. Levina, A.; Lay, P. A., Mechanistic studies of relevance to the biological activities of

chromium. Coordination Chemistry Reviews 2005, 249, 281-298.

29. Levina, A.; Lay, P. A., Chemical properties and toxicity of chromium(III) nutritional

supplements. Chemical Research in Toxicology 2008, 563-571.

30. Vincent, J. B., Beneficial effects of chromium(III) and vanadium supplements in diabetes.

Nutritional and Therapeutic Interventions for Diabetes and Metabolic Syndrome 2012, 381-

391.

31. Goldfine, A. B.; Patti, M. E.; Zuberi, L.; Goldstein, B. J.; LeBlanc, R.; Landaker, E. J.; Jiang,

Z. Y.; Willsky, G. R.; Kahn, C. R., Metabolic effects of vanadyl sulfate in humans with non-

insulin-dependent diabetes mellitus: in vivo and in vitro studies. Metabolism 2000, 49, 400-

10.

32. Anderson, R. A.; Bryden, N. A.; Polansky, M. M., Lack of toxicity of chromium chloride and

chromium picolinate in rats. Journal of the American College of Nutrition 1997, 16, 273-279.

33. Olin, K. L.; Stearns, D. M.; Armstrong, W. H.; Keen, C. L., Comparative retention absorption

of chromium-51 (Cr-51) from Cr-51 chloride, Cr-51 nicotinate and Cr-51 picolinate in a rat

model. Trace Elements and Electrolytes 1994, 11, 182-186.

34. Anderson, R. A.; Bryden, N. A.; Polansky, M. M.; Gautschi, K., Dietary chromium effects on

tissue chromium concentrations and chromium absorption in rats. Journal of Trace Elements

in Experimental Medicine 1996, 9, 11-25.

35. Kottwitz, K.; Laschinsky, N.; Fischer, R.; Nielsen, P., Absorption, excretion and retention of

Cr-51 from labelled Cr-(III)-picolinate in rats. BioMetals 2009, 22, 289-295.

36. Clodfelder, B. J.; Chang, C.; Vincent, J. B., Absorption of the biomimetic chromium cation

triaqua-mu3-oxo-mu-hexapropionatotrichromium(III) in rats. Biological Trace Element

Research 2004, 98, 159-69.

37. Sun, Y.; Mallya, K.; Ramirez, J.; Vincent, J. B., The biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+

decreases plasma cholesterol and triglycerides in rats: towards chromium-containing

therapeutics. Journal of Biological Inorganic Chemistry 1999, 4, 838-45.

38. Sun, Y. J.; Clodfelder, B. J.; Shute, A. A.; Irvin, T.; Vincent, J. B., The biomimetic

[Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma insulin, cholesterol, and triglycerides in

healthy and type 2 diabetic rats but not type 1 diabetic rats. Journal of Biological Inorganic

Chemistry 2002, 7, 852-862.

39. Kuryl, T.; Krejpcio, Z.; Wojciak, R. W.; Lipko, M.; Debski, B.; Staniek, H., Chromium(III)

propionate and dietary fructans supplementation stimulate erythrocyte glucose uptake and

beta-oxidation in lymphocytes of rats. Biological Trace Element Research 2006, 114, 237-248.

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CHAPTER 5

SURFACE CHARGE AND DOSAGE DEPENDENT DEVELOPMENTAL TOXICITY AND

BIODISTRIBUTION OF IRON OXIDE NANOPARTICLES IN PREGNANT CD-1 MICE

5.1: Introduction

The term nanoparticles (NPs) generally refers to small particles (1-100 nm in diameter) which

may exhibit unique size-dependent properties that are not present in bulk materials. These unique

properties, small size, as well as the large surface area to size ratio have generated ample interest

in NP technology. By 2015, the world market for nanomaterial-containing products is anticipated

to reach $2.6 trillion1 and 240 nano-enabled products are estimated to enter the pharmaceutical

pipeline.2 The increased use of NPs in consumer products and biomedicine has led to a significant

increase in human exposure to engineered nanomaterials, which has raised serious concerns about

the potential risk of nanomaterials, mainly NPs, to human health.3-6 For example, consumers may

find it difficult to avoid the titanium dioxide nanoparticles in sunscreen and silver nanoparticles in

food packing. Another growing area of NP research has been pharmaceutical or biomedical

research due in part to the small size and potentially increased biodistribution of NPs compared to

a bulk material. Some of the desirable properties of nanomaterials utilized for the biomedical field

include the photothermal transduction of gold nanorods, as well as the paramagnetism of iron oxide

NPs. A wide variety of NPs (gold, silver, platinum, iron, titanium dioxide, etc.) have been

investigated for many biomedical uses such as carriers in drug delivery systems, imaging contrast

agents, cancer treatments, contraceptives, and diagnostics.7, 8 The surface of NPs is commonly

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modified to tailor them to specific applications. For example, biomedical applications of iron oxide

NPs require a hydrophilic surface coating to increase water solubility as well as prevent NP

aggregation. Further studies are needed to examine how this surface modification may influence

the toxicity of the NPs.

