SAHLGRENSKA ACADEMY
The influence of circadian rhythm
and biological sex on the
biodistribution of iodine-131 in
mice
Mikael Elvborn
Essay/Thesis:
Program and/or course:
30 hp
Medical Physicist Programme
Level:
Semester/year:
Second Cycle
Spring 2016
Supervisor:
Examiner:
Eva Forssell-Aronsson, Britta Langen and Johan Spetz
Magnus Båth
Abstract
Essay/Thesis:
Program and/or course:
30 hp
Medical Physicist Programme
Level:
Semester/year:
Second Cycle
Spring 2016
Supervisor:
Examiner:
Eva Forssell-Aronsson, Britta Langen and Johan Spetz
Magnus Båth
131
Keyword:
I, circadian rhythm, biodistribution, biokinetics, C57BL, mice, sex
difference
Purpose:
The purpose of this study was to validate previous biodistribution data on circadian
rhythm (Andersson, 2015) and to investigate a novel early time point. A further aim
was to investigate the potential difference between the sexes concerning the
biodistribution and biokinetics of 131I in mice. In addition, the impact of iodine
concentration in animal food was studied.
Theory:
The thyroid is both a risk and target organ in radionuclide therapy. The gland takes up
iodine to synthesize thyroidal hormones which are important for various cellular
mechanisms throughout the body. 131I, a radioactive isotope, is used in nuclear
medicine, but can also be encountered from fallout of nuclear accidents. The circadian
rhythm affects many bodily functions, and has recently been shown to also influence
the biodistribution of 131I. Physiological differences between the sexes represent
another intrinsic variable that is thought to impact the biodistribution of 131I.
Method:
In total, 159 C57BL/6N mice (124 males and 35 females) were used in the
experiments and injected intravenously with 131I prepared in physiological saline. The
mice were administered with 140−175 kBq of 131I at different time points during the
day (8 am, 12 pm and 4 pm) and killed after 1, 4, 8, 18, 24, 72 or 168 h following
injection. Various organs, tissue samples, gastrointestinal contents, and the remaining
carcass were collected, weighed, and subjected to gamma counter measurement to
determine 131I activity concentration.
Result:
The results supported previous work showing that 131I biodistribution is influenced by
the time of day of administration. Furthermore, the results demonstrated a difference
in 131I biodistribution data between male and female mice, notably in the kidneys. The
results also showed that the extent of diurnal differences in biodistribution data is
influenced by the iodine concentration in animal food.
Table of content
Abbreviations .......................................................................................................................................... 1
Introduction ............................................................................................................................................. 2
Aims ........................................................................................................................................................ 4
Materials and Methods ............................................................................................................................ 5
Animal model ...................................................................................................................................... 5
Administration of radionuclides .......................................................................................................... 5
Biodistribution and biokinetics ........................................................................................................... 7
Radioactivity measurements................................................................................................................ 8
Detector calibrations............................................................................................................................ 8
Measurement corrections .................................................................................................................... 8
Statistical analysis ............................................................................................................................... 9
Results ................................................................................................................................................... 10
Detector calibrations.......................................................................................................................... 10
Measurement corrections .................................................................................................................. 11
Volume effect ................................................................................................................................ 11
Adsorption ..................................................................................................................................... 11
Biodistribution of 131I ........................................................................................................................ 12
Comparison between males at time point 8 am, 12 pm, and 4 pm ................................................ 17
Sex difference in 131I biodistribution ............................................................................................. 27
Influence of iodine concentrations in food at 12 pm ..................................................................... 31
Influence of iodine concentrations in food at 4 pm ....................................................................... 32
Effect of circadian rhythm at standard iodine concentration in food ............................................ 33
Discussion ............................................................................................................................................. 34
Conclusion and Outlook ........................................................................................................................ 37
Acknowledgements ............................................................................................................................... 38
Reference list ......................................................................................................................................... 39
Appendix ............................................................................................................................................... 41
Biokinetics for comparison with normal iodine concentrations in food for mice ............................. 41
Administration at 12 pm ................................................................................................................ 41
Administration at 4 pm .................................................................................................................. 43
Abbreviations
β-particle
- Electron or positron
cps
- Counts per second
eV
- Electron volt
127
I
- Iodine–127
131
I
- Iodine–131
i.p.
- Intraperitoneally
i.v.
- Intravenous
NIS
- Sodium-iodine symporter
SEM
- Standard Error of Mean
T3
- Triiodothyronine
T4
- Thyroxine
TSH
- Thyroid stimulating hormone
131
- Xenon–131
Xe
1
Introduction
All animals, including humans, are subject to basic needs such as eating or sleeping. These are
recurring and oscillating needs that are usually satisfied in a periodic manner across the day.
Periodicity is within us and all around us, with cycles of varying length: from day lengths of 24 h up to
seasonal changes. The cycle of day length is also referred to as the circadian rhythm. The circadian
rhythm affects functions in our bodies. Among these are the sleep/wake cycle, secretion of hormones
like TSH (Thyroid stimulating hormone), and body temperature (Schulz & Steimer, 2009).
These functions are regulated by biological clocks in the body with the SCN (suprachiasmatic nucleus)
in the hypothalamus of the brain as the main regulator. This regulator is reset by a range of factors,
called zeitgebers (time givers), with daylight being the most important one. Responsible for
maintaining circadian rhythm on the tissue level are certain genes expressed in the cells, called
molecular clock genes, that are stimulated by molecular signals from the SCN (Reppert & Weave,
2002).
Although circadian rhythm is an intrinsic factor in basically all biological activities, it is generally not
accounted for in current research or treatment schedules in nuclear medicine. There are a few
exceptions to this. One is the study of radiation-induced gene regulation with i.v. administration of 131I
in mice, where the outcome of gene regulation in tissues was dependent on the time of day when 131I
was injected (Langen et al., 2015). Recently, a biodistribution study on 131I in mice was performed
(Andersson, 2015), where mice were injected with 131I at different times of day. Several decades ago,
uptake of 131I in mice was also studied with consideration of varying time points of administration
(Walinder, 1971). That study, however, focused on the thyroid and thus did not consider the impact of
circadian variation on radionuclide biodistribution in other tissues.
The thyroid is an endocrine gland of approximately 20 grams, in humans, located at the sides of the
trachea, just below the larynx (Sand et al., 2007). This gland is subject to circadian variation of
follicular lumen size and concordantly weight variation due to change in colloid (water) volume
(Wright et al, 1995). The thyroid produces the thyroid hormones T3 (triiodothyronine) and T4
(thyroxine) that regulate important cellular mechanisms such as metabolic activity throughout the
body (Sand et al., 2007; Yen, 2001). T3 and T4 hormones are regulated by TSH (released from the
pituitary gland) and synthesized in the follicular structure of the thyroid, normally with stable iodine
(Sand et al., 2007). This nuclide, 127I, is the only stable isotope among the 37 known iodine isotopes
(The Lund/LBNL Nuclear Data Search, 1999). Thyroid hormones can also be synthesized with other
isotopes of iodine, e.g. 131I, since the molecular mechanisms cannot distinguish between isotopes.
