The DAGMAR Study. D-vitamin And Graves' Disease; Morbidity And Relapse Reduction: A Randomised, Clinical Trial.
Phase: NA
Conditions: Graves' Disease
Sponsor: University of Aarhus

Effect of vitamin D supplementation on Graves’ disease:  The DAGMAR trial 
 
Final protocol, version 8, May 11, 2017 [Danish]. 
First approval January 25, 2013. 
English translation August 16, 2022 
ClinicalTrials.gov Identifier: NCT02384668 
 
 
This supplement contains the following items:  
1. Final protocol, summary of changes (Table 1+2).  
2. Original statistical analysis plan, final statistical analysis plan. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Effect of vitamin D supplementation on Graves’ disease:  The DAGMAR trial 
Table 1 
Approved changes to original study protocol prior to study commencement 
November 27, 
2014 
Changes to study design 
From three to two study groups, equally 
randomized. 
Added exclusion criteria: pregnancy, abuse of 
alcohol or narcotics. 
The investigation program for a sub-cohort is 
extended with a visit at three months. 
Permission to expand planned biochemistry 
including flow-cytometry and to perform 
genetic investigations 
 
January 5, 2015 
Homepage for study participants. 
Number of questionnaires during study period 
increased to nine 
 
January 7, 2015 
Randomization by participating site. 
Collected blood volume with each sampling 
increased from 50 to 80 ml. 
HbA1c, glucose, and cholesterols analysed 
immediately and not as parts of batch analyses.  
 
 
Table 2 
Overview of approved changes to study protocol after study commencement 
 
May 15, 2015 
Case control study established with 
permission to recruit euthyroid controls 
(N=55) 
Frequency of questionnaires increased 
No changes concerning the 
primary outcome of DAGMAR 
July 31, 2015 
Permission to recruit euthyroid controls 
through social media 
Homepage created explaining about the 
DAGMAR study 
No changes concerning the 
primary outcome of DAGMAR 
August 5, 2015 
Permission to increase sampled blood 
volume at baseline, three, and nine months 
with 50 ml. 
No changes concerning the 
primary outcome of DAGMAR 
May, 11, 2017 
For a sub group of participants, extensive 
physical examination was performed at 
baseline, three, and nine months. Permission 
obtained to repeat examination at the end-
of-study visit.  
Permission to replace participant drop-outs. 
Upon the inclusion of participant number 
260, drop-outs will be counted and 
recruitment will continue until a similar 
number of new participants has been 
No changes concerning the 
primary outcome of DAGMAR 
 
 
 
Ensure sufficient power in 
primary analysis. 

enrolled or until the end of 2017. 
Replacement-participants will receive same 
randomization as drop-outs 
 
Importance of vitamin D supplementation in patients with Graves' disease: 
a randomized controlled trial 
Introduction 
Graves' disease is one of the most frequent causes of hyperthyroidism. The disease is 
characterized as an autoimmune disorder, caused by stimulating antibodies directed against the 
thyroid gland's TSH receptor. The disease is preferably treated medically, but with a high risk of 
relapse, as approx. 50% of patients experience a relapse of the disease, often within one year after 
the end of treatment. 
Vitamin D has been found to be important for the immune system and to be associated with other 
autoimmune disorders. The vitamin D level in the blood has been shown to be lower in patients 
with Graves’ disease than in healthy individuals. With this study, we want to investigate how 
supplementation with vitamin D affects the prognosis in patients with Graves' disease. 
Background 
Graves' disease 
Hyperthyroidism is a frequent disease in Denmark with approx. 5000 new cases per year(1). 
Approximately 40% of cases is caused by Graves' disease (GS), an autoimmune disease with 
potential reversibility. GS is characterized by the presence of autoantibodies (thyroid-stimulating 
immunoglobulin (TRAb)) directed against the TSH receptor, which cause increased production of 
thyroid hormones. Triiodothyronine (T3) is the active hormone. All the body's cells have nuclear 
receptors T3, and T3 regulates the transcription of many different genes. Hyperthyroidism 
therefore affects all organs in the body. 
 
T3 increases beta-adrenergic receptor activity, and cardiovascular symptoms are often 
predominant. Atrial fibrillation is one of the most serious complications of the disease, and is 
predominantly seen in patients over 40 years of age (2). Hyperthyroidism in general is associated 
with increased total mortality and cardiovascular mortality (3,4). 
 
Bone turnover is increased in hyperthyroidism, although the bone loss assessed by bone mass 
density measurement has been shown to be recoverable within a shorter number of years(5,6). 
During the hyperthyroid phase and for a number of years afterwards, the fracture risk in 
hyperthyroidism is increased (6). 
1.25 OH2 vitamin D is reduced as part of hyperthyroidism (7). 
Whether bone recovery can be accelerated by vitamin D treatment of GS is unknown. A newer and 
more advanced method for assessing bone density is high resolution peripheral quantitative 

computed tomography. There are no studies yet that use this method to describe the bone 
turnover in GS. 
 
In GS, muscle function is affected, described as proximal myopathy (8). The patients' catabolic 
state is important. A possible significance of the underlying immunological disorder and of a low 
vitamin D level is unknown. Myopathy symptoms may resemble the symptoms of vitamin D 
deficiency. The degree of this muscle weakening and its possible normalization are sparsely 
described in the literature. There are a number of case series where the patients' muscle 
weakness improves over 4-5 months after starting anti-thyroid medication, and then reaches a 
plateau. How pronounced the degree of muscle weakness is compared to healthy people has not 
previously been described. 
Applanation tonometry is a method used to assess vascular stiffness (9), which has been shown to 
be predict total and cardiac mortality and cardiac morbidity in an unselected population group 
(10). The method is well validated. Applanation tonometry has been used in very few studies on 
patients with GS and the studies suggest that vessel stiffness is increased in the hyperthyroid 
phase, but decreases again as the euthyroidism is restored. 
Generally, GS is treated with anti-thyroid drugs for 1.5 years, after which the treatment is 
discontinued. However, recurrence is frequent and occurs in approx. 50% (11). 
 
D vitamin 
Vitamin D deficiency occurs with high frequency in Denmark. In patients with Graves' disease, 
lower vitamin D levels have been found compared to the background population (12) and 
compared to patients with other causes of hyperthyroidism (13). Both in vitro studies and animal 
experimental studies suggest that vitamin D affects the course of GS.  
 
The effect can be caused by both a direct effect on gld. thyroid as of an effect on the autoimmune 
response in GS. 
 
Active vitamin D (1,25(OH)2D) exerts its effect through a binding to the vitamin D receptor (VDR), 
which belongs to the group of nuclear hormone receptors. Binding of the ligand to VDR leads to 
conformational changes in VDR which facilitate a heterodimerization with the retinoid X receptor 
(RXR), after which the complex can bind to vitamin D responsive elements (VDRE) on the DNA 
strand and thereby affect gene transcription (14). 
 