Iron oxide NPs have been widely explored in drug delivery,9, 10 as contrast agents in magnetic

resonance imaging (MRI),11 for soil and groundwater remediation,12 and as photocatalysts.13, 14 In

addition, iron oxide is a major potential product of zero-valence iron NPs, the most popular

metallic NPs in environmental remediation applications.15-19 This application has been

successfully commercialized in the United States with more than 50 established sites.20 All of these

applications lead to increased production of iron oxide NPs, subsequently increasing their levels

in the environment and human exposure to iron oxide NPs. Iron oxide NPs are generally believed

to be safe21 and can be potentially reabsorbed through normal iron metabolic pathways

(biodegradable).22, 23 In fact, iron oxide NPs have been in clinical use as MRI contrast agents.24

However, concerns remain about the potential longterm25 and developmental26 effects of iron

oxide NPs.

The risk to pregnant women and the possibility of NPs crossing the placenta and reaching the

developing fetus are of particular concern,27, 28 because fetuses are more sensitive to environmental

toxins than adults.29 Several studies on various NPs using perfused human placenta produced

mixed results; some NPs enter the placental tissue and fetal circulation while other NPs enter the

placental tissue but do not enter fetal circulation. Gold NPs30 were able to perfuse into the placental

tissue but were not found in fetal circulation; however, various quantum dot NPs perfused into the

placental tissue and then entered the fetal circulation.31, 32 The ability to enter fetal circulation

appears to be dependent on factors such as NP size and length of perfusion time.31

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Animal studies have shown that NP exposure can cause adverse effects on pregnant mice and

their offspring. Silica and titanium dioxide NPs were shown to cross the placenta and accumulate

in fetuses in pregnant mice.33 Titanium dioxide NPs transferred from the pregnant mice to their

offspring, resulting in brain damage, nerve system damage and reduced sperm production in male

offspring.34, 35 In another study of pregnant mice exposed to platinum NPs,36 NPs did not produce

fetal abnormalities, fetal death, or accumulation of NPs in maternal uterus, ovaries, or liver but

post-natally an increase in pup mortality and a decrease in growth rate were observed. Very little

information exists on the effects of iron oxide NPs on embryo-fetal development. In fact, only one

published study has been found examining the in vivo developmental toxicity of iron oxide NPs in

rodents.26, 37 Noori et al., using a 50 mg NP/ kg body mass intraperitoneal dose, observed decreased

infant growth as well as an alteration in testicular morphology in offspring who has been exposed

to NPs in utero26, 37 Therefore, further studies on the maternal and fetal effects of NPs are urgent

and critical.

Here, spherical iron oxide NPs, approximately 28-30 nm in hydrodynamic diameter, were

synthesized as reported previously,38-42 and the hydrophilic ligands polyethylenimine (PEI) or

poly(acrylic acid) (PAA) were attached to the surface of the NPs following previously reported

procedures, yielding NPs with positive and negative surface charges, respectively.40, 43 The aim of

this study was to determine whether the surface charge or chemistry of iron oxide NPs influences

their ability to cross the placenta and whether they will induce any negative effects on pregnant

dams and embryo-fetal development in CD-1 mice when given as a single, low dose or as eight

consecutive low doses during pregnancy via intraperitoneal injection. In particular, the intent of

the work described herein is to correlate the developmental toxicity and possible fetal

biodistribution of NPs with surface charge and dosage.

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5.2 Materials and Methods

5.2.1 Animals and Husbandry

Male and female CD-1 mice were obtained from Charles River Breeding Laboratories,

Wilmington, MA. Animals were acclimated for two weeks prior to mating. Individual animals

were uniquely identified by earpunch and cage cards. The temperature was maintained at 22 ± 2 °C

with 40-60 % relative humidity. The animals were maintained with a 12 h photoperiod, 12 h of

light then 12 h of darkness. Untreated animals were bred naturally, two females with one male.