Iodine is transported from the blood to the thyroid as iodide (I-) via the sodium-iodine symporter (NIS)
(Sand et al., 2007). Inside the follicular lumen of the thyroid, iodide is covalently bound to a tyrosinebased molecule, creating the T3 and T4 hormones (Yen, 2001). The hormones are then secreted
through the blood into various tissues in the body. The thyroid produces mostly the T4 hormone,
which is a prohormone and subsequently reduced to T3 in the body, in particular in the kidneys and
liver (Larsen et al., 2012; Gereben et al., 2008; Bianco et al., 2002).
Because of its high iodine uptake, the thyroid is both a risk organ as well as a target when it comes to
dealing with radioactive iodine such as 131I. This is utilized in e.g. targeted radionuclide therapy
treatment for hyperthyroidism, where free 131I is administered and taken up by the thyroid (Lee, 2012).
Patients with some types of cancer, e.g. breast carcinoma, where the tumor cells express sufficiently
high levels of NIS, can be treated in a similar fashion (Yao et al., 2015).
I has a half-life of 8.03 days (MIRD, 2006) and disintegrates via β- decay into 131Xe: this energy is
mostly absorbed locally due to the short range of respective β-particles (Berger et al., 1984). Because
of this, the surrounding tissues around the source organ are mostly spared from absorbed dose. Beside
the β-particles, 131I also has a gamma component with an energy of 364.5 keV (The Lund/LBNL
131
2
Nuclear Data Search, 1999), which enables detection with e.g. a gamma counter or gamma cameras in
clinical applications.
Exposure to 131I can occur from several sources. One example is radioactive iodine being released
from 131I-MIBG (Metaiodo-benzylguanidine) upon autoradiolysis. 131I-MIBG is used in treatment and
diagnostics of e.g. neuroblastomas, paragangliomas and pheochromocytomas, where the aim is to
target the cancer with a norepinephrine analogue bound to the radionuclide (Kayano & Kinuya, 2015).
Exposure to 131I can also occur upon nuclear accidents. An example is the nuclear accident that
occurred in Chernobyl, where nearly 1.8 ∙ 1018 Bq of 131I were released to the environment and caused
an increased incidence of thyroid cancer in Belarus in the following years (McLaughlin et al., 2012;
Kazakov et al., 1992).
Since iodine is distributed via the blood, it will reach all organs in the body and it is therefore
imperative to have a well-based knowledge about the potential risks and dose-responses in normal
tissues when dealing with radioactive iodine, regardless of the types of exposure. Biodistribution
studies are a good way of collecting these data. Animal models are often used to study biokinetics and
various species have been used: mice (Garg et al., 1990), rats (Spetz et al, 2013) and hamsters
(Peronace & Houssay, 1970), among others.
In clinical research, there is often reluctance towards using females in studies (Holdcroft, 2007). This
is of particular concern when considering that women are more prone to certain thyroid-related
diseases such as Grave’s disease and Hashimoto’s thyroiditis, with an incidence ratio of more than
7−10:1 (Beery & Zucker, 2011; Jacobson et al., 1997). There are many benefits to having data for
both biological sexes, especially when underlying processes are unknown (Beery & Zucker, 2011;
National Institutes of Health, 2001).
The purpose of this project was to study the biodistribution of 131I in various tissues in mice with three
different time points of administration, with special consideration of biological sex and iodine diet as
experimental variables.
3
Aims
The purpose of this work was to determine the biokinetics of 131I in mice in dependence of the time of
day of injection. To achieve this, a partial aim was to validate previous data from Charlotte
Andersson’s master thesis from fall 2015 (Andersson, 2015), i.e. to investigate the effect of circadian
rhythm for injections performed at 12 pm and 4 pm. Moreover, this work aimed to characterize 131I
biodistribution for an additional early time point of administration, at 8 am.
A further aim was to assess potential differences between the sexes concerning the biodistribution and
biokinetics of 131I in mice. In addition, the impact of iodine concentration in animal food on 131I
biodistribution was studied.
To minimize measurement errors of the gamma counters used for this work, the aim was also to
investigate and, if needed, correct for self-attenuation in samples and for adsorption in syringes due to
varying time spans between preparation of syringes and injection of subjects.
4
Materials and Methods
Animal model
In total, 159 C57BL/6N mice (124 males and 35 females) between 6 and 10 weeks of age, and
weighing 20.9–30.8 g for the males and 19.3–22.3 g for the females, were used in the study. The mice
were kept in groups of 5 individuals per cage. One group (24 h with administration at 4 pm) was
reduced to 4 individuals, since one mouse was unwell and had to be killed before start of the
experiments due to ethical guidelines.
Drinking water and food were given ad libitum. As standard, the mice were given breeding chow with
a reduced iodine concentration of 0.87 µg/g. The reduced iodine nutrition was chosen to limit the
individual variation of 127I amounts in the animals, which may have an effect on the uptake of 131I
during the experiments. This setup also matched the study design in previously performed experiments
(Andersson, 2015). An iodine concentration, in food, of 1.20 µg/g was given to 30 males in this work,
see Table 1.
Standard laboratory day and night cycle was maintained for the mice during the experiments, i.e. dark
from 6 pm to 6 am. The studies were approved by the Gothenburg Ethical Committee on Animal
Research (no. 146-2015).
Administration of radionuclides
131
I was obtained from GE Healthcare (Braunschweig, Germany) as Na131I, and diluted with saline
solution to the desired activity concentration for administration. Syringes (0.1-1 ml Braun syringes
with 0.4×20 mm cannulas) were prepared with an activity of approximately 165 kBq 131I from stock
solutions prepared before each injection time point. All the syringes were prepared according to
previous procedure (Andersson, 2015), i.e. were weighed after preparation as well as after injection to
determine the administered activity to each subject. At preparation, control syringes were prepared in
the same manner and emptied, in a 20 ml scintillation vial, after a similar time span to the waiting
period between preparation of animal syringes and injection of animals. The weight of the content of
each control syringe was then correlated to measurements in gamma counters, giving an activity/massratio. The time points and administered activity are given in Table 1.
5
Table 1: Experimental overview. The table shows mean values, with SEM (italics), for administered activity, weight,
and age for each group. It is also indicated if the diet was regular or iodine reduced.
