1. health and fitness
2. disease
3. diabetes

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
Copyright Kristin R. Di Bona 2014
ALL RIGHTS RESERVED
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
ii
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 positivelycharged 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.
iii
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.
iv
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
51
Chromium-51 labelled chromium chloride
g
Gram
h
Hour(s)
H2O2
Hydrogen peroxide
125
Iodine-125 labelled radio immunoassay
CrCl3
I RIA
kg
Kg
L
Liter
LMWCr
Low molecular weight chromium binding substance
v
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
vi
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.
vii
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
viii
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
ix
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
51
3.3.3
51
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
Cr Pharmacokinetics ...............................................................................61
Cr Absorption ..........................................................................................72
x
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
xi
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
xii
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
xiii
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
xiv
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: AIN93G without Cr added to the mineral mixture (low Cr); the standard AIN93G 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
xv
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
xvi
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
xvii
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
xviii
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
xix
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 selfselected 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
1
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 doseresponse 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
2
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 nonsupplemented.12-14 The effects of Cr supplementation in healthy rats residing in controlled metalfree 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.
3
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
4
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
5
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
6
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
7
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
8
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.
9
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Journal of Biotechnology 2011, 10, 1221-1227.
66. Li, J.; Chang, X.; Chen, X.; Gu, Z.; Zhao, F.; Chai, Z.; Zhao, Y., Toxicity of inorganic
nanomaterials in biomedical imaging. Biotechnology Advances 2014, 32, 727-743.
14
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
15
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
16
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
17
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
125
I 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
18
(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 Crsufficient 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 Crsufficient 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
19
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
20
stainless steel tubes. To prevent potential injury, the end of the tubing exposed to the rats was firepolished.
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)
21
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 (subboiling) 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
22
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.
23
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.
24
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
25
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
26
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
27
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
28
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
29
550
500
Body mass (g)
450
400
350
a
ab
300
250
200
b
b
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
aa
ab ab
ab
ab b
b
0
20
40
60
80
100
120
140
Time (day)
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
30
Non-Heme Fe (ug/100mL plasma)
350
300
250
200
150
100
50
0
Cr
Low
t
ien
ffic
u
S
Cr
r/kg
gC
0u
+20
r/kg
gC
u
00
+10
Figure 2.3: Non-heme plasma Fe levels for Zucker lean rats on the standard and modified AIN93G 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,
31
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.
32
400
350
a
Glucose (mg/dL)
300
250
ab
b
200
b
150
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
100
50
0
20
40
60
80
100
120
Time after glucose injection (min)
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
33
40000
Glucose area under curve
a
30000
ab
ab
b
20000
10000
0
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
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
34
400
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
Glucose (mg/dL)
300
200
a
100 ab ab
b
0
0
20
40
60
80
100
120
Time after insulin injection (min)
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
35
Glucose area under curve
40000
30000
20000
10000
0
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
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.
36
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
× 100
𝐺𝑙𝑢𝑐𝑜𝑠𝑒0
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
37
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.
38
Insulin (micro insulin units/mL)
120
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
a
100
a
80
a
ab
60
ab
b
ab
40
ab
b
b
20
b
b
0
0
20
40
60
80
100
120
Time after glucose injection (min)
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
39
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
40
12000
Insulin area under curve
10000
a
8000
ab
6000
bc
4000
c
2000
0
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
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
41
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.
42
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
43
140
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
120
ng Cr/h
100
80
60
40
20
0
0
2
4
6
8
10
12
Time (h)
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 AIN93G 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
44
140
Low Cr
Cr Sufficient
+ 200 ug Cr/kg
+ 1000 ug Cr/kg
120
ng Cr/h
100
80
60
40
20
0
0
2
4
6
8
10
12
Time (h)
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 AIN93G 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
45
500
Area Under the Curve
400
300
200
100
0
Cr
Low
ffic
Su
Cr
t
ien
r/kg
gC
u
00
+2
+
r/kg
gC
u
0
100
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
46
500
Area Under the Curve
400
300
200
100
0
Cr
Low
t
ien
ffic
Su
Cr
r/kg
gC
u
00
+2
+
r/kg
gC
u
0
100
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
47
250
200
ng Cr/h
150
100
50
0
0
2
4
6
8
10
12
14
Time (h)
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
48
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
49
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.
50
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 “nonresponders.” Further investigation is necessary to confirm the existence of responders versus nonresponders. 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.
51
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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.
53
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.
54
40. Sun, Y.; Ramirez, J.; Woski, S. A.; Vincent, J. B., The binding of trivalent chromium to lowmolecular-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, 11841193.
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.
55
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 prediabetic obesity, respectively.
56
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
57
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
51
CrCl3 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
58
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
59
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
60
and route of administration must be utilized. Cr was given orally in the form of
51
CrCl3 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 postdose, 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
61
120
% of Applied Dose
100
b,c
80
60
Stomach
Small Intestine
Large Intestine
Feces
40
20
b
0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
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.