The vitamin D receptor is identified in thyroid follicular cells and 1,25(OD)2D has been shown to 
dose-dependently inhibit both TSH-stimulated iodine uptake and growth of follicular cells from 
rats (15,16). 
Hyperthyroidism is associated with symptoms and findings that also occur with vitamin D 
deficiency, including decreased muscle strength and decreased bone mineral density. It is 

unknown to what extent this can be attributed to the fact that vitamin D levels are often reduced 
in this group of patients. 
Vitamin D has also been shown to be important for the development of autoimmune disease. In 
clinical epidemiological studies, a lack of vitamin D has been shown to be associated with an 
increased incidence of various autoimmune diseases, such as multiple sclerosis and type I diabetes 
mellitus (17). 
VDR has been detected in most of the cell types of the immune system, including in antigen 
presenting cells such as macrophages, dendritic cells and CD4+ and CD8+ T-lymphocytes (18-20). 
Both in vivo and in vitro studies have shown that 1,25(OH)2D through its binding to VDR is able to 
affect the function of antigen presenting cells (21), including inhibiting differentiation, inhibiting 
IL12 synthesis, increasing IL10 synthesis, downregulating the synthesis of co-stimulatory cytokines, 
increasing the apoptosis tendency and inducing immunological tolerance (21-26). Of particular 
importance is probably the ability of 1,25(OH)2D to inhibit the synthesis of IL-12 simultaneously 
with an increased synthesis of IL-10, thereby reducing T-cell activation (27,28). In addition, 
1,25(OH)2D has been shown to affect the differentiation and proliferation of B lymphocytes (29). 
In addition to counteracting the activation of T cells, studies suggest that vitamin D can 
downregulate the expression of HLA-II molecules on the follicle cells in gld. thyroid (30) and 
thereby possibly reduce the risk of developing an autoimmune reaction. 
 
Several studies have looked at the association between polymorphisms in VDR and the risk of 
developing GS. The results from most of these studies have recently been combined in a meta-
analysis which showed that among Asians the risk of developing GS is significantly associated with 
the ApaI, BsmI and FokI polymorphisms, while a similar association did not occur in Caucasians 
(31). In an animal experimental study in which hyperthyroidism was induced in mice fed either a 
vitamin D-poor or vitamin D-rich diet, no effect was found on plasma levels of TSI, but vitamin D 
deficiency led to persistent hyperthyroidism ( 32). 
There are very few studies of the possible clinical significance of vitamin D on the course of GS. 
In an unblinded study, 30 patients with GS, in addition to conventional anti-thyroid treatment with 
methimazole, were randomized to a daily supplement of 1.5 micrograms alfacalcidol or no 
alfacalcidol (33). After 24 weeks of treatment, the mean dose of methimazole was similar in the 
two groups. Treatment with alfacalcidol led to a significantly lower plasma concentration of total 
and free T3 and thyroxin (T4), while TSH was significantly higher in those who had received 
treatment with alfacalcidol. There was no difference in the plasma level of TRAb. Treatment with 
alfacalcidol led to a significant decrease in the plasma concentration of alkaline phosphatase (33). 
However, the clinical use of 1,25(OH)2D has been limited by hypercalcemic side effects. The fact 
that the activation of vitamin D from 25-hydroxyvitamin D to 1,25(OH)2D has now been shown to 
take place not only in the kidneys but also locally in a number of tissues, including the cells of the 
immune system, has increased interest in vitamin D's possible immunomodulatory properties (34). 
In an unpublished review of 90 medical charts from patients with GS treated at the Regional 
Hospital Silkeborg or the Department of Internal Medical and Endocrinology, Aarhus University 

Hospital, a low plasma concentrations of 25OHD are associated with a significantly increased risk 
of recurrence of GS after cessation of anti-thyroid treatment. Compared to P-25OHD < 45nmol/l, a 
concentration > 45nmol/l was associated with a 60% reduced recurrence risk (RR 0.40; 95% CI, 
0.22-0.71), while a P-25OHD concentration above compared to below 60 nmol/l was associated 
with a 21% (RR 0.79; 95% CI, 0.50-0.99) decreased recurrence risk. In contrast, there was no 
significant effect at a threshold value of 80 nmol/l (RR 1.08; 95%CI, 0.46 – 2.32). The study also 
showed a non-significantly reduced relapse risk in patients who used vitamin D supplements 
compared to those who did not take such a supplement (RR 0.71; 95%CI, 0.42 – 1.21). In the 
population, the overall recurrence rate was 47%. 
 
The following describes the randomized study i). 
The controlled cohort study ii) is described separately on page 18) 
 
Purpose 
To clarify the importance of vitamin D for the course of GS, as we want to test the following 
hypotheses: 
 
Primary hypothesis. 
Treatment with vitamin D supplementation will reduce the number of treatment failure of Graves' 
disease in the intervention group by 33% compared to the placebo group. 
 
Secondary hypotheses. 
Treatment with vitamin D supplements will accelerate the normalization of bone turnover and 
muscle balance function and shorten the time to symptom freedom. 
Vitamin D supplementation reduces vascular stiffness in patients with GS. 
 
Design 
The study is conducted as a double-blind, randomized and placebo-controlled trial with a clinical 
follow-up time of up to three years. The participants are patients with newly diagnosed Graves' 
disease recruited from the hospitals in the Central Denmark Region. The study proceeds with two 
parallel study groups. The participants in each of the study groups take 1 tablet daily. Each tablet is 
either placebo or 70 micrograms of cholecalciferol. The placebo and cholecalciferol tablets are 
identical in appearance. 
All subjects are randomized to one of the following two groups: 
Group 1: 1 placebo tablet daily (50% of participants) 
Group 2: 1 tablet of cholecalciferol of 70 micrograms (50% of the participants) 
The study proceeds as shown in Figure 1. 
After randomisation, a smaller cohort will also undergo a study program as outlined below. 
Register-based follow-up studies will be carried out after 5 and 10 years on the entire cohort. 

When 260 subject have been randomized, drop-outs will be replaced until the end of 2017 when 
the inclusion period ends. Study closure is when the last participant has completed the study 
program as described in figure 1, alternatively has stopped participation in the study. 
The individual participant stops taking the project medication at the latest 12 months +/- 1 month 
after the end of anti-thyroid treatment - possibly earlier (cf. section on withdrawal). For the 
individual participant, the maximum length of treatment will be 36 months. 
A control cohort of 55 healthy sex- and age-matched individuals is established. 
 
Primary outcome measures: 
The primary effect measure is assessed based on whether the participants have benefited from 
the treatment as assessed by: 
• Treatment success: No signs of hyperthyroidism 12±1 months after the end of anti-thyroid 
treatment. Criteria for treatment failure not met. 
• Treatment failure is defined as: referral to radioiodine therapy, surgery, failure to taper off anti-
thyroid treatment after a maximum of 24 months of treatment, or recurrence of hyperthyroidism 
within one year after cessation of anti-thyroid treatment. 
 
Secondary outcome measures: 
 
• Relapse after ending anti-thyroid treatment, defined as recurrence of hyperthyroidism within 
one year after cessation of anti-thyroid treatment (TSH<0.1) or new prescription of anti-thyroid 
drugs in the same period. 
• Plasma TRAb level 
• Biochemical vitamin D status 
• Dose-response relationship with supplementation with vitamin D 
• Occurrence of TAO/GO 
• Occurrence of side effects to anti-thyroid treatment (agranulocytosis, drug rash, hepatitis, 
vasculitis) 
• Weight changes 
• Quality of life (SF36v2) + ThyPRO. 
• Effects on muscle and balance function 
• Effects on calcium homeostasis and bone turnover 
• Effects on blood pressure and arterial stiffness measures 
• Heart rate variability 
• Frequency of infection (incl. consumption of antibiotics) 
• Hospital admissions / number of doctor visits 
 