Group
Number of
animals
Injected activity
[kBq]
Weight [g]
Age [weeks]
Reduced iodine diet
(Y/N)
Males
Injection at 8 am
1h
n=5
175
3
23.0
0.4
9-10
Y
4h
n=5
170
3
24.6
1.1
9-10
Y
8h
n=5
170
1
25.5
1.2
9-10
Y
18 h
n=5
165
1
25.2
0.5
9-10
Y
24 h
n=5
170
2
24.6
0.4
9-10
Y
72 h
n=5
170
2
23.7
0.3
9-10
Y
168 h
n=5
165
2
23.7
0.2
9-10
Y
1h
n=5
170
2
22.9
0.1
9-10
Y
4h
n=5
165
2
23.6
0.2
9-10
Y
8h
n=5
165
2
23.6
0.2
9-10
Y
18 h
n=5
160
2
23.8
0.4
9-10
Y
24 h
n=5
165
2
23.9
0.2
9-10
Y
72 h
n=5
165
1
22.4
0.2
9-10
Y
1h
n=5
170
3
24.0
0.4
6-7
N
4h
n=5
170
3
22.2
0.5
6-7
N
72 h
n=5
140
0
23.6
0.3
8-9
N
1h
n=5
170
4
23.9
0.3
9-10
Y
4h
n=5
160
1
24.2
0.5
9-10
Y
8h
n=5
165
2
23.7
0.2
9-10
Y
18 h
n=5
160
1
23.9
0.4
9-10
Y
24 h
n=4
160
1
23.5
0.6
9-10
Y
72 h
n=5
165
2
27.6
1.2
9-10
Y
1h
n=5
170
1
22.4
0.7
6-7
N
4h
n=5
170
1
23.6
0.5
6-7
N
72 h
n=5
140
5
22.6
0.3
8-9
N
1h
n=5
170
2
20.5
0.3
9-10
Y
4h
n=5
170
2
20.4
0.5
9-10
Y
8h
n=5
170
2
20.7
0.4
9-10
Y
18 h
n=5
175
2
21.4
0.3
9-10
Y
24 h
n=5
170
1
21.0
0.2
9-10
Y
72 h
n=5
175
1
20.9
0.2
9-10
Y
168 h
n=5
170
2
20.6
0.3
9-10
Y
Injection at 12 pm
Injection at 4 pm
Females
Injection at 8 am
6
Table 2: Sampling overview. Organs, part of organs, as well as gastrointestinal contents were excised or sampled and put
into 20 ml scintillation vials for measuring in gamma counters. Gastrointestinal contents were excised from the sampled parts
of their respective organ.
Organ
Method of sampling
Blood
Thyroid
Neck
Lungs
Heart
Liver
Kidney left
Kidney right
Salivary glands
Spleen
Small intestine
Large intestine
Stomach
Small intestine content
Large intestine content
Gastric content
Intestines rest
Carcass
Sampled
Excised
Excised
Excised
Excised
Excised
Excised
Excised
Excised
Excised
Excised
Excised
Excised
Sampled
Sampled
Sampled
Excised
As is after excisions
Biodistribution and biokinetics
The subjects were injected intravenously (in the tail vein) at 8 am, 12 pm, or 4 pm, and then killed
after 1, 4, 8, 18, 24, 72 or 168 h following injection, as shown in Table 1. Animals were injected i.p.
with sodium pentobarbithal (APL, Sweden) and killed via cardiac puncture, which was the designated
time of death. Organs, or part of organs, as well as gastrointestinal contents were then excised or
sampled and put into 20 ml scintillation vials (see Table 2). The large intestines were sampled starting
from the sigmoid colon, the small intestines were sampled starting from the duodenum, and the
contents of each intestine were sampled from the excised parts. The carcass was also committed to a
20 ml scintillation vial after excision of the remaining gastrointestinal tract. The thyroid was, after
weighing, embedded in 4% formaldehyde solution to enable validation at a later point. All vials were
weighted before and after adding the samples and subsequently measured in the gamma counters.
Some of the thyroid samples were of unreasonably high weight (up to ~6.5 mg) in comparison with
literature. These weights were corrected according to previous procedures, i.e. the weights above 4.4
mg were reduced to 3.9 mg (Andersson, 2015).
7
Radioactivity measurements
A CRC-15R dose calibrator ion chamber, produced by Capintec, IA, USA, with known calibration
was used as a reference detector to measure 131I stock solutions of activity above 1 MBq for
preparation of syringes and calibration samples. Injected activity was also determined using between 4
and 5 control syringes for each stock solution. A mean value from the control syringes was used to
reduce weighing errors from the scale.
Activities of 131I in organs, tissue samples, and control syringes were measured ex-vivo using two
Wallac 1480 Wizard® 3" NaI(Tl) gamma counters, produced by Wallac Oy, Turku, Finland. Gamma
counter with serial number 4800173 is hereinafter referred to as gamma counter 1 and gamma counter
with serial number 4800361 is referred to as gamma counter 2. For measurements, pre-installed
protocols for 131I were used with the energy window of 260‒430 keV for both detectors.
Percentage of administered activity of 131I per organ mass (%IA/g) was determined based on weight
measurements and activity concentration in the samples, decay-corrected to the time of 131I
administration.
Detector calibrations
To establish the relationship between sample activity and measured counts per second (cps) in the
gamma counter, a calibration of the gamma counters was performed. Calibration samples were
prepared with a constant volume of 500 µl and decreasing concentrations of 131I from 800 kBq to 1.6
kBq by performing a two-fold serial dilution. The ion chamber with known calibration was used as a
reference detector.
By plotting measured cps vs. sample activity and making a linear fit to the curve, a calibration factor
was obtained. The calibration factor was then used to determine 131I activity in the samples from
gamma counter measurements.
Measurement corrections
Due to self-attenuation in large samples and limitations in sensitivity of the gamma counter, an
investigation of potential volume effects was performed. An activity of 20 kBq 131I was pipetted into a
20 ml scintillation vial and then diluted with ultrapure water (Merck Millipore, Darmstadt, Germany)
to a total volume of 1, 2, 3, 4, 5, 7.5, 10, 12.5, 15, 17.5 and 20 ml. The activity was chosen with regard
to the sensitivity of the gamma counter and expected activity of 131I in organ samples.
Due to varying time spans between preparation of syringes and injection of mice (ranging from 1 h to
8 h after preparation), an investigation of adsorption effects, i.e. the binding of activity on the inside
surface in the syringe resulting in a lower activity concentration than originally prepared, was
performed to ensure correct estimation of the individual administered activities. The tests were made
with the same activity concentration as the one administered to animals.
Measurements were corrected for background radiation and dead time. Measurement time was
adjusted so that each organ had a minimum of 1000 counts over background and the measured
activities were corrected to the time of 131I administration.
