Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy
Conditions: Type 2 Diabetes, Bone Disease, Osteoporosis, Diabetic Neuropathy Peripheral, Autonomic Neuropathy, Diabetic
Sponsor: Aalborg University Hospital

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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Research Protocol 
 
Deep Phenotyping of Bone Disease in Type 2 
Diabetes and Relations to Diabetic Neuropathy 
 
 
 
 
 
 
 
Ansøgning v/ Regional Videnskabsetisk Komité:  
Signed:   
 
______________________________________________________  
Principal investigator: Julie Lindgård Graversen, Medical doctor, Ph.d.-student    
Steno Diabetes Center Nordjylland & Department of Endocrinology, Aalborg University Hospital 
Søndre Skovvej 3E & Mølleparkvej 4, 9000 Aalborg 
Aalborg, 27.06.23 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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Table of Contents 
1. PROJECT GROUP ............................................................................................................................................ 3 
2. BACKGROUND AND SIGNIFICANCE................................................................................................................ 4 
2.1. Background ............................................................................................................................................. 6 
2.2. State of the art ........................................................................................................................................ 6 
2.3. Significance and perspectives of the project to the research area ........................................................ 8 
3. STUDY OBJECTIVES ........................................................................................................................................ 9 
4. METHODS ...................................................................................................................................................... 9 
4.1. Study Design ........................................................................................................................................... 9 
4.2. Study population .................................................................................................................................... 9 
4.3. Assessment of resources ...................................................................................................................... 11 
4.4. Recruitment .......................................................................................................................................... 11 
4.5. Study procedures .................................................................................................................................. 12 
5. DATA COLLECTION ....................................................................................................................................... 20 
6. DATA ANALYSIS ............................................................................................................................................ 21 
6.1. Sample size considerations ................................................................................................................... 21 
6.2. Statistical methodology ........................................................................................................................ 21 
6.3. Statistical significance ........................................................................................................................... 21 
6.4. Missing data .......................................................................................................................................... 21 
6.5. Procedures in change ........................................................................................................................... 21 
7. DATA AND SAFETY MONITORING PLAN ...................................................................................................... 21 
7.1. Data ...................................................................................................................................................... 21 
7.2. Establishing of research biobank .......................................................................................................... 21 
8. STUDY LIMITATIONS .................................................................................................................................... 22 
9. ETHICAL CONSIDERATIONS .......................................................................................................................... 22 
9.1. Informed consent ................................................................................................................................. 23 
9.2. Risks and side effects ............................................................................................................................ 24 
9.3. Benefits to subjects .............................................................................................................................. 25 
9.4. Costs to subject .................................................................................................................................... 25 
9.5. Compensation to subjects .................................................................................................................... 25 
9.6. Provision for vulnerable subjects ......................................................................................................... 25 
10. ECONOMY .................................................................................................................................................. 25 
11.PLAN FOR DISSEMINATION OF FINDINGS .................................................................................................. 26 
12. INSURANCE ................................................................................................................................................ 26 
13.REFERENCES ............................................................................................................................................... 26 
 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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1. PROJECT GROUP 
Principal Investigator: 
Julie Lindgård Graversen, MD, Ph.D. student 
Steno Diabetes Center North Jutland (SDCN) 
Aalborg University Hospital & Aalborg University 
E-mail: j.lindgaard@rn.dk 
Phone: + 45 30 24 10 79 
 
Secondary Investigator: 
Marie Balle Sjøgaard, MSc, Ph.D. student 
Steno Diabetes Center North Jutland (SDCN), Aalborg University Hospital 
Department of Clinical Medicine, Faculty of Medicine, Aalborg University 
Danish Pain Research Centre, Aarhus University 
Core Centre for Molecular Morphology, Aarhus University 
Aalborg University 
E-mail: m.sjoegaard@rn.dk 
Phone: + 45 30 11 77 34 
 
Main Supervisor: 
Peter Vestergaard, MD, Ph.D., dr. med 
Professor of Endocrinology 
Head of Research SDCN 
Department of Endocrinology / SDCN 
Aalborg University Hospital 
 
Co-supervisors: 
Nicklas Højgaard-hessellund Rasmussen, MD, Ph.D. 
Department of Endocrinology 
Aalborg University Hospital 
 
Johan Røikjer, MD, Ph.D. 
SDCN & Department of Health Science and Technology (HST)  
Aalborg University Hospital & Aalborg University 
 