Statistical analysis plan (Original) 
The data analysis will be performed in accordance with "guidance on Statistical Principles for 
Clinical Trials" (IHC guidance E9). Primarily, differences between the placebo and vitamin D groups 

are analysed. The primary effect measure is assessed based on whether the participants have 
benefited from the treatment as assessed by: 
• Treatment success: No signs of hyperthyroidism 12±1 months after the end of anti-thyroid 
treatment. 
• Treatment failure is defined as: referral to radioiodine therapy, surgery, failure to taper off anti-
thyroid treatment after a maximum of 24 months of treatment, or recurrence of hyperthyroidism 
within one year after cessation of anti-thyroid treatment (TSH<0.1 or new prescription of anti-
thyroid drugs). 
Predefined subanalyses: 
We will investigate the effect of vitamin D supplementation in relation to the vitamin D level at 
inclusion. Furthermore, we will examine the effect of vitamin D supplements on the group of 
participants who have not used supplements with vitamin D on their own initiative. 
In a pre-defined subanalysis, subjects who continue anti-thyroid medication beyond 24 months 
due to Graves' orbitopathy will be excluded. 
Differences between groups will be assessed with Fisher's Exact test or chi square test. 
Other effect measures are calculated with t-test for two independent groups or a corresponding 
non-parametric test, depending on the distribution of the material. A significance level of 0.05 will 
be used. Primarily, data will be analyzed according to the "intention-to-treat" principle, although 
secondarily an "on treatment" analysis will be carried out, where the degree of compliance is 
corrected. 
The intention-to-treat population consists of all participants who have been randomized to the 
study and who have received at least one dose of the study medication. In the event of missing 
data in the Intention-to-treat analysis, "last value carried forward" will be used. In the final 
protocol, there will be an account of unused or omission of inauthentic data, as well as an account 
of reasons for any deviations from the originally planned statistical analyses. 
It is planned to carry out interim analyzes when the sub-cohort of 100 subjects (which includes 
mainly from Aarhus University Hospital and Regional Hospital Silkeborg) has completed the entire 
study program. This is to ensure that this population can be included in a data analysis which will 
form the basis of a PhD thesis 
 
Study population 
260 patients, men and women, with newly diagnosed GS where anti-thyroid treatment is planned. 
Potential study participants are recruited from hospitals in Central Denmark Region in connection 
with patients' attendance at the outpatient clinic. 
 
Inclusion criteria 
• Newly diagnosed Graves' disease verified by TSH below the reference range and thyroxin and/or 
triiodothyronine (T3 and/or T4) above the upper reference range. 
• Age 18 years or older 
• Elevated plasma level of thyroid stimulating immunoglobulin (TRAb) 

• Planned or started (< 3 months) medical anti-thyroid treatment 
• Speaks and reads Danish. 
• Written consent after oral and written information 
 
Exclusion criteria 
• Daily intake of vitamin D supplements of more than 10 micrograms, which the participant wishes 
to continue. 
• A previously diagnose of hyperthyroidism 
• Planned treatment with radioactive iodine or thyroidectomy 
• Chronic granulomatous disease 
• Significant hypercalcemia (ionized plasma calcium > 1.40 mmol/l) 
• Reduced kidney function (eGFR < 45 ml/min) 
• Regular intake of medication with known immuno-modulating effects, including systemic 
glucocorticoids (>5mg/day for 3 months), imurel, methotrexate, TNFα-inhibiting agents. 
• Active malignant disease 
• Alcohol abuse resulting in the need for treatment or hospitalization 
• Lack of willingness or desire to participate in the study 
• Abuse of euphoric substances 
• Pregnancy at the time of inclusion 
 
Patients who meet all inclusion criteria and none of the exclusion criteria will be invited to 
participate in the study. 
Withdrawal 
Participants will be asked to cease treatment with the project medicine when an exclusion criteria 
is met: 
• Changed treatment strategy for hyperthyroidism (radioiodine therapy, surgery). 
• Hypercalcemia or reduced kidney function 
• Onset of diseases or initiation of medication intake as listed under exclusion criteria (except 
systemic glucocorticoid treatment due to thyroid-associated ophthalmopathy). 
• Recurrence of hyperthyroidism after discontinuation of anti-thyroid treatment. 
 
The investigator can stop the treatment for a participant if this is deemed necessary for the 
participant's safety. 
In case of pregnancy, the participant can continue with the project medication. Participants in the 
intensively studied sub-cohort (n=100) will be excluded from further studies during pregnancy, 
due to the radiation risk associated with the scans. However, the participants can continue in the 
main study (n=260). 
 
Population size 
Vitamin D and relapse risk in Graves' disease 

There are no previous human studies with treatment with vitamin D3 supplements. In a medical 
record review of 90 medical records from patients with GS treated at Region Hospital Silkeborg or 
Aarhus University Hospital, it was found that P-25OHD > 45 nmol/L was associated with a 60% 
reduced recurrence risk compared to P-25OHD < 45 nmol/L ( RR 0.40, 95% CI 0.22–0.71). The 
study also showed a non-significantly reduced recurrence risk in patients who used vitamin D 
supplements compared to those who did not take such a supplement (RR 0.71; 95% CI 0.42-1.21). 
Power calculation: In order to show a difference in relapse risk for intervention with vitamin D 
compared to placebo of 33% (from 50% to 33%), a total of 260 participants must be included in 
the study, with 50% randomized to placebo and 50% to supplementation with vitamin D. (α=0.05; 
β=0.20). Drop-outs will be replaced by the inclusion of new participants: Upon the inclusion of 
participant no. 260, drop-outs will be counted and replaced by the inclusion of new participant 
until the end of year 2017 when the Inclusion Period ends. 
 
Vitamin D and bone turnover, muscle function tests and cardiovascular indices 
Bone turnover. 
Based on the experience from other intervention studies with vitamin D, it is considered 
reasonable to expect that treatment with vitamin D will cause at least a 2% difference between 
the intervention and placebo groups. With measurements carried out on the lumbar spine (L1-L4), 
where it can be assumed that the mean BMD is 0.920 g/cm2 (SD 0.03 g/cm2), the required 
number of participants can be calculated - as it is desired to be able to demonstrate a 2% 
difference between the 2 groups - based on the following formula (α= 0.05; C2α = 1.96. β= 0.20; 
Cβ = 0.84): 
N = (C2α + Cβ)2 x (2x SD/ (Mean x 0.02))2 = 72 participants. 
Since 100% compliance cannot be expected and since some of the participants may drop out 
during the study period, it is reasonable to increase the number of participants. Thus, a total of 
100 participants will be included, randomized equally to either treatment with placebo or vitamin 
D supplementation. 
There are no applicable data for strength calculation for the effect of vitamin D supplementation 
on muscle function tests and cardiovascular indices in patients with Graves' disease. 
Pulse wave velocity. A recent meta-analysis has shown that an increase in pulse wave velocity of 1 
m/s leads to a 15% increase in total and cardiovascular mortality41. With 50 patients in each 
group, and given that the standard deviation in each group is 1.5 m/s, it will be possible to detect a 
difference of 1 m/s with a power of 92% (α=0.05). 
 
Intervention and randomization 
 
Vitamin D supplements 
In the study, vitamin D tablets of 70 µg and a corresponding placebo are used, which are supplied 
free of charge by Orkla Health. 

The project medication is packaged and labeled according to the Danish Medicines Agency's 
regulations by the Hospital Pharmacy at Aarhus University Hospital. 
Vitamin D supplements and placebo tablets will be packed in quantities corresponding to 3-9 
months' consumption and handed out to the participants upon attendance or forwarded by 
agreement at the regular contacts. (figure 1) 
Procedures for randomization 
The hospital pharmacy, Aarhus University Hospital, will carry out block-randomization, so that for 
every 4-16 participants included, there will be an equal number in each of those treated with 
either placebo or vitamin D supplementation. The exact size of the blocks will be unknown to the 
investigator. The participants are allocated consecutively. Site-randomization will be used to 
ensure equal randomization of study subjects at each participating department. 
 