8
Statistical analysis
Statistical uncertainties in measurement analysis are represented as Standard Error of Mean, 𝑆𝑆𝑆𝑆𝑀𝑀𝑥𝑥̅ ,
calculated with the following equation:
� 1 ∑𝑛𝑛𝑖𝑖=1(𝑥𝑥𝑖𝑖 − 𝑥𝑥̅ )2
𝑛𝑛 − 1
=
,
𝑆𝑆𝑆𝑆𝑆𝑆𝑥𝑥� =
√𝑛𝑛
√𝑛𝑛
𝑆𝑆𝑆𝑆
where SD is the sample standard deviation, n is the sample number in the group, {𝑥𝑥1 , 𝑥𝑥2 , … , 𝑥𝑥𝑛𝑛 } is the
value of the observed sample items and 𝑥𝑥̅ is the mean value of these observations.
Determination of statistically significant differences in radioactivity concentration in organs between
administration time points was performed with a Student's t-test where probabilities above 95%
(p<0.05) were considered statistically significant.
Student's t-test was also used to determine statistical significance of potential volume effects in
radioactivity measurements, where probabilities above or equal to 99% (p≤0.01) were considered
statistically significant in this case.
9
Results
Detector calibrations
The results from the calibrations showed similar relationships between measured cps and 131I activity
in the two gamma counters. Both gamma counters display the same dead time factor for 25 kBq for all
observations, i.e. 11%. Thus were measurements with dead time factors under 11% accepted, and
tissues with higher dead time factors were left to decay until meeting this criterion. The measurements
that met the dead time criterion were corrected by the dead time factor acquired for that measurement.
The activity calibration factors were acquired by linear curve fit to data points up to 25 kBq and were
found to be k1=426.29 cps/kBq (with an R2-value of 0.99987) and k2=424.95 (with an R2-value of
0.99994) cps/kBq for gamma counters 1 and 2, respectively, see Figure 1. The calibration curve can be
considered to be linear up to about 11 000 cps.
a
b
Figure 1: Activity calibration of the gamma counters. The relationship between measured cps and 131I activity for gamma
counter 1 (a), and 2 (b), is shown in the graphs. The activities were prepared by means of linear two-fold serial dilution, with
the ionization chamber as a reference detector, creating linear decreasing concentrations with equal volumes of 0.5 ml. The
line is a linear curve fit to the data points up to 25 kBq. Data points are mean values of three dilution series and the error bars
indicate SEM; error bars not visible are smaller than the data symbol.
10
Measurement corrections
Volume effect
Results from the self-attenuation and sensitivity investigation are shown in Figure 2. The figure shows
a decreasing count ratio with increasing sample volume for a constant 131I activity of 20 kBq. The
sample sizes 15, 17.5 and 20 ml show a statistically significant difference compared to the 1 ml
sample used as a reference. The count rate of a 20 ml phantom is considered to be underestimated by
20 % and measured count rates for the carcass were therefore corrected with a factor 1.2 to account for
this.
Figure 2: Count rate ratio between samples of different volumes and a sample of 1 ml at constant 131I activity.
Statistically significant differences compared with measurements of 1 ml samples (p≤0.01) were found at 15, 17.5 and 20 ml
sample volumes. Error bars display SEM; n=2.
Adsorption
Results from the adsorption tests are shown in Figure 3. The presented data are mean values
normalized to the control syringes (n=5). The investigation showed no statistically significant
difference between control syringes and the syringes where ejection was delayed by 4 or 8 hours.
Therefore, no adsorption correction was made in the animal experiments.
Figure 3: Investigation of adsorption effect in syringes. The mean values of activity concentration in syringes with 4 or 8 h
delay between preparation and ejection are shown, normalized to the activity concentration in control syringes. The x-axis
shows the time delay (compared to the control syringe) that would have been administered at the nominal time point 8 am.
Error bars display with SEM, n=5.
11
Biodistribution of 131I
Biodistribution studies were performed for time spans 1–168 h after administration with
administrations performed at 8 am, 12 pm or 4 pm. For injections at 8 am, the highest activity
concentration in tissues was found in the thyroid (5800 %IA/g) at 18 h after administration. High
activity concentrations were also found in gastric content and stomach at earlier time points. For
administration at 12 pm and 4 pm, the highest activity concentration in tissues was found, also in the
thyroid, to be 2400 %IA/g and 2100 %IA/g respectively (both at the time point 8 h following
injections). For the administration series of female mice, performed at 8 am, the highest activity
concentration in tissues was also found in the thyroid (6000 %IA/g) 18 h following administration of
131
I. Please note that two out of the approximately 3000 samples in this work, i.e. two samples of small
intestine content in males after 168 h with administration at 8 am, were not able to meet the criterion
of obtaining 1000 counts over background, due to very low activity.
Activity concentrations in tissues and gastrointestinal contents for males with administration at 8 am
can be seen in Figure 4a and in Figure 4b; for administration at 12 pm in Figure 5a and Figure 5b; and
for administration at 4 pm in Figure 6a and Figure 6b. For all tissues in male mice, with exception for
the thyroid and large intestine content, the highest activity concentration was found 1 h after
injections. for large intestine this happened at 4 h after injections.
Activity concentrations in tissues and gastrointestinal contents for females with administration at 8 am
can be seen in Figure 7a and in Figure 7b. For the female mice, the highest activity concentrations
were found at 1 h after injections, with exceptions for thyroid, salivary gland and intestinal contents.
The activity concentration levels in the salivary glands, reaching a maximum 8 h following
administrations of 131I, were found maximally at 24 %IA/g. In comparison, the maximum for the male
mice was found 1 h following administration for all time of day of injection, and reached a maximum
at almost 14 %IA/g (with administration at 12 pm).
12
a
b
Figure 4: Biodistribution of 131I activity concentration in male mice at 1 h up to 7 days after administration of 165–175
kBq, performed at 8 am. Error bars display SEM; n=5 for all data points. Please note the change of scale on the vertical
axis.
13
a
b
Figure 5: Biodistribution of 131I activity concentration in male mice at 1 h up to 3 days after administration of 160–170
kBq, performed at 12 pm. Error bars display SEM; n=5 for all data points. Please note the change of scale on the vertical
axis.
14
a
b
Figure 6: Biodistribution of 131I activity concentration in male mice at 1 h up to 3 days after administration of 160–170
kBq, performed at 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration (n=4). Please
note the change of scale on the vertical axis.
15
a
b
Figure 7: Biodistribution of 131I activity concentration in female mice at 1 h up to 7 days after administration of 170–
175 kBq, performed at 8 am. Error bars display SEM; n=5 for all data points. Please note the change of scale on the
vertical axis.
16
Comparison between males at time point 8 am, 12 pm, and 4 pm
Figure 8–24 show the biokinetics for each tissue for 131I administration in males at 8 am, 12 pm, and 4
pm. The time points where statistically significant differences (p<0.05) were found between the three
times of administration are noted in the figures. In general there were large deviations observed during
time points from 1 h up to 18–24 h, especially for gastrointestinal content, see Figure 21–23.