Mating was confirmed with the observation of a copulation plug, which indicated gestation day

(GD) 0. Females were randomly assigned to treatment groups immediately after mating and

individually housed in polycarbonate shoe-box style cages (29 cm x 19 cm x 13 cm) with hardwood

bedding. Mice were provided Teklad LM-485 rodent diet (Harlan Teklad, Madison, WI) and tap

water ad libitum throughout the study. All procedures performed on the animals were reviewed

and approved by The University of Alabama’s Institutional Animal Care and Use Committee

(IACUC) and were in accordance with established guidelines. These guidelines include

institutional guidelines, International Council of Harmonisation (ICH) guidelines, and the AVMA

Guidelines for the Euthanasia of Animals.44, 45

5.2.2 Nanoparticle Preparation and Characterization

Iron oxide NPs were prepared by following a modified "heat-up" method, where

trioctylphosphine oxide (TOPO) was added during synthesis as a weak binding co-surfactant.38-43

In brief, the previously described iron oleate complex (2.5 g, 2.8 mmol) was heated up to 320 °C

(over 2.5 h) with the surfactants oleic acid/TOPO (OA- 0.22 mL, 0.7 mmol, TOPO- 0.2 g,

0.5 mmol) in 1-octadecene (10 mL). After the reaction mixture cooled down (20 °C), the as-

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prepared NPs were separated from the solvent by centrifugation and dried under vacuum. To

render NPs water soluble, the hydrophobic surfactants around NP surface were directly replaced

by hydrophilic molecules (PAA and PEI) via a ligand-exchange method, as described previously.40

Briefly, well-dried NP powder was redissolved into chloroform to achieve a stock solution

(5 mg/mL). Stock solution (1 mL) was then mixed well with PAA or PEI into 49 mL of dimethyl

sulfate oxide (DMSO) by sonication. The NP surface iron atoms to exchange ligands molar ratio

was set roughly at 1:5. After 48 h mixing, the water soluble NPs were precipitated out by

centrifugation, washed with and redispersed into nanopure H2O (18 mΩ) (1 mg/mL). NPs were

then examined by transmission electron microscopy (TEM) to confirm uniformity of size and

distribution in water. Zeta potential was measured using a Zetasizer nano series dynamic light

scattering (DLS) instrument to ensure the stability and charge of the NPs. No precipitation was

observed after months of storage for these water soluble NPs.

5.2.3 Treatments

Mated female CD-1 mice were randomly assigned into one of the following treatment groups:

(1) a control group, 8 doses distilled H2O (n = 14) , (2) 1 dose (10 mg NPs/kg body mass) PEI-NP

(n = 18); (3) 1 dose (10 mg NPs/kg body mass) PAA-NP (n = 16); (4) 8 doses (10 mg NPs/kg body

mass) PEI-NP (n = 16), and (5) 8 doses (10 mg NPs/kg body mass) PAA-NP (n = 16). The

concentration of the exposure solutions were 1 mg NPs/mL in nanopure water. All doses of NPs

were 10 mg NPs/kg body mass which equates to 2.5 mg Fe/kg body mass. Ferumoxtran-10

(Combidex) is an intravenous iron oxide NP MRI contrast agent which has been used in many

studies and clinical trials.46, 47 The dose of 10 mg NP/kg body mass (2.5 mg Fe/kg body mass) was

chosen to represent the approximate dose of iron oxide NPs one would receive due to undergoing

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MRI. Male CD-1 mice were euthanized at the completion of the mating period. Clinical

observations were recorded daily and females were weighed on GD 0, as well as before each

dosing. Treatments were delivered by intraperitoneal injection(s) during gestation. Animals in

groups (2) and (3) were administered a single dose of the test material on GD 9, while animals in

groups (4) and (5) were administered the test material once daily from GD 9 through GD 16. The

dosage volume was 0.01 mL/g body weight. The control group received an equivalent volume of

the vehicle (H2O).

Treatments n

(1) Controlx8 8 doses of DI H2O given on GD 9 through GD 16 14

(2) PEIx1+ 1 dose (10 mg NPs/kg body mass) PEI-NP given on GD 9 18

(3) PAAx1- 1 dose (10 mg NPs/kg body mass) PAA-NP given on GD 9 16

(4) PEIx8+ 8 doses (10 mg NPs/kg body mass) PEI-NP given on GD 9

through GD 16 16

(5) PAAx8- 8 doses (10 mg NPs/kg body mass) PAA-NP given on GD 9

through GD 16 16

Table 5.1: Treatment groups and number of animals per group (n).

5.2.4 Data Collection

Throughout the gestation period, pregnant females were monitored daily for signs of morbidity,

behavioral changes, changes in general appearance, and mortality. For treatment groups (2) and

(3), maternal body weights were measured on GD 0, GD 9, and GD 17 (without the gravid uterus).