For each randomization number, the pharmacy supplies an opaque, sealed envelope numbered 
with the study's randomization number. The envelope can be opened if there is an urgent need to 
obtain information about which study medication the participant in question is receiving, as the 
envelope contains information about the contents of the tablets that the subject is taking. In this 
case, this is noted in the CRF, including the reason why the randomization code for the patient in 
question has been broken. The envelopes will be stored at Aarhus University Hospital. 
The hospital pharmacy keeps a randomization list indicating the treatment group for all 
randomization numbers. The license is handed over to the Investigator when the experiment has 
been completed. 
Data collection 
The studies are carried out as outlined in Figure 1. 
Baseline information, randomization and initiation of project medicine must begin within three 
months of anti-thyroid drug treatment. 
For a sub-cohort of 100 patients (the first 100 subjects recruited from the outpatient clinics at the 
Medical Endocrinology Department, Aarhus University Hospital as well as interested participants 
from the regional hospitals in the central region) baseline information and the study program will 
be collected within four weeks after diagnosis of GS. Randomization and start-up of the project 
medication will take place immediately afterwards for this group. 
In accordance with section 43, subsection 1 of the Health Act, participants’ consent will be 
obtained to, via the medical charts, to collect information about treatment choice and course of 
GS, duration of treatment, occurrence of complications and side effects, including Graves' 
orbitopathy, side effects to the anti-thyroid treatment, as well as information about infections, 
other medical treatment, new illnesses including contact with a doctor and admissions. 
 
Baseline 
Based on medical record information, interview and questionnaire, the following will be registered 
in the CRF: 

Age, conditions mentioned under criteria for participation in the study, current and previous 
illness, history of fractures, occurrence of fractures and illness in 1st generation relatives, 
consumption of dairy products, coffee, tea, tobacco and alcohol. Diet and sun-bathing habits. 
Medical history, including use of calcium and/or vitamin D supplements, vitamin supplements, 
dietary supplements and use of natural preparations. Use of hormonal contraceptives. The 
participants will be requested that the Investigator may obtain information from any previous 
admissions. Participants who have the opportunity to do so will be asked to complete an 
electronic version of the questionnaires via the internet (the website will be created in relation to 
the project being launched). 
Follow-up 
Up to nine times during the course of study, as indicated in figure 1, questionnaires on health- and 
disease-specific quality of life will be repeated (appendices 9, 10 and 11). Changes in treatment 
strategy, occurrence of side effects, complications and unintended events will be recorded based 
on interview and medical record information. Information on other illness during the study period 
is obtained from patients’ medical chart. After five and ten years, register-based follow-up studies 
are carried out including vital status and discharge diagnoses based on data from the CPR register 
and the National Patient and Cause of Death Register. 
 
Biochemistry 
Blood samples will be taken as shown in Figure 1. At each blood sample, approx. 80 ml will be 
taken. For participants recruited from Aarhus University Hospital and for participants in the 
intensive examination program, 130 ml of blood will be taken during the first three of a total of 
four blood samplings. This will apply to 50 participants. The plasma concentration is determined 
immediately for the following parameter: albumin, ion calcium, phosphate, creatinine, 
magnesium, thyrotropin (TSH), T3, and T4. HbA1c, glucose and cholesterols. As part of the initial 
clinical investigation, TRAb is also determined. 
Plasma will be frozen (-80 °C) at each blood sampling for later batch analysis of: TRAb, PTH, 
25OHD, 1,25(OH)2D, vitamin D binding protein (DBP), bone-specific alkaline phosphatase (BSAP), 
osteocalcin and N-terminal extension peptide of procollagen (P1NP), CTX and antibodies. 
Furthermore, adiponectin, leptin, insulin. Markers of the renin-angiotensin-aldosterone system. 
Organ-specific antibodies, including TPO antibody, transglutaminase antibody IgA, total IgA, 
deamidated gliadin peptide antibody IgG, and markers of immune system activity, including high 
resolution CRP and immunoglobulins. 
Furthermore, selected inflammatory and pro-inflammatory markers as well as adipokines. DNA is 
taken from leukocytes for genetic studies, see below. 
Flow cytometry will be performed on fresh blood at the Department of Clinical Immunology, 
Aarhus University hospital, where lymphocyte subpopulations and cell differentiation will be 
examined. In cell culture studies, dendritic cells and their co-stimulatory factors will be examined. 
In addition, measurement of soluble HLA-G as well as functional studies of the regulatory T cells' 
suppressor function will be performed. 

 
Surveys 
A sub-cohort of 100 test subjects will complete the study program below, see figure 2. Separate 
information material has been prepared for this, see appendix 5. 
Baseline examinations will be carried out within 14 days of referral. 
Examinations at three and nine months will be carried out with a precision of +/- four weeks. 
The examinations (including biopsies) will be repeated in connection with the end-of-study blood 
test after a renewed information interview and informed consent. Only participants who have 
previously participated in this research program and who continue to participate in the DAGMAR 
study will be able to participate. Pregnancy is an exclusion criterion. The background for the 
repeated examinations is that at the nine month investigations, especially the muscle strength 
testing, there are signs of continued improvement. Long-term clinical studies in Graves' disease on 
these aspects are currently limited or non-existent. It is therefore unclear when final restitution 
will be achieved, which these studies will be able to help shed light on. 
 
Osteodensitometry 
Bone scans will be performed as a "Dual-Energy X-ray absorptiometry" (DEXA) scan with a Hologic 
QDR Discovery scanner. BMC and BMD will be measured in the lumbar spine (L1-L4), the hip 
region (femoral neck, trochanter, Ward's triangle and intertrochanteric region), the forearm 
(proximal, middle 1/3 and ultra-distal) and the whole body. Furthermore, body composition will be 
determined. 
 
HRpQCT scan 
High resolution peripheral quantitative computed tomography (HRpQCT) scan of the distal radius 
and distal tibia (Xtreme CT, Scanco Medical AG, Bruüttisellen, Switzerland). The scans are 
performed on the non-dominant side. For a person who is right-handed, the scan is performed on 
the left arm and leg and vice versa. In the case of a metal implant or previous fracture in an area 
that is desired to be scanned, the scan is performed on the opposite side. 
Muscle function and balance studies 
Isometric voluntary muscle strength is measured on the dominant leg with a dynamometer 
(Meititur Ltd, Finland) that can measure isometric muscle strength in flexion and extension with 
the knee bent at an angle of 60° and 90°. In a similar way, muscle strength in the hand (grip 
strength) is measured. 
Balance function is determined by measuring dynamic stability on a stadiometer (Meititur Ltd, 
Finland). In addition, two smaller physical tests are carried out, the "Get up and sit down test" 
(where the participants have to get up and sit on a chair ten times) (35) and the "Timed Up and Go 
test" (where the participants have to get up from a chair, walk three meters and turn around to go 
back and sit on the chair again) (36). 
 
Arterial stiffness and blood pressure 

Arterial stiffness is investigated with the commercially available tonometry-based equipment 
SphygmoCor® and Arteriograph24, determining aortic pulse wave velocity (APWV) and central 
aortic pressure (CAP). The tests are non-invasive, well-validated and without risks or discomfort. 
Expected time consumption is approx. 45 min. With the Arteriograph24, 24-hour blood pressure 
and 24-hour tonometry measurements are also determined. 
Muscle and fat biopsies 
Thirty participants are requested to have a muscle and fat biopsy performed at the time 0+3+9 
months with a precision of +/- 4 weeks. 
 