 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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List of abbreviations 
aBMD 
 
Areal bone mass density 
AGE 
 
Advanced glycation end products   
ALAT  
 
Alanin-aminotransaminase  
AP 
 
Anterior-Posterior   
BI 
 
Business intelligence 
BMD 
 
Bone mineral density  
BMI 
 
Body mass index  
BMSi 
 
Bone Material Strength index 
CAN 
 
Cardiac autonomic neuropathy 
CDT 
 
Cold detection threshold 
CI 
 
Confidence Interval  
CoP 
 
Center of pressure   
COPD 
 
Chronic Obstructive Pulmonary Disease 
CST 
 
Chair Stand Test   
CT 
 
Computer Tomography   
DD2 
 
Danish Center for Strategic Research in Type 2 Diabetes 
DM 
 
Diabetes Mellitus   
DPN 
 
Diabetic Polyneuropathy Neuropathy   
DXA 
 
Dual-energy X-ray absorptiometry  
ECG 
 
Electrocardiogram   
EDTA 
 
EthyleneDiamineTetraAcetic acid  
eGFR 
 
estimated Glomerular Filtration Rate  
EPJ  
 
Electronic Patient Journal  
GABA 
 
γ-aminobutyric acid 
GDPR 
 
General Data Protection Regulation 
GGT 
 
Gamma-Glutamyltransferase  
GLP-1 
 
Glucagon Like Peptide 1 
Gy 
 
Grays   
HbA1c 
 
Hemoglobin A1c  
HR-pQCT 
 
High-resolution peripheral quantitative computed tomography 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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ICH GCP 
 