In the thyroid, see Figure 9, a statistically significant difference between administration at 8 am and 12
pm was found after 4 and 18 h, with a global maximum after 18 h for administration at 8 am. A
maximum of activity concentration was found after 8 h for administration at 12 pm and 4 pm. For the
neck and thyroid combined, plotted in %IA, see Figure 10, the maximum was observed more clearly
after 8 h for administration at 12 pm; interestingly, for administration at 4 pm, two peaks were
observed after 4 and 18 h.
At 1 h after administration, there was a statistically significant difference for the small intestine
between 8 am and 12 pm injections (Figure 18). Statistical significance was found 4 h after
administration between 8 am and 12 pm in all tissues with exception for small intestine content.
Between administrations performed at 12 pm and 4 pm, statistical significance was found after 4 h in
all tissues except small intestine and thyroid combined with neck. No statistically significant
difference was found 4 h after administration between 8 am and 4 pm. Statistical significance was
found after 1 h only in small intestine between the 8 am and 12 pm groups (Figure 18).
Significant difference was found between all three administration time points at 18 h for salivary gland
and small intestine (Figure 16 and Figure 18). At 24 h after administration, the results showed no
statistical significance in tissues, with the exception of stomach (between 8 am and 4 pm, Figure 20)
and gastric content (between 8 am and 4 pm, and between 12 pm and 4 pm, Figure 23). At 72 h after
administration, significance was found only between 8 am and 12 pm (in salivary gland, small
intestine, gastric content and carcass, Figure 16, 18, 23 and 24). Between administrations at 8 am and
4 pm, statistically significant differences were found only after 8 and 18 h, with the exception of 24 h
for stomach and gastric contents. Gastric contents showed statistical significance between 8 am and 12
pm for the time points 4 and 72 h; between 12 pm and 4 pm for the time points 4, 8 and 18 h; between
12 pm and 4 pm for the time points 4, 8, 18 and 24 h, see Figure 23.
All organs, with the exception of thyroid, thyroid combined with neck, and large intestine content,
showed the highest %IA/g at 1 h, after which the activity concentration declined monotonically. An
exception was found in the kidneys, however, which showed a local maximum at 72 h with a
statistically significant rise between 24 h and 72 h for all injection series (and a statistically significant
rise between 18 and 24 h in the left kidney for the administration series performed at 12 pm and 4 pm,
as well as the right kidney for the administration series performed at 12 pm), see Figure 14–15. The
injection series at 12 pm depicted a slower decline in activity concentration than injections at 8 am and
4 pm, 1‒18 h after administration.
17
Figure 8: 131I concentration in blood for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was
found after 4 and 18 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and 4 pm.
Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
Figure 9: 131I concentration in thyroid for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was
found after 4 and 18 h between 8 am and 12 pm; after 4 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data
points except for 24 h after administration at 4 pm (n=4).
18
Figure 10: 131I concentration in neck and thyroid for administrations at 8 am, 12 pm, or 4 pm. Statistical significance
(p<0.05) was found after 4 and 18 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 8 h between 12
pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4). Please note
that the graph display %IA.
Figure 11: 131I concentration in lungs for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was
found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and 4 pm.
Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
19
Figure 12: 131I concentration in heart for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was
found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm.
Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
Figure 13: 131I concentration in liver for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was
found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm.
Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
20
Figure 14: 131I concentration in left kidney for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05)
was found after 4 and 18 h between 8 am and 12 pm; after 8 h between 8 am and 4 pm; after 4 h between 12 pm and 4 pm.
Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
Figure 15: 131I concentration in right kidney for administrations at 8 am, 12 pm, or 4 pm. Statistical significance
(p<0.05) was found after 4 h between 8 am and 12 pm; after 8 h between 8 am and 4 pm; after 4 h between 12 pm and 4 pm.
Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
21
Figure 16: 131I concentration in salivary glands for administrations at 8 am, 12 pm, or 4 pm. Statistical significance
(p<0.05) was found after 4, 18 and 72 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 18 h
between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
Figure 17: 131I concentration in spleen for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05) was
found after 4 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 18 h between 12 pm and 4 pm. Error
bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
22
Figure 18: 131I concentration in small intestine for administrations at 8 am, 12 pm, or 4 pm. Statistical significance
(p<0.05) was found after 1, 4, 18 and 72 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 18 h between
12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
Figure 19: 131I concentration in large intestine for administrations at 8 am, 12 pm, or 4 pm. Statistical significance
(p<0.05) was found after 4 h between 8 am and 12 pm; after 8 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and
4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
23
Figure 20: 131I concentration in stomach for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05)
was found after 4 h between 8 am and 12 pm; after 8, 18 and 24 h between 8 am and 4 pm; after 4 and 18 h between 12 pm
and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
Figure 21: 131I concentration in small intestine content for administrations at 8 am, 12 pm, or 4 pm. Statistical
significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm. Error bars
display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
24
Figure 22: 131I concentration in large intestine content for administrations at 8 am, 12 pm, or 4 pm. Statistical
significance (p<0.05) was found after 4 h between 8 am and 12 pm; after 8 and 18 h between 8 am and 4 pm; after 4, 8 and
18 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm
(n=4).
Figure 23: 131I concentration in gastric content for administrations at 8 am, 12 pm, or 4 pm. Statistical significance
(p<0.05) was found after 4 and 72 h between 8 am and 12 pm; after 8, 18 and 24 h between 8 am and 4 pm; after 4, 8, 18
and 24 h between 12 pm and 4 pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4
pm (n=4).
25
Figure 24: 131I concentration in carcass for administrations at 8 am, 12 pm, or 4 pm. Statistical significance (p<0.05)
was found after 4 and 72 h between 8 am and 12 pm; after 18 h between 8 am and 4 pm; after 4 and 8 h between 12 pm and 4
pm. Error bars display SEM; n=5 for all data points except for 24 h after administration at 4 pm (n=4).
26
Sex difference in 131I biodistribution
Biokinetics for both male and female mice, injected with 131I at 8 am, are presented in Figure 25–41.
The time points where statistically significant differences were found between the sexes are indicated
in the figures. In general, statistically significant differences were found at least at three time points in
all samples. Only three samples showed fewer or no significant differences: for the thyroid (see Figure
26) and large intestine content (see Figure 39), significance was found at two time points, whereas for
thyroid combined with neck (see Figure 27) no statistical significance was observed. The samples for
which the highest number of statistically significant differences were found were the carcass (see
Figure 41; statistical significance at all time-points), the spleen (see Figure 34) and the kidneys (see
Figure 31–32). In total, statistically significant differences between male and female mice were
demonstrated in 70 out of 119 observations for the 17 samples studied.