A biopsy will be taken laterally from the thigh, as local anesthesia with lidocaine and adrenaline is 
applied to the skin, subcutaneous tissue and muscle fascia under a sterile procedure. A minimal 
incision is then made in the skin and fascia, after which 100 mg of muscle tissue can be taken from 
the vastus lateralis muscle with a special needle (Bergstrøm's needle). The tissue is subsequently 
frozen in liquid nitrogen. The muscle biopsy is analyzed for protein expression using western 
blotting and subjected to gene expression analysis using quantitative PCR (qPCR). Furthermore, 
fiber type and cross-sectional area, myosin heavy chains including their relative distribution, 
various markers for and actors in muscle contractility are investigated. 
 
The fat biopsy is taken after disinfection and local infiltration analgesia from the abdominal 
subcutaneous fat depot lateral to the umbilicus. Using a suction needle, approx. 1 gram of adipose 
tissue. The tissue is immediately frozen in liquid nitrogen and stored at -80 C until analyses. The 
adipose tissue will be examined for adipocytes and their content of vitamin D metabolites and 
vitamin D receptors as well as selected markers of inflammation and proinflammation and other 
markers of immunological activity. 
 
Genetic determinants ALL 
Polymorphisms in genes of importance for the risk of developing GS / the risk of relapse after 
treatment will be investigated. This will be carried out as a "genome wide search" where all 
polymorphisms are identified including polymorphisms in the vitamin D receptor (VDR), Group-
specific component (vitamin D binding protein; GC), CTLA4 (CytotoxicT-lymphocyte-associated 
protein 4), Interferon inducedwithhelicase C domain 1 (IFHI1), Thyroglobulin gene (TG), Thyroid 
stimulating hormone receptor (TSHR). 
Gene analyzes will be made that can reflect specific up- and down-regulation of different parts of 
the inflammatory response. 
 
Data processing 
Case record forms (CRF) 
For each participant, a case record form (CRF) is kept, on which the date of the examinations that 
the participant has undergone, medication that has been dispensed/returned, deviations in the 
treatment, side effects and incidents are stated. In the case of "drop-outs" and "withdrawals", this 

will be noted and explained in the CRF, including the time of cessation of intake of study 
medication. 
All subsequent data processing takes place at the study site. 
Investigator allows direct access to source data/documents (including patient records) during 
monitoring, auditing and inspection from the Research Ethics Committee for Central Region and 
the GCP unit for Aarhus University Hospital and its partners. The participants are made aware of 
this and specific consent is obtained in the form of a power of attorney. 
All information obtained will be handled in accordance with the Act on the Processing of Personal 
Data. 
 
Research biobank 
A research biobank will be set up where plasma taken from each subject will be stored at -80 
degrees Celsius. The material will be analyzed as described under "biochemistry". 
After the end of the experiment, remaining material will be transferred to a biobank, where it will 
be stored for up to 15 years before being destroyed. Participants will be asked to consent to this. 
The biobank will be notified to the Danish Data Protection Authority in accordance with applicable 
legislation. 
 
Medicine accounting 
The investigator will keep careful records of who the study medication has been delivered to, incl. 
date and amount of medicine dispensed. In addition, the amount of returned medication will be 
recorded. In each participant's CRF, the quantity, batch number, and date of dispensing of study 
medication are noted. 
For each participant, treatment compliance will be calculated as the difference between the 
number of dispensed and returned doses. Regardless of degree of compliance, participants will be 
encouraged to continue in the trial. If a participant stops taking the project medicine during the 
course of the study, the time of cessation of taking the study medicine will be noted together with 
the reason why the participant has stopped taking the medicine. However, the participants will be 
asked to continue to come to planned control visits, which will improve the quality of subsequent 
intention-to-treat analyses. 
 
Risks, side effects and inconvenience 
 
Risks 
In several studies, supplementation with vitamin D has been shown to be largely harmless. In one 
study, it was thus found that a daily supplement of 250 micrograms over 3 months did not lead to 
side effects, including that no signs of hypercalcemia were found as a result of the treatment [33]. 
There are no epidemiological data for treatment with high doses of vitamin D in patients with 
Graves' disease. In the literature, hypercalcaemia has been described in relation to 
supplementation with vitamin D in granulomatous diseases such as sarcoidosis, tuberculosis and 

silicosis, which is why patients with these diseases cannot participate in the trial. Symptoms of 
hypercalcemia include constipation, headache, arrhythmia, confusion, and dehydration with 
polyuria. Hypertension can also occur with overtreatment. 
During scanning, a total radiation dose of 0.9 mSV is applied to each patient, which corresponds to 
approx. 90-120 days of normal background radiation. Exposure to this amount of radiation can 
theoretically be assumed to increase the lifetime risk of dying from cancer by 0.01%; from approx. 
25% to 25.01%. 
When participating in the additional examination program in connection with the end-of-study, 
each patient will be exposed to a total radiation dose of 0.45 mSv, which corresponds to approx. 
45-60 days of normal background radiation. Theoretically, this radiation dose must be assumed to 
increase the lifetime risk of dying from cancer by 0.005%. 
Pregnancy and breastfeeding 
It has been shown in a recent intervention study that vitamin D supplements in doses up to 4000 
IU/day (100 micrograms) are non-toxic for mother and fetus (37). The pregnant women 
simultaneously receive regular vitamin supplements including 10 micrograms of vitamin D. No 
hypervitaminosis D was found, and no adverse events or other events were recorded. In 
breastfeeding mothers, doses up to 164 micrograms/day have been shown to be safe for the 
breast-fed child (38). 
Pregnant and breastfeeding women will therefore be able to continue treatment with project 
medicine. However, pregnancy and breastfeeding will result in exclusion from the subcohort study 
due to the radiation risk associated with DXA and HRpQCT scanning. 
 
Safety measures 
Upon inclusion, all test subjects will be informed orally and in writing about any symptoms of 
hypercalcaemia, and will be asked to contact the investigator immediately. The investigator will, 
by continuous contact with the test subjects, every 3-6 months, inquire about side effects and 
possible symptoms of hypercalcaemia. If this is suspected, the subject will be requested to have a 
blood test carried out as soon as possible to determine serum creatinine and serum ionized 
calcium. Persistently elevated s-calcium or s-creatinine, which does not decrease on repeated 
checks, when other possible calcium and vitamin D are discontinued and the patient is 
biochemically well treated for his Grave's disease, will lead to discontinuation of study medication 
and exclusion from the study. If the hypercalcemia persists thereafter, the patient will be offered a 
referral for investigation for hypercalcemia if necessary. 
In the toxic phase of Graves' disease, mild hypercalcemia is a frequent and transient phenomenon 
and in this phase, elevated values will be acceptable if they show a stagnant/declining trend in a 
repeated control blood test: The subject will be asked to continue the project medication and 
simply repeat ionized calcium and p-creatinine after another week and as needed. 
No biochemical abnormalities are expected as a result of the treatment. Should participants 
develop symptoms compatible with hypercalcemia (thirst, polyuria, dehydration, confusion), they 

will be examined for this in accordance with current clinical guidelines. If hypercalcemia is found 
to be the cause of the symptoms, the treatment will be discontinued. 
Participants are advised not to start supplemental treatment with vitamin D, in addition to the 
supplement they may receive through the project medication. Participants who, despite this, start 
taking a supplement of more than 10 micrograms/day will be excluded. Participants will be asked 
to inform the Investigator if they are put on medical treatment or start taking nutritional 
supplements or using herbal remedies. 
In relation to the subjects' participation in the study, the Investigator will ensure that the 
participants are informed about and receive/are referred to relevant treatment for 
diseases/conditions that arise during the study period, including that any side effects to the 
treatment are handled/treated in the best interest of the trial participant. 
 