For treatment groups receiving 8 doses, groups (1), (4), and (5), maternal body weights were

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measured on GD 0, GD 9 through GD 16, and on GD 17 after the fetuses were removed. Dams

were sacrificed on GD 17, one day prior to parturition which occurs on GD 18. Animals were

euthanized by CO2 inhalation in accordance with institutional guidelines and the AVMA Guidelines

for the Euthanasia of Animals.45 Presumed pregnant females were euthanized by CO2

asphyxiation, their uteri were exposed, and the uterine contents were examined for the numbers of

live and dead fetuses, early or late resorptions, and total implantation sites. If no implantation sites

were observed, the female was considered not to have been pregnant. Live fetuses were removed

from the uterus, weighed individually, and examined for changes in external morphology.

Maternal body weight, minus the gravid uterine weight, was then obtained. Maternal body weight

gain was calculated by subtracting the maternal body weight on GD 0 from the maternal body

weight on GD 17 minus the gravid uterus.

Placenta, fetal liver, and fetal kidney were collected from each treatment group on GD 17 in

order to measure the ability of the positively and negatively surface-charged coated NPs to cross

the placenta and enter the fetus during pregnancy. In order to qualitatively observe changes in iron

concentration, tissue samples were fixed in 4 % paraformaldehyde prior to histological sectioning

and stained with Prussian Blue, an iron selective stain. Increases in iron content were visualized

by an increase in blue pigment when viewed with an optical microscope, indicating increased iron

oxide NPs. These visual results were quantified by assaying the samples for iron using an

ultraviolet/visible spectrophotometer by the colorimetric ferrozine method.48

Live fetuses were euthanized via intraperitoneal administration of Euthasol and fixed in 70 %

ethanol in compliance with IACUC standards. Fetuses were subsequently eviscerated, cleared with

KOH, and stained with Alcian blue and Alizarin red (Sigma Aldrich, St. Louis, MO) using the

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double-staining technique described by Webb and Byrd.49 Bony structures and cartilage of all

fetuses were examined for malformations and variations using a dissecting microscope.

5.2.5 Statistical Analysis

The litter or the pregnant female were used as the experimental unit for statistical analysis. This

study was performed in multiple replicates. The data from each replicate were calculated

independently, tested for homogeneity of variance by means of the Levene statistic using SPSS

(SPSS, Inc., Chicago, IL), and then pooled and analyzed to give the results reported. All tabular

data are presented as the mean ± standard error (SEM). Data were analyzed by one-way analysis

of variance (ANOVA) or Kruskal-Wallis one-way ANOVA followed by a least significant

difference (LSD) or Dunn’s post-hoc test, respectively, to determine specific significant

differences (p ≤ 0.05).

5.3: Results and Discussion

5.3.1 Nanoparticle Synthesis and Characterization

Figure 1 shows the transmission electron microscopy (TEM) images of the PAA and PEI coated

iron oxide NPs. The TEM images show the NPs are spherical in shape with a uniform, narrow size

distribution. The groups of NPs on the image were the result of NPs that fell on top of each other

during sample preparation, not NP aggregates. The water dispersity of these NPs were previously

determined by DLS analyses where the hydrodynamic sizes of the PAA and PEI coated NPs were

about 28 and 30 nm, respectively.43 Zeta potentials were measured to determine surface charges

of the polymer-coated NPs. Zeta potential values above 30 mV or below -30 mV indicate stability

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Figure 5.1: TEM images of (A) PEI-NPs and (B) PAA-NPs in H2O.

Figure 5.2: Zeta potentials of (A) PEI-NPs and (B) PAA-NPs in H2O.

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of a colloid system.43 The measured zeta potential values (Figure 2) of 51 mV for PEI-NPs and -

52 mV for PAA-NPs indicate high stability of these NPs as their absolute values are well above

30 mV. This high stability of the colloid system should lead to a resistance towards aggregation,

which was confirmed with TEM.

5.3.2 Effect of Surface-Charged NPs on Dams

Maternal body weight gain during gestation is an indicator of maternal health during pregnancy

and can have long term effects on the developing fetus.50, 51 A single, low dose of either the

positively or negatively coated NPs when given on GD 9 (treatments (2) and (3)) had no effect on

maternal weight gain. Maternal body weight gain significantly decreased about 40 % (p ≤ 0.05)

during gestation when the animals received the positively charged PEI-NPs for eight consecutive

days (4) when compared to the control group (1), indicating an apparent treatment effect (Figure 3).

This effect was not observed in animals receiving the negatively charged PAA-NPs for eight

consecutive days (5), indicating a difference in toxicity with different charged polymeric coatings.