In the thyroid, statistically significant differences were found after 24 and 72 h between male and
female mice. The activity concentration peaked after 18 h for both sexes, yet presumably with a
somewhat higher activity concentration in females (about 200 %IA/g higher mean value), see Figure
26. For male mice, there was a global maximum after 18 h with a statistically significant difference
compared with 24 h after administration. For neck and thyroid together, the biokinetics also depicted a
statistically significant maximum at 18 h (showing statistically significant difference between 8 and 18
h, and between 18 and 24 h for both sexes) for both males and females, although there was no
statistically significant difference between the sexes, see Figure 27.
For the kidneys, see Figure 31–32, distinctly different curve behavior was observed between the sexes:
In male mice, there was a faster decrease in activity concentration in the time span between 1 and 18
h, followed by an increase with a local maximum after 72 h and subsequent monotone decrease. In
contrast, activity concentration in female mice did not display a local maximum but monotone
decrease as observed for various other samples. After 72 and 168 h, there was a comparatively large
difference in activity concentration for male and female mice. Statistically significant difference was
found at all time-points, with the exception of 1 h.
A majority of the remaining tissues showed a fast decrease of activity concentration within the first 18
h for both sexes (where female mice showed a slightly less steep decrease in the time span between 1
and 18 h), with some exceptions: For salivary glands (see Figure 33) and small intestines (see Figure
35), the curve depicted an increase of activity concentration for female mice after 1–4 h reaching a
maximum after 4 h.
In the large intestine content, see Figure 39, the activity concentration in females depicted a distinct
maximum after 8 h, which was about twice as high as the observed maximum for males; statistically
significant differences for the large intestine content were observed at 8 and 168 h.
27
Figure 25: 131I concentration in blood for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 24,
72 and 168 h. Error bars display SEM; n=5.
Figure 28: 131I concentration in lungs for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 24
and 72 h. Error bars display SEM; n=5.
Figure 26: 131I concentration in thyroid for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 24 and
72 h. Error bars display SEM; n=5.
Figure 29: 131I concentration in heart for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 18,
24 and 72 h. Error bars display SEM; n=5.
Figure 27: 131I concentration in neck and thyroid for
administrations at 8 am for male and female mice. No
statistical significance was found Error bars display
SEM; n=5.
Figure 30: 131I concentration in liver for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 18
and 72 h. Error bars display SEM; n=5.
28
Figure 31: 131I concentration in left kidney for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 18,
24, 72 and 168 h. Error bars display SEM; n=5.
Figure 34: 131I concentration in spleen for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 1, 4, 8,
18, 24 and 72 h. Error bars display SEM; n=5.
Figure 32: 131I concentration in right kidney for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 18,
24, 72 and 168 h. Error bars display SEM; n=5.
Figure 35: 131I concentration in small intestine for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 18,
24 and 72 h. Error bars display SEM; n=5.
Figure 33: 131I concentration in salivary glands for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 1, 4, 8,
18 and 72 h. Error bars display SEM; n=5.
Figure 36: 131I concentration in large intestine for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8, 24
and 72 h. Error bars display SEM; n=5.
29
Statistical significance (p<0.05) was found after 8 and
168 h. Error bars display SEM; n=5.
Figure 37: 131I concentration in stomach for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 18, 24
and 72h. Error bars display SEM; n=5.
Figure 40: 131I concentration in gastric content for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 1, 4, 24
and 72 h. Error bars display SEM; n=5.
Figure 38: 131I concentration in small intestine content
for administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found after 4, 8 and
24 h. Error bars display SEM; n=5.
Figure 41: 131I concentration in carcass for
administrations at 8 am for male and female mice.
Statistical significance (p<0.05) was found at all timeintervals. Error bars display SEM; n=5.
Figure 39: 131I concentration in large intestine content
for administrations at 8 am for male and female mice.
30
Influence of iodine concentrations
in food at 12 pm
Biokinetics for 131I administration at 12 pm
with either normal or reduced levels of iodine
in food is presented for blood, thyroid, liver
and kidneys in Figure 42–46.
Statistical significance was found at 1 h for all
five tissues, with the exception of thyroid
where no statistical significance was found.
The thyroid showed a variation of activity
concentrations at 1 and 4 h, but no statistical
significance was found. The data for the
remaining samples are shown in the Appendix,
Figure 57–67.
Figure 44: 131I concentration in liver for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 42: 131I concentration in blood for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 45: 131I concentration in left kidney for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 43: 131I concentration in thyroid for
administrations at 12 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 46: 131I concentration in right kidney for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
31
Influence of iodine concentrations
in food at 4 pm
Biokinetics for 131I administration at 4 pm with
either normal or reduced levels of iodine in
food is presented for blood, thyroid, liver and
kidneys in Figure 47–51.
The results showed no significant differences
for these tissues, with the exception of thyroid
for which significant difference was found
after both 1 and 4 h. The data for the remaining
samples are shown in the Appendix, Figure
68–78.
Figure 49: 131I concentration in liver for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 47: 131I concentration in blood for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 50: 131I concentration in left kidney for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 48: 131I concentration in thyroid for
administrations at 4 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1, 4 and 72 h. Error bars display
SEM; n=5.
Figure 51: 131I concentration in right kidney for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
32
Effect of circadian rhythm at
standard iodine concentration in
food
The comparison between 131I administration at
12 pm or 4 pm with standard levels of iodine in
food is shown for blood, thyroid, liver and
kidneys in Figure 52–56. The activity
concentration curves in blood, liver and
kidneys intersect between 1 and 4 h and show
statistically significant differences at time
points 1 and 4 h.
Figure 54: 131I concentration in liver for
administrations at 4 pm and 12 pm for normal iodine
concentration in food. Statistical significance (p<0.05)
was found after 1 and 4 h. Error bars display SEM; n=5.
Figure 52: 131I concentration in blood for
administrations at 4 pm and 12 pm for normal iodine
concentration in food. Statistical significance (p<0.05)
was found after 1 and 4 h. Error bars display SEM; n=5.
Figure 55: 131I concentration in left kidney for
administrations at 4 pm and 12 pm for normal iodine
concentration in food. Statistical significance (p<0.05)
was found after 1 and 4 h. Error bars display SEM; n=5.
Figure 56: 131I concentration in right kidney for
administrations at 4 pm and 12 pm for normal iodine
concentration in food. Statistical significance (p<0.05)
was found after 1 and 4 h. Error bars display SEM; n=5.
Figure 53: 131I concentration in thyroid for
administrations at 4 pm and 12 pm for normal iodine
concentration in food. No statistical significance was
found. Error bars display SEM; n=5.