Disadvantage 
Some of the participants may find it uncomfortable to have a blood test. There is a minimal risk of 
infection at the injection site. During blood sampling, a total of around 320 ml of blood will be 
drawn for participants from the regional hospitals and 470 ml of blood for participants in the 
intensive examination program and for participants recruited from Aarhus University Hospital, 
which is not expected to cause discomfort. 
The local anesthetic for the fat and muscle biopsy may be associated with pain, and there may be 
tenderness in the area afterwards. The muscle test will leave a small scar on the thigh and the fat 
test may cause a bruise. There is a small risk of infection and, in rare cases, a small collection of 
blood may occur, causing additional soreness for a few days. 
For the sub-cohort of 100 subjects, a time consumption of approx. three hours for each of the 
three study days. The time consumption for the planned forth examination is also approximately 
three hours. 
This examination does not require additional attendance, as the examinations will be able to be 
done in connection with the participant being in for the planned end-of-study blood test anyway. 
Completing questionnaires is assumed to take 30 minutes. 
 
Side effects and events 
No side effects are expected for the project drug. 
At the start of the study, the participants will be informed orally and in writing to contact the 
Investigator if they experience incidents and side effects, including serious ones during the 
treatment. At each control visit, the participants will be asked about this. In the specific situation, 
it will be assessed whether the incident / side effect in question is of such a nature that the 
participant must withdraw from the study. In each case, this must be explained in the CRF. To 
ensure that all events / side effects are reported to the relevant authorities, a list will also be kept 
on which the randomization number and date of registration of the event / side effect are stated, 
each time such an event is registered. In addition, it is recorded on the list whether the incident / 
side effect in question has been immediately reported. 

Serious incidents and serious side effects related to the treatment will be immediately reported to 
the Scientific Ethics Committee for Central Denmark Region. All events and side effects will also be 
reported on a form at the end of the trial. 
Time and place 
The participants are recruited from the medical departments in the Central Denmark Region, as 
randomization, handling of study drug and specific examinations as described above will take 
place at the Department of Endocrinology and internal Medicine, Aarhus University Hospital. As a 
starting point, the inclusion interview will take place at the patients' home hospital. Blood 
sampling as well. In cases where the patients have not planned attendance at the home hospital 
(have switched to "letter responses"), the patients will be invited to an inclusion interview at 
Aarhus University Hospital. In selected cases, ie. patients who come from afar, the inclusion 
interview will be able to take place via Skype (video transmitted telephone conversation over the 
Internet with image and sound). 
It is planned that the study will start in early 2015. The inclusion is expected to be completed 
during 2017, so that the entire study is expected to be completed during 2021. 
 
Confidentiality, access to documents, right of appeal, and possibility of compensation 
All information about the subjects' health conditions, other purely private matters and other 
confidential information is subject to confidentiality. The test subjects are protected - and all 
personal data, including tissue, blood samples etc. are stored - in accordance with the Act on the 
processing of personal data and in accordance with the Health Act. The participants have the 
opportunity to inspect the trial protocol in accordance with the provisions of the Public Disclosure 
Act. 
In connection with participation in the study, in the event of damage related to errors/negligence, 
the participants have the opportunity to complain and seek compensation in accordance with the 
Act on Access to Complaints and Compensation within the Health Service. Furthermore, the 
participants are covered by the patient insurance of the participating hospitals. In the event of 
harmful effects related to medicines, the participants are covered according to the Law on 
Compensation for Medicine Damages. 
Recruitment and consent 
According to the Health Act section 46, subsection 1, potential test subjects will be sought based 
on a review of referrals and patient records, obtaining information on referral diagnosis 
(thyrotoxicosis without specification, thyrotoxicosis, and Graves' disease), biochemical results for 
p-thyrotropin (TSH) and total or free triiodothyronine , total or free thyroxine, and thyroid 
stimulating immunoglobulin, TRAB. 
Potential participants will be informed about the study by contacting the patient in writing and 
making inquiries in connection with attending an outpatient check-up. The letter sent will contain 
participant information (appendix 5 or 6) and the pamphlet "Subject's rights in a biomedical 
research project" (appendix 7). In addition, a cover letter (appendix 3 or 4). It will appear from the 

written information material that further information about the project can be obtained by 
contacting the investigator whose telephone number is given. 
A brochure about the project will be available in the waiting rooms of the participating 
endocrinology clinics. This is to make potential participants aware of the project. The brochure 
contains relevant contact information for the project coordinator, and information is provided 
about the project's website address (www.dagmar.auh.dk). On this website you can read more 
about the project and see all relevant documents that will be available online. You will also be able 
to see copies of the questionnaires and supplementary information about the examination 
program (DXA). 
Patients indicate an interest in the project by contacting the investigator directly by telephone or 
by informing the attending physician about this, after which the investigator contacts the patient 
by telephone. A time for oral information is agreed here. The participant is informed about the 
possibility of bringing a companion. The information interview will take place at Aarhus University 
Hospital or at the treating hospital and will be conducted by the investigator or someone else from 
the project group. The conversation will take place in an undisturbed setting behind closed doors 
and without interruptions. Sufficient time is set aside for conducting the interview (minimum 30 
min). 
From both written and oral information, it will appear that this is a scientific experiment. The 
design of the survey will be carefully reviewed. The project's purpose, potential risks and 
discomfort as well as expected benefits will also be stated. Furthermore, the participant will be 
made aware that there may be unpredictable risks and burdens linked to participation in a 
biomedical research project. It will be emphasized that participation is voluntary and that the 
person has the right to withdraw their commitment to participate in the study at any time without 
further justification and without this affecting the rest of the treatment at the hospital. 
The project participant will be informed of the right to reflection time ("can go home and think 
about things") before giving a final answer. If the person concerned wishes to participate in the 
study, the person concerned can give oral and written consent to this at the end of the interview. 
If the patient asks for time to think, the person is requested to give feedback within 2 weeks, in 
the event that the person decides to participate in the study. In that case, a new time will be 
agreed for a repeated information interview under a similarly undisturbed framework and with 
the possibility of co-sitters. Verbal and written consent to participate in the study will then be 
obtained. The project participant receives a copy of this. 
After obtaining consent, the investigator will inform each subject's general practitioner that he or 
she is participating in the study. 
 
 
Especially regarding the controlled cohort study ii) 
 
Purpose 

Examining the muscle balance function in patients with newly diagnosed Graves' disease, etc. to 
clarify whether this has normalized three months after starting anti-thyroid treatment. 
 
Hypothesis. 
Muscle and balance function in newly diagnosed patients with Graves' disease will be reduced 
compared to healthy sex- and age-matched controls. 
Three months after starting anti-thyroid treatment, there is no longer a difference between 
controls and the patients who have received vitamin D supplementation, whereas patients who 
have not received vitamin D supplementation continue to have reduced muscle and balance 
function compared to the controls. 
 
Design 
Controlled prospective cohort study of patients with newly diagnosed Graves' disease vs healthy 
sex- and age-matched controls. 
 
Population 
Fifty-five healthy controls, matched for sex and age (+/- 2 years) and menopause status. 
  
Inclusion criteria 
• Between 18 and 80 years old 
• Euthyroidism 
• Speaks and reads Danish 
• Written consent after oral and written information 
 
Exclusion criteria 
• Current or previous metabolic disease 
• Significant other disease, including renal impairment (eGFR <60 ml/min, hypercalcemia, 
treatment with the glucocorticoid system. 
• Known neuromuscular disorders 
• Pregnancy 
• Alcohol abuse resulting in the need for treatment or hospitalization 
• Abuse of euphoric substances 
Statistics/strength calculation 
In our research group, in a previous study on healthy adults, we have found that the mean value 
for maximum knee flexion at 90 degrees is 463N with a standard deviation of 170 N. To detect a 
difference between groups of 20% with a power of 0, 80 given a significance level of 0.05, 53 
participants are required in each group. 
 