62
120
% of Applied Dose
100
80
60
Stomach
Small Intestine
Large Intestine
Feces
40
a,c
a
20
0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
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
51
Cr 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,
63
120
% of Applied Dose
100
80
60
Stomach
Small Intestine
Large Intestine
Feces
40
a
b
20
0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
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
51
Cr 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 postadministration. Blood plasma levels of
51
Cr 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.
64
2.0
Blood
Urine
% of Applied Dose
1.5
1.0
0.5
0.0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
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.
65
2.0
% of Applied Dose
1.5
Blood
Urine
1.0
0.5
a,c
c
0.0
0
c
c
500
1000
1500
2000
2500
3000
Time(Minutes)
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
66
2.0
Blood
Urine
% of Applied Dose
1.5
a,b
1.0
a,b
a,b
0.5
a,b
0.0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
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
67
1.2
% of Applied Dose
1.0
Right Femur
Heart
Muscle
Testes
Epid. Fat
0.8
0.6
0.4
0.2
0.0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
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
51
CrCl3 followed the results presented herein with liver containing the
second highest Cr concentrations.9 Unlike this study, the Cr nicotinate resulted in higher kidney
68
1.2
% of Applied Dose
1.0
Right Femur
Heart
Muscle
Testes
Epid. Fat
0.8
0.6
0.4
0.2
c
0.0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
Figure 3.3.B: Concentration of 51Cr measured in the right femur, heart, skeletal muscle, testes, and
epididymal fat after an oral dose of
51
CrCl3 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
69
1.2
Right Femur
Heart
Muscle
Testes
Epid. Fat
% of Applied Dose
1.0
0.8
0.6
0.4
0.2
b
0.0
0
500
1000
1500
2000
2500
3000
Time (Minutes)
Figure 3.3.C: Concentration of 51Cr measured in the right femur, heart, skeletal muscle, testes, and
epididymal fat after an oral dose of
51
CrCl3 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 postadministration 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
70
0.20
Pancreas
Spleen
Liver
Kidney
% of Applied Dose
0.15
0.10
0.05
0.00
0
500
1000
1500
2000
2500
3000
Time (Minutes)
Figure 3.4.A: Concentration of
oral dose of
51
51
Cr measured in the pancreas, spleen, liver and kidney after an
CrCl3 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
71
0.20
Pancreas
Spleen
Liver
Kidney
% of Applied Dose
0.15
0.10
0.05
0.00
0
500
1000
1500
2000
2500
3000
Time (Minutes)
Figure 3.4.B: Concentration of
oral dose of
51
51
Cr measured in the pancreas, spleen, liver and kidney after an
CrCl3 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,
72
0.20
% of Applied Dose
0.15
Pancreas
Spleen
Liver
Kidney
0.10
0.05
0.00
0
500
1000
1500
2000
2500
3000
Time (Minutes)
Figure 3.4.C: Concentration of
oral dose of
51
51
Cr measured in the pancreas, spleen, liver and kidney after an
CrCl3 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 CrCl 3) was
73
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
51
CrCl3.17 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 insulindependent 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,
74
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 alloxaninduced diabetic rats.19 Alloxan induces a type 1 diabetic model by selectively destroying insulinproducing 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 alloxaninduced 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
75
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
76
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.
77
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 lowmolecular-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.
78
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.
79
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
80
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 highfat 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
81
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
82
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
83
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
85
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
86
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
87
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.
89
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
90
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.
91
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 CrCl 3
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
92
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).
93
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).
94
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.
95
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.
96
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.
97
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.
98
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.
99
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.
100
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.
101
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).
102
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.
103
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).
104
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).
105
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
106
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
107
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 highfat 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
108
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 highfructose 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 CrCl 3 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
109
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.
110
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, 12432.
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.
111
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.
112
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, 381391.
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 noninsulin-dependent diabetes mellitus: in vivo and in vitro studies. Metabolism 2000, 49, 40010.
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.
113
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
114
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
115
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.
116
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-
117
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
119
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
120
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
121
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.
122
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.
Maternal Weight
Gain, g
Controlx8
PEIx1+
PAAx1-
PEIx8+
PAAx8-
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
123
Maternal Weight Gain, g
12
10
8
*
6
4
2
0
Controlx8 PEIx1+
PAAx1-
PEIx8+
PAAx8-
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
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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
125
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
126
30
*
Resorptions, %
25
20
*
15
10
5
0
Controlx8 PEIx1+
PAAx1-
PEIx8+
PAAx8-
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.
127
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 PEINPs 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
128
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
129
1.5
*
Absorbance
1.2
0.9
0.6
0.3
0.0
Controlx8 PEIx1+
PAAx1-
PEIx8+
PAAx8-
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
130
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
131
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.
132
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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 CrCl 3 (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.
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