Controlx8 PEIx1+ PAAx1- PEIx8+ PAAx8-

Maternal Weight

Gain, g 8.1 ± 0.6 8.8 ± 0.8 9.1 ± 0.4 5.9 ± 0.5* 8.4 ± 0.7

Table 5.2: Maternal weight gain (g ± SEM) for treatment groups as follows (1) Controlx8, (2)

PEIx1+, (3) PAAx1-, (4) PEIx8+, and (5) PAAx8-, n = 14-18. *indicates significant differences

compared to all other groups (p < 0.05).

No evidence of morbidity, mortality, changes in behavior, or changes in general appearance were

observed for any treatment group. Decreased maternal weight gain observed in treatment (4) with

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Figure 5.3: Maternal weight gain assessed by subtracting the female body mass measured on GD 0

from the final body mass minus gravid uteri on GD 17, n = 14-18, *indicates significant differences

compared to all other groups (p < 0.05).

multiple maternal exposures across several days to positively charged iron oxide NPs indicates

that these NPs may be accumulating in the mother, negatively affecting maternal health. These

results were not observed in dams dosed with negatively charged NPs (5), indicating a difference

in toxicity based on surface charges. As the health of the mother has a direct influence on the

health of the fetus, maternotoxicity could translate into adverse effects on fetal development such

as decreased fetal weight, skeletal anomalies and post-implantation loss.52

Controlx8 PEIx1+ PAAx1- PEIx8+ PAAx8-

Mate

rnal W

eig

ht

Gain

, g

0

2

4

6

8

10

12

*

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5.3.3 Effects of Charged NPs on Litter Values

The number of implantations did not differ significantly between treatment groups. The

percentages of resorbed or dead fetuses was significantly higher in the PEI-NP and PAA-NP

treatment groups ((4) and (5)) that were treated with 10 mg/kg body mass daily for eight

consecutive days (GD 9-16) when compared to the control ((1), H2O only) (Figure 4). The animals

dosed only once with NPs ((2) and (3)) did not show increased resorptions or fetal death and were

comparable to the control group. No effect was observed on litter size or fetal weight among all

treatment groups. Few changes in external morphology were observed in treatment groups exposed

to 8 doses of NPs ((4) and (5)). One fetus in treatment (5), PAAx8-, exhibited signs of talapes (club

foot) in combination with a shortened, bent tail. In treatment (4), PEIx8+, four mice from two

litters exhibited altered external morphology. In the first litter, one fetus was observed to have a

bent tail. The second litter contained three abnormal fetuses, one exhibited talapes (club foot), a

second exhibited talapes with a short tail, while a third exhibited exencephaly and a curly tail. The

dam which gave rise to three offspring with external malformations also gave birth prematurely.

No changes in external malformation were observed in mice in the control group, or either

treatment given a single dose of either NP (treatment (1), (2), or (3)). A slight increase was

observed in the number of skeletal variations such as supernumerary ribs in treated litters as shown

in Table 5.3, but these increases were not statistically significant. Incidence of supernumerary ribs

appeared highest in offspring of females treated with positively charged PEI-NPs (single (2) or

multiple dose (4)).

Perhaps the most troubling result is that multiple low-dose exposures of either charged NP ((4)

or (5)) lead to significant increases in post-implantation loss, specifically resorptions (both early

and late depending on surface coating, Figure 5.4 and Table 5.4). The average resorption incidence

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Controlx8 PEIx1+ PAAx1- PEIx8+ PAAx8-

Litter Size 15.6 ± 0.7 14.3 ± 0.4 14.1 ± 0.6 14.1 ± 0.6 14.1 ± 0.5

Fetal Body Mass GD 17,

g ± SEM 0.99 ± 0.03 0.98 ± 0.02 1.06 ± 0.03 1.02 ± 0.02 1.00 ± 0.05

Resorptions, % ± SEM 7.3 ± 1.4 5.8 ± 2.2 4.2 ± 1.4 21.5 ± 6.2* 13.5 ± 3.7*

Supernumerary Ribs,

% ± SEM 12.4 ± 3.5 18.8 ± 5.1 14.8 ± 4.1 21.8 ± 5.5 16.4 ± 4.8

Table 5.3: Litter values for treatment groups as follows (1) Controlx8, (2) PEIx1+, (3) PAAx1-,

(4) PEIx8+, and (5) PAAx8-, n = 14-18, *indicates significant difference versus control and single

dosed treatment groups (p < 0.05).

for the controls (1) and single-dosed groups ((2) and (3)) ranged from 4-7.3 ± 1.6 % resorptions,

as a small number of resorptions are often observed in control groups. To compare, the average

resorption incidence for multiple doses of positively charged PEI-NPs (4) and negatively charged