During thyrotoxicosis, the loss of bone mass has been estimated at 9% per remodeling cycle. 
Assuming that the average lumbar bone mass density is 0.92 g/cm2 (SD 0.11 g/cm2), with the 

stated population size we will be able to demonstrate a difference between groups of 10% at 
baseline with a power of 0.97 (α=0 .05). 
 
A recent meta-analysis found that the risk of all-cause mortality and cardiovascular death 
increased by 15% for an increase in pulse wave velocity (PWV) of 1 m/s. Given a standard 
deviation of 1.5 m/s in each group, the study size will be able to demonstrate a difference 
between groups at a baseline of 1 m/s with a power of 0.94 (α=0.05). 
 
Data collection 
Survey program 
The participants complete an examination program similar to the baseline examinations for the 
intensively examined cohort in the randomized clinical trial: 
 
Baseline 
Health information 
Blood tests 
Questionnaire about i.a. quality of life 
DXA scan 
HRpQCT scan 
Arterial stiffness and blood pressure meassurement 
Muscle and balance function examination 
 
Follow-up, carried out with a precision of +/-4 weeks 
Blood tests 
questionnaire 
Muscle and balance function 
Arterial stiffness and blood pressure meassurement 
 
Health information and questionnaire 
Participants answer the same baseline, ThyPRO and follow-up questionnaire as approved for the 
main study. 
Based on the interview and questionnaire, the following will be registered in the CRF: 
Age, conditions mentioned under criteria for participation in the study, current and previous 
illness, fracture history, consumption of dairy products, coffee, tea, tobacco and alcohol. Diet and 
sunbathing habits. Medical history, including use of calcium and/or vitamin D supplements, 
vitamin supplements, dietary supplements and use of natural preparations. Use of hormonal 
contraceptives. Participants who have the opportunity to do so will be asked to complete an 
electronic version of the questionnaires via the internet (the website will be created in relation to 
the project being launched). 
 

Biochemistry 
At each blood sample, approx. 80 ml will be taken. Immediate analyzes as mentioned under 
"Biochemistry" p. 12. 
Plasma will be frozen (-80 °C) at each blood sampling for later batch analysis as stated under 
"Biochemistry" on p. 12. 
Osteodensitometry 
Bone scans will be performed as a "Dual-Energy X-ray absorptiometry" (DEXA) scan with a Hologic 
QDR Discovery scanner. BMC and BMD will be measured in the lumbar spine (L1-L4), the hip 
region (femoral neck, trochanter, Ward's triangle and intertrochanteric region), the forearm 
(proximal, middle 1/3 and ultra-distal) and the whole body. Furthermore, body composition will be 
determined. 
 
Muscle function and balance studies 
Isometric voluntary muscle strength is measured on the dominant leg with a dynamometer 
(Meititur Ltd, Finland), which can measure isometric muscle strength in flexion and extension with 
the knee bent at an angle of 60° and 90° as well as flexion and extension above the elbow joint at 
90°. In a similar way, muscle strength in the hand (grip strength) is measured. 
Balance function is determined by measuring dynamic stability on a stadiometer (Meititur Ltd, 
Finland). In addition, two smaller physical tests are carried out, the "Get up and sit down test" 
(where the participants have to get up and sit on a chair ten times) (35) and the "Timed Up and Go 
test" (where the participants have to get up from a chair, walk three meters and turn around to go 
back and sit on the chair again) (36). 
 
Arterial stiffness and blood pressure 
Arterial stiffness is investigated with the commercially available tonometry-based equipment 
(SphygmoCor® and Arteriograph24), determining aortic pulse wave velocity (APWV) and central 
aortic pressure (CAP). The tests are non-invasive, well-validated and without risks or discomfort. 
Expected time consumption approx. 45 min. With Arteriograph24, 24-hour blood pressure and 24-
hour tonometry measurements are also determined. 
Data processing 
Case record forms (CRF) 
Detailed CRFs are kept for all participants, in which the results of the above examinations are 
stated. 
Investigator allows direct access to source data/documents (including patient records) during 
monitoring, auditing and inspection from the Research Ethics Committee for Central Region and 
the GCP unit for Aarhus University Hospital and its partners. The participants are made aware of 
this and specific consent is obtained in the form of a power of attorney. 
All information obtained will be handled in accordance with the Act on the Processing of Personal 
Data. 
 

Risks, side effects and inconvenience 
The blood test may be associated with discomfort. There is a minimal risk of infection at the 
injection site. When taking a blood sample, 80 ml will be drawn each time, i.e. around 160 ml of 
blood in total. This is not expected to cause discomfort. 
During the scans, a total so-called effective radiation dose of an estimated 26 microSv is applied to 
each patient, which corresponds to approx. four days of normal background radiation. 
 
The total time consumption is estimated to be four hours. 
It is the investigator's belief that any disadvantages, risks and side effects are minimal and are out-
weighed by the new knowledge that the study will be able to provide. 
 
Time and place 
Participants are recruited by letter from the Aarhus area, see under recruitment. Inclusion 
interviews and examinations take place at the Department of Endocrinology and Internal 
Medicine, The Osteoporosis Clinic, Aarhus University Hospital, Tage-Hansens Gade 2, 8000 Aarhus 
C. 
It is planned that inclusion will begin in August 2015. The inclusion is expected to take place over 1 
year, so that the entire study is completed within 1.5 years. 
Confidentiality, access to documents, right of appeal and possibility of compensation 
All information about the subjects' health conditions, other purely private matters and other 
confidential information is subject to confidentiality. The test subjects are protected - and all 
personal data, including tissue, blood samples etc. are stored - in accordance with the Act on the 
processing of personal data and in accordance with the Health Act. The participants have the 
opportunity to inspect the trial protocol in accordance with the provisions of the Public Disclosure 
Act. 
In connection with participation in the study, in the event of damage related to errors/negligence, 
the participants have the opportunity to complain and seek compensation in accordance with the 
Act on Access to Complaints and Compensation within the Health Service. Furthermore, the 
participants are covered by the patient insurance of the participating hospitals. In the event of 
harmful effects related to medicines, the participants are covered according to the Act on 
Compensation for Medicine Damages. 
 
Recruitment 
There will be an advertisement for test subjects via the daily press as well as via notice boards at 
e.g. educational institutions and larger workplaces (see appendix 15), as well as via the social 
media: Facebook. Also via www.forsøgspersonner.dk (see appendix 16). 
A Facebook page called "Subjects for the "DAGMAR project" will be created. On this page, under 
the "About" tab, there will be more specific information about the project, see appendix 17. There 
will be a fixed notice on the page, see appendix 18, and there will be more specific notices on age 
and gender, see appendix 19. There advertising for both the Facebook page and for small postings 