Harmonised Tripartite Guideline for Good Clinical Practice  
IGF-1  
 
Insulin-like growth factor-1   
IC 
 
Informed consent    
IENF 
 
Intra epidermal nerve fibers 
IQC 
 
International Quality Control   
MARD 
 
Mild age-related diabetes 
ML 
 
Medial-lateral 
MNSI 
 
Michigan Neuropathy Screening Instrument 
MOD 
 
Mild obesity-related diabetes 
N 
 
Sample requisition numbered  
NBV 
 
National treatment guidelines in Endocrinology  
OCT 
 
Optical Coherence Tomography 
PTT 
 
Perception threshold tracking 
QST 
 
Quantitative sensory testing  
SAF 
 
Skin autofluorescence 
SGLT-2 
 
Sodium-Glucose Cotransporter-2 
SIDD 
 
Severe insulin-deficient diabetes 
SIRD 
 
Severe insulin-resistant diabetes 
Sv 
 
Sieverts  
T1D 
 
Type 1 Diabetes  
T2D 
 
Type 2 Diabetes  
TBS 
 
Trabecular bone score  
TZD 
 
Thiazolidinedione   
TUG  
 
Timed Up and Go   
UGS 
 
Usual gait speed 
vBMD 
 
Volumetric bone mineral density 
VFA 
 
Vertebral Fracture Assessment   
VPT 
 
Vibration Perception Threshold  
WDT 
 
Warm detection threshold 
WHO 
 
World Health Organization   
 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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2. BACKGROUND AND SIGNIFICANCE 
2.1. Background 
Type 2 diabetes (T2D) is a prevalent disease currently affecting 422 million people worldwide (2014), leading 
to a global prevalence of 8.5 % among adults1. The total number of people living with diabetes (T1D and T2D) 
is projected to rise to 783 million by 20452. Thus, prevention and intervention of complications is important. 
A less well-known complication of T2D is diabetic bone disease. T2D is associated with an normal or even 
increased bone mineral density (BMD)3,4, which in theory should lead to a decreased risk of fractures. 
However, T2D is associated with an increased risk of any fractures, with relative risks reported up to 1.75–8. 
A number of meta-analyses reports an 1.08 to 1.7-fold increased risk of hip fractures in patients with T2D3,5–
7,9,10. Evidence of vertebral fractures is more ambiguous, with the meta-analysis by Moayeri et al.7 reporting 
an increased risk, while Jia et al.6 did not find an significantly increased risk of vertebral fractures in T2D. 
Another meta-analysis by Koromani et al.11 showed a lower risk of prevalent but increased risk of incident 
vertebral fractures in T2D.  Furthermore, there seems to be an increased risk of fractures at more distal sites 
such as the humerus8,12,13, distal forearm12, ancle8 and foot7 but the evidence is more limited. The reason for 
this increased fracture risk is not fully understood, but may result from a combination of a decreased bone 
biomechanical competence and an increased risk of falls14. Several associations thus need more detailed 
examination and characterization in the relationship between T2D and skeletal health.  
2.2. State of the art 
The increased fracture burden in diabetes might partially be explained by an increased risk of falls and by 
alteration in the fall mechanism. A meta-analysis by Yang et. al. 15 showed that older persons with diabetes 
has increased risk of falls compared with healthy controls. Several risk factors that contribute to falls in 
diabetes exists. These include impaired postural control and gait, diabetic neuropathy, impaired vision, 
vestibular disorder, cardiovascular disease, comorbidity including hypertension, drugs and hypoglycemia16. 
Furthermore, patients with T2D has a higher risk of sarcopenia17 and decreased muscle function18, which has 
been proposed as a contributor to increased fall tendency19 and increased risk of fractures20. Several studies 
of diabetes and fracture risk have included data on falls. In these studies, higher fracture risk associated with 
T2D persisted even after adjustment for increased frequency of falls21–23. This suggest that additional 
underlying mechanisms such as decreased bone quality is involved in the increased fracture risk in T2D. Bone 
quality is affected by bone mass, bone turnover and bone material properties. 
It is suggested that advanced glycosylation end products (AGEs) produced in response to hyperglycemia are 
incorporated in the bone structure, hence reducing the material and biomechanical properties of bone by 
stiffening the bone collagen and reducing bone strength24. Furthermore, the hyperglycemia triggers 
hypermineralization in the bone causing high BMD25. The interplay between bone and glucose metabolism is 
complex and not fully understood. An interaction is present between osteocalcin, which is a bone formation 
marker, but also interacts with insulin sensitivity, thus forming a bone-beta-cell axis26,27. T2D is associated 
with decreased bone turnover caused by osteocyte dysfunction28,29. It has been suggested that the low bone 
turnover in T2D cause microcracks, thereby increasing fracture risk30. Bone Material Strength index (BMSi) is 
measured by microindentation and is reported to be decreased in T2D31,32, which is considered to reflect 
decreased ability of bone to resist microcrack generation and propagation33. 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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Alterations in bone microarchitecture may be present among T2D patients, resulting in an increased fracture 
risk. Trabecular bone score (TBS), a textural parameter that can be applied to DXA, has been reported to be 
significantly decreased in patients with T2D in a recent meta-analysis34. TBS may even be decreased in 
prediabetes, indicating that the degradation of bone microarchitecture may occur in early stages of the 
disease35. Higher HbA1c levels might be related to lower TBS values35–38, as well as the presence of 
microvascular disease39. Additionally, low TBS values has been associated with adiposity (estimated by the 
relative fat mass) and insulin resistance40, and one study found that  reduction of visceral fat could be related 
to improvement of TBS41. High resolution peripheral quantitative computed tomography (HRpQCT)  is a non-
invasive 3D imaging modality that permits the assessment of bone microarchitecture, including the 
measurement of volumetric cortical and trabecular bone mineral density (vBMD) and cortical 
thickness/porosity, bone strength as well as other parameters in the peripheral skeleton such as distal radius 
and tibia42. In some studies, but not all, cortical porosity is reported to be higher in T2D compared to controls 
in postmenopausal women43–45. Furthermore, T2D patients are reported to have lower cortical vBMD and 
cortical thickness45,46. The changes in microarchitecture results in a decreased bone quality, and this 
decreased peripheral bone quality might attribute to fractures in T2D. 
Shanbhouge et al.45 found that the observed cortical deficits were a characteristic of the T2D patients with 
microvascular complications rather than all T2D patients.  However, the study was not able to perform an 
adequate assessment of the separate roles and severity of neuropathy, retinopathy, and nephropathy in 
bone structure due to limited sample size. A meta-analysis by Liu et al.47 reported that T2D patients with 
diabetic neuropathy have an increased risk of developing osteoporosis and fragility fractures with an OR of 
2.15 (95% CI 1.56-2.97). Contrastingly, a Danish study by Khan et al.48 using patients enrolled in the Danish 
Center for Strategic Research in Type 2 Diabetes (DD2) cohort were not able to find a significant association 
between diabetic neuropathy and fractures. However, they did find an increased risk of falling. A well-
powered retrospective study by Lee et. al49 found that older male Veterans with diabetes (>98% had T2D) 
have an increased risk of any fractures and hip fractures, and diabetic neuropathy was reported to be  the 
most important mediator of fracture risk observed in their cohort. A histomorphometry study in humans with 
high bone turnover showed that the nerve profiles density was 5-fold higher in the intracortical pores 
compared to bone marrow and periosteum, and the authors suggest an anatomical link between innervation 
and bone remodeling50. Overall, there seems to be a higher fracture risk in subjects with T2D and diabetic 
neuropathy, which is a very common complication of T2D. However, the exact mechanisms behind this 
remain to be elucidated.  
Multiple studies have investigated the effects of antidiabetic medication on fracture risk. Several studies 
found an association on glitazones and increased fracture risk51–53. Evidence of the effect on insulin on 
fracture risk in T2D is more conflicting, as insulin use have shown both increased13,54,55, neutral56 and 
decreased57 outcome on fracture risk. Also sulphonylureas are reported to have both neutral53,58,59, 
negative60,61, or perhaps even protective57 effect on fractures risk. Other medications have been associated 
with a decreased or neutral effect on the risk of fractures in T2D, such as Metformin51,53,57,58, DDP-4 
inhibitors56,62,63, GLP-1 receptor agonists62,64 and SGLT-2 inhibitors65,66. In addition, concomitant medications 
related to comorbidities might also play a role in fracture risk in T2D. Thiazides have been associated with a 
decreased fracture risk, while loop diuretics are associated with higher fracture risk67. An updated meta- 
analysis revealed no significant effect of statin treatment on the risk of fractures68.  Also drugs commonly 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
Project-ID: N-20220038 
Research protocol_v5.1_270623 
 