33
Discussion
In this work, animal experiments were conducted to reproduce and further extend previous
investigations on the influence of circadian rhythm on 131I biodistribution. Furthermore, potential
differences between male and female mice and the impact of iodine concentration in animal food were
investigated, concerning the biodistribution and biokinetics of 131I. In addition, several technical
investigations were made to minimize measurement errors. These investigations concerned selfattenuation in samples and the sensitivity of the gamma counter when measuring large samples, and
the decrease of activity concentration over time by means of adsorption on the inside surface of
syringes. The latter is of concern when the experimental schedule necessitates that syringes are
prepared early in the morning, while the injections for different groups are distributed across the day,
which leads to that certain syringe batches are used approximately four or eight hours after
preparation. In addition to this, calibrations of the gamma counters were performed to translate the
counted pulses of the samples in the gamma counters to an activity in kBq.
Calibration factors of the gamma counters were acquired for 131I by plotting measured count rates from
samples between 1.6 kBq to 25 kBq, measured in the two gamma counters available, and making a
linear curve fit to these data points. The results show that a count rate up to approximately 11 000 is
acceptable for both gamma counters. With obtained R2-values ≥0.99987, the linear curve fitting was a
good approximation and the count rate response could be considered linear up to 25 kBq for 131I,
which corresponded to acceptable dead time factors of up to 11%.
A volume effect was observed for large sample sizes, which would lead to underestimation of cps if
not corrected for. Although only the volume sizes of 15, 17.5 and 20 ml could be proven statistically
significant (p≤0.01), the trend indicated that the effect may appear at 10 ml or even smaller volumes.
In particular, this issue concerned measurement of the mouse carcass, which constitutes a very large
sample that fills nearly the entire volume of a 20 ml scintillation vial. The loss of counts due to this
volume effect was estimated to be 20 %, and a correction for this was made to more accurately
estimate the activity in the carcass. It should be noted that the activity in the carcass is an important
quantity, for instance when designing a compartment model for 131I biodistribution in future work. The
correction, however, was only a rough approximation based on homogenous density of the water
phantom in the scintillation vial. A more accurate approximation for the loss of pulses would require a
tissue-equivalent phantom with non-homogenous density distribution corresponding to a mouse
carcass, or a computer simulation of respective scintillation vials with radiation transport codes.
The investigation of 131I adsorption in syringes (brand "Braun") showed that adsorption did not occur
to a significant extent (p>0.01) when injection was delayed up to 8 h. This finding is of particular
importance for planning these types of animal studies, since the nominal administered activity is valid
even if syringes are used several hours later. This means that all syringes can be prepared once from
the same stock solution without the necessity to correct for adsorption with additional (increased)
stock concentrations.
In previous work, various time points following 131I administration showed statistically significant
difference in %IA/g between administration at 12 pm or 4 pm and a number of these instances was
also reproduced in the present studies. For instance, in the lungs after 4 and 8 h following injection,
statistically significant differences were demonstrated in both previous and present work. Regarding
the thyroid, circadian variation in 131I biodistribution was also demonstrated with statistical
significance, although at another time point after administration compared to previous work
(Andersson, 2015). Interestingly, the novel time point of administration revealed a distinctly different
biodistribution curve behavior for the thyroid: for the earliest administration time point (8 am), the
peak of activity concentration occurred at a much later time point after administration compared with
later administrations (12 pm or 4 pm). Moreover, the effective day times for respective peaks did not
34
match. This finding may appear counter-intuitive, but results for the respective time point were
statistically significant, indicating the complexity of iodine biodistribution kinetics in the body.
Overall, these studies demonstrated that differences in %IA/g due to circadian variation are large
enough to be detected even with comparatively small populations. However, when considering that
significant differences could not be demonstrated, or reproduced, at certain time points following 131I
administration, it is recommendable to increase group size in future studies to better account for
biological variation in 131I uptake. For all tissues except the thyroid in the male cohort, the activity
concentration retention was higher, up to 18 h after 131I administration, for the mice administered at 12
am compared to the mice at the two other time points. This would lead to a higher absorbed dose in
these tissues and, in a clinical perspective, would suggest that another time point is chosen if the
lowest possible absorbed dose to these tissues is desirable.
Comparing biodistribution data between male and female mice, the typical behavior was that the curve
for female mice was less steep than for male mice over the first time points following injection.
Interestingly, the curve behavior for female mice (injected at 8 am) resembled the curve behavior of
male mice when injected at 12 pm, i.e. that male mice showed a slower decrease in activity
concentration after injection at 12 pm compared with the other administration times. The retention in
female mice could be less pronounced if 131I was administered at a later time during the day, which in
turn would indicate a diurnal shift in iodine metabolization between male and female mice.
The kidneys indicated another sex-specific difference in 131I biodistribution. The male mice showed
higher levels of activity concentration in the kidneys compared with females, and a distinctly different
curve behavior, i.e. a local maximum at 72 h following 131I administration, with the trend starting after
18 h. In contrast, female mice showed a monotonously decreasing activity concentration over time.
The local maximum after three days was observed for all male cohorts, meaning also for the other
administration series. This curve behavior was also demonstrated in the previous study for 131I
administrations at 12 pm and 4 pm (Andersson, 2015). The prohormone thyroxine (T4) is partially
reduced to triiodothyronine (T3) in the kidneys and liver (Larsen et al., 2012; Gereben et al., 2008;
Bianco et al., 2002), which could explain the rising levels after three days following 131I
administration. What is especially interesting in this context is the observed sex difference for 131I
biodistribution over time in the kidneys, which may imply a physiological difference in iodine
metabolization.
In total, there were statistically significant differences in about 60 % of all investigated time points
when comparing administration at 8 am between male and female mice. When excluding the throat
sample, the thyroid and intestinal content, the number lies close to 80 %. Overall, the female mice had
somewhat increased levels of activity concentration in almost all organs compared with male mice.
This could be explained by the mean weight difference between the sexes. The female mice, having a
mean weight of approximately 20 g, would be given a slightly higher activity-to-mass ratio than the
male mice, having a mean weight of approximately 24 g, i.e. about 20–25 % higher. However, the
number of stable iodine atoms (127I) available in a daily serving of the iodine reduced breeding chow
are about 75000-times the 131I atoms injected (based on an intake of 3 grams of breeding chow and 170
kBq of injected 131I). The difference between the injected iodine atoms and the iodine atoms in a daily
serving is so large, and the deviation of a daily intake of breeding chow is likely to affect more, that a
validating study with the same activity-to-weight ratio would probably not give much different results,
and would not have a great ethical value. A higher activity concentration may instead be a result from
physiological differences in iodine metabolization. This interpretation is supported by the difference in
activity concentrations in the kidneys at time points from 24 h and later following 131I administration.
Although, this interpretation would be harder to account for since physiological differences between
male and female mice are complex and not documented in sufficient detail in the literature. Hence,
these differences would advocate the use of sex-specific biodistribution data when calculating
absorbed dose to certain tissues.