(appendix 20) on the Facebook page (not for app. 19) is purchased. The announcement will 
contain the text in appendices 18 and 20 respectively and will be targeted at gender, age and 
place of residence corresponding to the inclusion criteria for the project (age, sex, place of 
residence in the Aarhus area). These ads will be visible on the target group's Facebook home page 
and will be listed as "sponsored ad". If you choose to follow the project's Facebook page (by 
"liking" it), future posts on the Facebook page (both appendices 19 and 20) will be displayed on 
your Facebook homepage as a regular post, i.e. not as a sponsored ad. In connection with all posts 
and announcements as stated, the project will be called "DAGMAR euthyroid" corresponding to 
the name on the Facebook page. 
If you contact us regarding the above advertisements, you will be sent written information about 
the project before any informational interview. 
“Forskerservice” will be requested a data file with name, address, age, and gender, and social 
security number for 25,200 people aged 18-80 from the Aarhus area (200 per age per gender). 
Every time a patient with Graves' disease is included in the intensive study cohort, a letter will be 
sent via e-box to 20 gender- and age-matched (+/- 2 years) people from this data extract. 
The letter sent will contain a cover letter (appendix 12) as well as participant information 
(appendix 13) and the pamphlet "Subject's rights in a biomedical research project" (appendix 7). In 
the cover letter, there will be a link to an internet-based mixed response (appendix 14), in which 
the participant can state whether he or she is interested in participating in the survey. If interest is 
expressed, the participant will be asked to answer a short questionnaire regarding menopause 
status (women only) and exclusion criteria. The questionnaire is answered pseudo-anonymized 
without directly identifiable information using a username and codes in the sent covering letter. It 
will appear from the written information material that further information about the project can 
be obtained by contacting the investigator whose telephone number is given. If there is no 
response, information about the project will be re-sent after 1 week. If no response is obtained 
from the potential trial participants, a letter is sent out to another 20 people who are matched in 
the same way on age and gender. The above procedure is repeated if there is no response. 
Interested potential participants are requested to come to an interview about the project. The 
participant is informed about the possibility of bringing a companion. The information interview 
will take place at Aarhus University Hospital and will be handled by the investigator or someone 
else from the project group. The conversation will take place in an undisturbed setting behind 
closed doors and without interruptions. Sufficient time is set aside for conducting the interview 
(minimum 30 min). The procedure for this information interview will follow the guidelines as 
stated under the randomized study i) above. 
Participant information (appendix 13) and the pamphlet "Subject's rights in a biomedical research 
project" (appendix 7) will be reviewed and handed out. From both written and oral information, it 
will appear that this is a scientific experiment. The design of the survey will be carefully reviewed. 
The project's purpose, potential risks and discomfort as well as expected benefits will also be 
stated. Furthermore, the participant will be made aware that there may be unpredictable risks and 
burdens linked to participation in a research project. It will be emphasized that participation is 

voluntary and that the person has the right to withdraw their commitment to participate in the 
study at any time without further justification and without this affecting future treatment at the 
hospital. 
The project participant will be informed of the right to reflection time ("can go home and think 
about things") before giving a final answer. If the person concerned wishes to participate in the 
study, the person concerned can give oral and written consent to this at the end of the interview. 
If the person asks for time to think, the person is requested to give feedback within 1 week in the 
event that the person decides to participate in the investigation. In that case, a new time will be 
agreed for a repeated information interview under a similarly undisturbed framework and with 
the possibility of co-sitters. Verbal and written consent to participate in the study will then be 
obtained. The project participant receives a copy of this. 
After obtaining consent, the investigator, with the patient's consent, will inform each subject's 
general practitioner that he or she is participating in the study. 
Regarding both study i) and ii) 
 
Ethical considerations 
The study is carried out in accordance with the conditions mentioned in the Helsinki II declaration 
regarding biomedical research involving humans. 
The guidelines in accordance with GCP (International Conference on Harmonization (ICH): 
guidance E6) will be followed. The GCP unit at Aarhus University Hospital has been requested to 
monitor the study. 
The project has been notified to the Danish Data Protection Authority via the region's joint 
notification. 
The trial will be registered at www.clinicaltrial.gov. 
Informed consent will be obtained from all participants (Appendix 8), and the study will be carried 
out in accordance with the applicable recommendations. Participation in the study is voluntary, 
and any participant can withdraw from the study at any time if they wish to do so, and the 
participant cannot be required to provide any explanation in this regard. All trial participants will 
be given a copy of the consent form. 
 
Ethical considerations 
It is the investigator's belief that any risks and side effects are minimal and are outweighed by the 
new knowledge that the study will be able to provide. The individual participant has no immediate 
benefit from his participation. Should there be a demonstrable vitamin D deficiency at the end of 
the experiment, the participant will be informed and guided about this. 
During the planned examinations, unexpected findings may arise, e.g. non-symptom-producing 
diseases and conditions. If diseases and conditions requiring treatment are observed during the 
examinations carried out, the project participant will be offered a referral to relevant treatment. 

When the study is finally completed, the participants will be sent a brief description of the study's 
most important results with information on whether they were treated with active medication or 
placebo during the study period. 
After the end of the experiment, the investigator will inform the Scientific Ethics Committee for 
Central Denmark Region about the results obtained, observed side effects, number of patients 
treated, doses used and duration of dosing. 
Lack of vitamin D is widespread in Denmark. The present study will elucidate the importance of a 
vitamin D supplement for the prognosis and symptomatology of patients with Graves' disease. If 
the study shows a significant effect of intervention on the recurrence rate in the cohort, this may 
have an impact on the future treatment of patients with Graves' disease. 
 
Publication 
The results from the study will be sought for publication in international medical journals, as both 
positive, negative and inconclusive results will be published. The publications are designed 
exclusively by the trial manager and investigator together with the other co-investigators. There 
are no companies or individuals who may be presumed to have commercial interests in the 
interests of the study that influence the results statement or the interpretation of the trial results. 
 
Economy 
The study was created on the initiative of doctors from the Department of Medical Endocrinology, 
Aarhus University Hospital. No one from the project group has financial interests in the study. 
Expenses for the investigation will be financed by the departments involved, as fund support is 
also applied for. 
No financial remuneration is provided to the test subjects, but possibly a transport allowance. 
Study medication in the form of vitamin D and placebo is provided free of charge by the food 
manufacturer and dietary supplement manufacturer Orkla Health. 
 

Statistical analysis plan, final 
 
Analyses of treatment effects will be based on the intention-to-treat principle. The first analysis will 
compare baseline characteristics by randomized treatment assignment to ensure that balance was 
achieved by the randomization. Characteristics to be examined include known risk factors for treatment 
failure including age, sex, smoking habits, severity of thyrotoxicosis, former use of vitamin D and baseline 
vitamin D status. 
Differences between groups will be evaluated using a Chi Square test for categorical variables and a two-
sample t test or Wilcoxon ranksum test, as appropriate, for continuous variables. For the primary outcome, 
the risk of treatment failure will be calculated as risk ratio (RR) with 95% confidence interval (CI).  
With the intention-to-treat (ITT) approach, all subject who had been randomized and had received at least 
one dose of study medication will be included in the analysis. If study outcome is unknown, e.g. study 
withdrawal or exclusion, participants will be included in the ITT assuming either a worst-case scenario (all 
had treatment failure) or a best-case scenario (all experienced a treatment success) 
In per-protocol analyses, only participants in whom outcomes can be assessed will be included.  
In a sub-analysis, the risk of relapse at any given time after ATD discontinuation until 12 ± 1 months will be 
examined using Cox regression.  
In pre-specified analyses, the interaction between the intervention and baseline 25(OH)D status 
(insufficient (<50 mmol/L) or replete) and prior use of vitamin D supplementation (yes or no) will be 
assessed using logistic regression.  
in addition to the original SAP, we will explore the interaction between the intervention and risk factors for 
relapse such as sex, age (above or below 40 year), time since diagnosis (above or below 35 days), and 
smoking status (current vs not). In case of significant interaction, the effect of intervention will be tested in 
each group separately.  
 
  
References 
(1) Cerqueira C, Knudsen N, Ovesen L, Perrild H, Rasmussen LB, Laurberg P, et al. Association of 
iodine fortification with incident use of anti-thyroid medication--a Danish Nationwide Study. J Clin 
Endocrinol Metab 2009 Jul;94(7):2400-2405. 
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