PAA-NP (5) were 21.5 ± 6.2 % and 14.8 ± 4.1 %, respectively. The increase in the percentage of

resorptions for both the negatively and positively charged NPs when given a small dose for eight

consecutive days indicates that the NPs are negatively effecting embryo-fetal survival. The

observed increase in resorption incidence may be a result of a single small dose of NPs on a specific

gestation day or an accumulation effect from multiple exposures to NPs. More studies are needed

in order to acertain which is occuring. The resorption incidence appeared higher in the positively

charged PEI-NPs (4) compared to the negatively charged PAA-NPs (5) given for eight consecutive

days, but they were not found to be significantly different from each other, though they are both

significantly different from the control (1). Though the percentage of resorptions between females

given both the positively (4) and negatively (5) charged NPs 8 times were comparable, the stage

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Figure 5.4: Percent resorbed fetuses, n = 14-18, *indicates significant difference versus control

and single dosed treatment groups (p < 0.05).

in pregnancy in which the resorptions occurred varied. Approximately 76 % of the resorptions

observed in treatment (4), PEI-NPx8+, occurred as early resorptions while approximately 74 % of

the resorptions observed in treatment (5), PAA-NPx8-, occurred late during pregnancy. This

difference in the occurence of resorptions indicates that the mechanism of toxicity of the positively

and negatively charged NPs may be affecting the fetus at different stages of gestation.

Controlx8 PEIx1+ PAAx1- PEIx8+ PAAx8-

Reso

rpti

on

s, %

0

5

10

15

20

25

30*

*

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Controlx8 PEIx1+ PAAx1- PEIx8+ PAAx8-

Total Resorptions,

% ± SEM 7.3 ± 1.4 5.8 ± 2.2 4.2 ± 1.4 21.5 ± 6.2* 13.5 ± 3.7*

Early Resorptions, % 40.0 57.1 100 75.6 25.9

Late Resorptions, % 60.0 42.9 0 24.4 74.1

Dead Fetuses, % 0.5 0.5 0 0.9 0.5

Table 5.4: Resorptions and dead fetus distribution. Total resorptions and dead fetuses are expressed

as the average percentage in each litter. Early and late resorptions are presented as a percentage of

the total resorptions. n = 14-18, *indicates significant difference versus control and single dosed

treatment groups (p < 0.05).

5.3.4 Biodistribution of Surface-Charged NPs in Fetal Tissues

Iron concentrations were measured in samples taken from the mother and fetus to determine if

the NPs were able to cross the placenta into the fetus. No differences were observed in the level of

iron in the kidneys of fetuses from dams given PEI-NPs ((2) and (4)) or PAA-NPs ((3) and (5)) on

GD 9 or GD 9 through GD 16 compared to controls (1). In addition, when mice received only one

dose of NPs with either coating ((2) or (3)), no differences were observed in the level of iron in

the fetal liver or placental samples compared to controls (1). Significant increases in iron content

were observed in the fetal liver and placenta in the animals treated with positively charged PEI-

NPs for eight consecutive doses (4), but not in other treatment groups (Figure 5 and Figure 6). This

sharp increase in iron indicates an increased concentration of iron oxide NPs. The observation of

increased iron concentration in the mice dosed with the positively charged PEI-NPs (4), but not in

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Figure 5.5: Fetal livers stained for iron content using Prussian Blue (blue indicates presence of

iron) in (A) Control (H2O treated) (1), (B) 1 dose of PEI NPs (2), and (C) 8 doses of PEI coated

NPs (4).

the negatively charged PAA-NPs (5) indicates that the surface charge of the NPs may play a role

in bioaccumulation in the developing fetus. Increased iron concentrations in the liver and placenta

of fetuses dosed with NPs were only observed in the treatment group receiving multiple doses of

NPs (4).

The results observed throughout this study exhibit similarities to other studies of metal oxide

NPs as well as differences. Similarly to the study herein, investigations into the developmental

toxicity of another metal oxide, TiO2, observed an increase in fetal resorptions as well NPs present

in the fetal livers and placentae with exposure to TiO2 NPs on GD 16 and GD 17.33 The TiO2

particles were uncoated and the negative developmental effects observed were ameliorated with

addition of a charged surface coating (-COOH or -NH4). TiO2 NPs were several times larger than

the iron oxide NPs being discussed herein, and size is a very important factor in NP toxicity and

biodistribution. A 2011 study into the developmental toxicity of anionic dimercaptosuccinic acid

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Figure 5.6: Fetal liver iron content, n = 9, *indicates significant differences compared to all other

groups (p < 0.05).