35
This work also showed a higher activity concentration in the thyroid when using standard iodine levels
in animal food. This finding suggests that standard food is to be preferred if high 131I uptake in the
thyroid is desirable. However, this finding is not in agreement with the literature in the sense that the
study of Walinder on this matter indicated that the uptake decreases as the iodine concentration in food
is increased (Walinder, 1971). The study of Walinder did, however, include several lower
concentrations of iodine in animal food than used in this study, ranging from 0.33 μg/g to 0.74 μg/g. It
should be noted that the mice in this work have been given food ad libitum, in agreement with
standard laboratory care, and it was therefore not possible to monitor how much food, and hence stable
iodine, each mouse had ingested during the day. Individual eating behavior may have an even greater
impact in this context than the actual iodine concentration in food, since a deviation of 1 gram from
the estimated 3 grams daily serving of food would give a range of iodine atoms ingested between 50
000–100 000-times the amount of injected iodine atoms. In order to exclude potential bias from
individual food intake, it is advisable to feed the animals in a controlled fashion in future studies, and
concordantly, estimate stable iodine intake more accurately.
The blood, liver and kidneys showed the same trend for normal levels of iodine in food, i.e.
biodistribution curves intersected between 1 and 4 h following injection, which was also statistically
significant at these time points. Moreover, the data points of 1 and 4 h showed that for 131I
administration at 12 pm, the uptake is lower for all of these tissues (with the exception of thyroid)
which then retained the activity concentration to a greater extent. The deviations within the groups of
these tissues are relatively small which renders finding statistical significance. However, this supports
Walinders data that statistically significant differences can be detected with different iodine
concentration levels in food (Walinder, 1971).
Age is another important aspect in these types of studies. The mice used in this work were fairly
young corresponding to young adolescents in man (Dutta & Sengupta, 2015). The results on iodine
uptake in the kidneys compared between male and female mice suggested that physiological
differences play a role in metabolization of iodine. Since physiology changes with age, e.g. the
regression of the thymus in man as children turn into adults (Sand et al., 2007), it is reasonable to
assume that biodistribution data for young mice would not necessarily be the same as for older mice. It
is also reasonable to believe that if mice enter puberty at different ages, as humans do (Abreu &
Kaiser, 2016), the male and female mice could, due to their young age, therefore be in different stages
of biological maturation. During puberty there are numerous of hormones in the body, expressed in
larger quantities than otherwise (Abreu & Kaiser, 2016), and the levels of transcript in mice of e.g.
transforming growth factor-α, TGFα, that is involved in the pubertal onset in mice and rats are
different between the sexes of the same age (Koshibu & Levitt, 2005). Since the effects of TGFα are
many, including e.g. cell migration, cell differentiation and stem cell proliferation it is reasonably to
believe that different stages of maturation could themselves lead to a difference in uptake mechanisms
and activity concentrations. However, the results of this study are still to be considered vital pieces of
information, since a broad data base is necessary when estimating doses from e.g. fallout from nuclear
accidents, especially between children or adolescents of the same age. Age should be considered as an
experimental variable when using biodistribution data, and should also be considered critically in the
clinical setting and for absorbed dose estimation in hazard exposure settings. Further studies on 131I
biodistribution are needed, using a larger age range for both male and female mice.
36
Conclusion and Outlook
The results from these animal experiments demonstrated several sex-specific differences in the
biodistribution and biokinetics of 131I. Consequently, biological sex may be an important variable
when calculating absorbed dose, especially concerning absorbed dose to the kidneys. In this regard,
circadian variation should be investigated in further detail with female mice and not only using male
mice.
In summary, this work validated that time of administration of 131I is an essential factor in
biodistribution studies and that circadian rhythm is an important experimental variable in this context.
Moreover, the biological sex appears to have a significant impact on biodistribution data. Furthermore,
factors such as animal weight and iodine concentrations in animal food should also be considered as
experimental variables and ought to be reported clearly in biodistribution studies and absorbed dose
calculations. The extent of these factors should be further investigated in future work.
37
Acknowledgements
I would like to express my sincerest gratitude to all the people without whom this work would not
have been accomplished.
First and foremost, I would like to thank my supervisors Eva Forssell-Aronsson, Johan Spetz and
Britta Langen. You have been truly inspiring and I am grateful for your support and encouragements,
as well as for including me in the hallway chit-chats.
I would like to thank Ann Wikström and Emman Shubbar for helping with animal-handling and organ
extraction procedures. I hope you enjoyed all our hours in the animal lab as much as I did. I would
also like to thank you both for reminding me of all the children’s songs I thought forgotten, and for
putting up with me singing(?) them to you.
Thank you Johanna Dalmo, Lars Gunnar Månsson and Ylva Surac for supplying me with
radionuclides and Sture Lindegren and Tom Bäck for helping me with the gamma counters and for all
the interesting things you showed me.
I would also like to thank Hana Hameed and Petra Bergström at Central Radionuclide Pharmacy for
helping me with the radioactive waste.
I am also truly grateful for the help, support, comments and perspectives I have received from my
fellow students.
38
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40
Appendix
Biokinetics for comparison with
normal iodine concentrations in food
for mice
Administration at 12 pm
Figure 59: 131I concentration in salivary glands for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 and 4 h. Error bars display
SEM; n=5.
Figure 57: 131I concentration in lungs for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 60: 131I concentration in spleen for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 58: 131I concentration in heart for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 61: 131I concentration in small intestine for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 62: 131I concentration in large intestine for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 h. Error bars display SEM;
n=5.
Figure 63: 131I concentration in stomach for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 and 4 h. Error bars display
SEM; n=5.
Figure 64: 131I concentration in small intestine content
for administrations at 12 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 65: 131I concentration in large intestine content
for administrations at 12 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 66:131I concentration in gastric content for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 4 h. Error bars display SEM;
n=5.
Figure 67: 131I concentration in carcass for
administrations at 12 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 1 and 72 h. Error bars display
SEM; n=5.
Administration at 4 pm
Figure 68: 131I concentration in lungs for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 69: 131I concentration in heart for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 71: Spleen 131I concentration in spleen for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 72: 131I concentration in small intestine for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 73: 131I concentration in large intestine for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 70: 131I concentration in salivary glands for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 74: 131I concentration in stomach for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 75: 131I concentration in small intestine content
for administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 76: 131I concentration in large intestine content
for administrations at 4 pm for normal and reduced
Figure 77: 131I concentration in gastric content for
administrations at 4 pm for normal and reduced
iodine concentration in food. No statistical significance
was found. Error bars display SEM; n=5.
Figure 78: 131I concentration in carcass for
administrations at 4 pm for normal and reduced
iodine concentration in food. Statistical significance
(p<0.05) was found after 72 h. Error bars display SEM;
n=5.