(DMSA) coated Fe3O4 NPs monitored pups after prenatal exposure to a single, intraperitoneal dose

of NPs on GD 8.26 Fetuses were examined at GD 13 for iron accumulation in the liver using

Prussian Blue staining. Aggregates of iron oxide NPs were observed in the placentae as well as

the sinusoids and hepatocytes of the fetal liver as in this study.26 Noori et al. went on to observe

pup weights and testes development, indicating abnormal development of the seminiferous tubules

when given at higher doses (> 50 mg NP/kg body mass). The observations presented throughout

this study as well as other studies of metal oxide NPs support the data that NPs have the ability to

Controlx8 PEIx1+ PAAx1- PEIx8+ PAAx8-

Ab

so

rban

ce

0.0

0.3

0.6

0.9

1.2

1.5*

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cross the placenta, accumulate in the fetus, and cause detrimental effects on development in a dose

and surface coating dependent manner.26, 33

5.4: Conclusions

Due to their small size, customizability, and unique properties, NPs may be beneficial in many

fields, including biomedicine, but particular care is needed to evaluate their toxicity. Increased

toxicity due to exposure and charge were observed here. Following 8 consecutive days of dosing,

PEI-NPs (4) reduced maternal body weight gain and increased the level of iron in placentae and

fetal livers. These observations were not observed in groups that were given 8 consecutive doses

of PAA-NPs (5), a single dose of either PEI-NPs (2) or PAA-NPs (3), or the control (1). Increased

postimplantation loss was observed in treatment groups receiving 8 consecutive doses of either

PAA-NPs (5) or PEI-NPs (4). Though the NPs are composed of the same core material, when

their surface charge is changed by varying the polymer coating they interact and accumulate in the

mother and fetus differently. Through multiple exposures, positively charged NPs (4) appear to

accumulate in the fetal liver, while accumulation of the negatively charged NPs (5) was not

observed. Overall, the positively charged PEI-NPs (4) induced greater toxic effects when given

multiple times; increasing postimplantation loss significantly (21.5 ± 6.2 % versus 7.3 ± 1.4 % of

controls), significantly decreasing maternal weight gain, and crossing the placenta to accumulate

in the fetal liver. Though these differences were observed between charged NPs, multiple

exposures of either charged NPs ((4) or (5)) induced significantly increased fetal death.

No negative developmental effects were observed when dams were given a single, low-dose of

iron oxide NPs with either charged coating ((2) or (3)), but when given multiple doses ((4) and

(5)), increased fetal death and decreased maternal weight gain was observed dependent on the

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polymeric coating. Thus, pregnant women and their offspring exposed to such NPs may be at risk

with multiple exposures.

These results bring up a more pressing issue which is the regulation and toxicity of NPs. Though

the core material (iron oxide) is consistent, the functionalization of the surface with different

polymers with different charges induces different developmental toxicity. Surface charge should

be considered when evaluating new NPs, especially for consumer or biomedical applications.

These preliminary studies indicate an increased risk of maternotoxicity and fetotoxicity with

multiple exposures to positively charged NPs compared to negatively charged NPs. More in depth

studies are needed to elucidate the role of surface charge in the developmental toxicity of NPs.

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CHAPTER 6

OVERALL CONCLUSIONS

Careful examination of the effects of Cr supplementation on parameters of carbohydrate

metabolism in healthy, Zucker lean rats indicated that extended Cr supplementation (> 5 months)

in healthy individuals results in increased insulin sensitivity at high levels and lower fasting plasma

insulin levels. No differences in glucose metabolism, body mass, or insulin sensitivity were

observed between rats given the lowest possible Cr-containing diets and rats receiving standard

rat chow. These results further supports the role of Cr in carbohydrate metabolism as a

pharmaceutical supplement and not a nutritional element necessary for proper glucose metabolism.

No differences were observed in body mass, food intake, non-heme plasma Fe levels, or urinary

Cr loss in response to an insulin challenge. Increased insulin sensitivity would be beneficial to

models of insulin resistance such as pre-diabetic obesity and type 2 diabetes. Further studies are

necessary in controlled environments to elucidate the mechanisms of this increase in insulin

sensitivity in both healthy and insulin-resistant models.

Unfortunately the rate of urinary Cr loss in response to an insulin or glucose challenge does not

correlate with the amount of Cr supplemented in the diet. The group supplemented with the largest

concentration of Cr did not display the same response to insulin as the other groups (a light increase

in urinary Cr loss followed by a sharp rate decrease, not fully recovering to pre-challenge levels

by 12 h). Instead the urinary Cr loss in the group given the highest concentration of Cr (+ 1,000 μg

Cr/kg diet) remained constant throughout the insulin and glucose challenges, indicating a possible

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saturation of Cr transport systems by the presence of excess Cr.

Further examination into the pharmacokinetics of a single, orally administered dose of 51CrCl3

in healthy Zucker lean rats, as well as models of insulin resistance (pre-diabetic Zucker obese rats

(ZOB) and type 2 diabetic Zucker diabetic fatty rats (ZDF)) indicate differences in Cr absorption

and excretion in diabetic rats compared to controls. Type 2 diabetic ZDF rats absorbed increased

amounts of Cr from the gastrointestinal tract compared to healthy Zucker lean rats, but they also

lost significantly more Cr in the urine. These results match similar studies looking into the

absorption and excretion of 51CrCl3 in STZ-induced type 1 diabetic rats.1 Increased urinary Cr loss

in diabetic models does not appear to be due to the increased urinary output present in diabetic

states, but is attributed to an increased Cr absorption in the gastrointestinal tract.

Tissue metal concentrations were also measured in Zucker lean, ZOB, and ZDF rats in order to

examine strain differences as well as the result of supplementation of various Cr complexes. Tissue

Cu, Zn, Fe, Mg, and Ca were compared and contrasted from liver, kidney, heart, and spleen

samples collected from Zucker lean, ZOB, and ZDF rats as well as kidney and liver Cr

concentrations. ZOB rats displayed the most strain differences as the result of the inability to

receive leptin signals. Liver concentrations for Cu, Zn, Fe, and Mg were significantly reduced per

g of tissue compared to the Zucker lean and ZDF models due primarily to the substantial increase

in adipose tissue present in ZOB rats as observed in previous studies.2 Other differences observed

in the ZOB rats were reduced splenic Fe levels and increased kidney Ca levels. Elevated kidney

Ca levels have been observed in ZOB rats before and implicated in contributing to hypertension

observed in the models.3 ZDF rats displayed approximately 4 times higher kidney Cu levels than

Zucker lean and approximately 8 times higher kidney Cu levels than ZOB rats as a function of

their disease.

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Extended daily Cr supplementation resulted in few tissue metal alterations throughout the

models. In Zucker lean and ZOB rats, only the highest doses of Cr3 or CrCl3 (1 mg Cr/kg body

mass), but not Cr(pic)3, resulted in increased kidney Cr. Increased kidney Cr was not observed in

the ZDF rats. Interestingly, the same doses of the same compounds (1 mg Cr/kg body mass of Cr3

or CrCl3) resulted in the beneficial effect of decreased kidney Cu levels in the ZDF rats, suggesting

a beneficial effect of pharmaceutical Cr supplementation in type 2 diabetes.

Also investigated was the developmental toxicity of surface-charged iron oxide NPs in pregnant

CD-1 mice. Iron oxide NPs given multiple times throughout pregnancy were able to cross the

placenta and accumulate in the fetal liver, resulting in toxicity. NPs examined were approximately

the same size and same core material, so the observed developmental toxicity and fetal

biodistribution seems dependent on the surface characteristics of the NPs. Positively charges

polyethylenimine-coated NPs (PEI-NPs) induced greater toxicity than negatively-charged

poly(acrylic acid)-coated NPs (PAA-NPs) resulting in increased accumulation in the fetal liver,

greater post-implantation loss (~22 %), and decreased maternal weight gain. No negative effects

were observed when mice were given a single, low dose (dose needed for approximately one MRI)

of NPs with either positive or negatively charges surface coatings. Results from this study can aid

in the design of future NPs to which pregnant women may be exposed. Preliminary results indicate

positively-charges NPs are more toxic toward the developing fetus when exposed in utero. Future

studies are needed to further explore the influence of charge on developmental toxicity of NPs.

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6.1 References

1. Feng, W. Y.; Ding, W. J.; Qian, Q. F.; Chai, Z. F., Study on the metabolism of physiological

amounts of Cr(III) intragastrical administration in normal rats using activable enriched stable

isotope Cr-50 compound as a tracer. Journal of Radioanalytical and Nuclear Chemistry 1998,

237, 15-19.

2. Rolland, V.; Roseau, S.; Fromentin, G.; Nicolaidis, S. V.; Tome, D.; Even, P. C., Body weight,

body composition, and energy metabolism in lean and obese Zucker rats fed soybean oil or

butter. American Journal of Clinical Nutrition 2002, 75, 21-30.

3. Zemel, M. B.; Sowers, J. R.; Shehin, S.; Walsh, M. F.; Levy, J., Impaired calcium metabolism

associated with hypertension in Zucker obese rats. Metabolism 1990, 39, 704-8.