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used for diabetic neuropathy such as tricyclic antidepressants69,70 and γ-aminobutyric acid (GABA) analogs69 
are associated with increased risk of falls and fracture. 
 
 
Figure 1: Factors leading to increased fracture risk in T2D 
2.3. Significance and perspectives of the project to the research area  
Osteoporosis is a major public health problem. Hip fractures are associated with increased mortality71,72, and 
the risk is even higher in diabetic patients compared to non-diabetics73. The cost of osteoporosis in the EU in 
2010 was estimated at €37 billion with the highest estimated cost per capita in Denmark. Thus, osteoporosis 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
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has major economic consequences for society74. As the risk of fractures will increase with the expected 
increasing prevalence of T2D, it is important to identify risk factors and mechanisms underlying diabetic bone 
disease and fracture risk to enhance treatments and prevention. 
This study will provide new knowledge on T2D bone phenotyping and the crosstalk between bone, nerves, 
muscle, fat and beta cells. Furthermore, the study contributes with a characterization of the interplay 
between diabetic neuropathy and bone disease which could play a pivotal role in advancing the field of 
neuroskeletal biology.  
The study will include novel and advanced technologies in the assessment of the T2D bone phenotype 
covering bone microarchitecture, bone strength and bone remodeling markers. In addition, the study will 
include comprehensive functional assessments spanning from motor to neuronal assessments with inclusion 
of sensory assays for both large and small fibers and evaluation of autonomic function. 
3. STUDY OBJECTIVES 
We hypothesize that a crosstalk between beta cells, bone-, nerve-, muscle- and fat tissues exists, and our 
main goal is to attain a better understanding of these relations and describe the T2D bone phenotype.  
Hence, our aims are to:  
• 
Compare bone microarchitecture, bone biomechanical competence, and bone turnover markers in T2D 
with and without fractures. 
• 
Examine how different phenotypes of diabetic neuropathy affects bone microarchitecture, bone 
material strength and bone turnover markers as well as postural control and physical function. 
• 
To examine how insulin resistance effects bone microarchitecture, bone material strength and bone 
turnover. 
• 
To examine the relationship between T2D phenotype (classical/insulinopenic/hyperinsulinemic or SIDD 
[severe insulin deficient diabetes]/SIRD [severe insulin resistant diabetes]/MOD [mild obesity-related 
diabetes]/MARD [mild age-related diabetes]) and body composition (fat and muscle mass and fat 
distribution, i.e. visceral adipose tissue) 
• 
To investigate how microvascular complications (neuropathy, retinopathy, and nephropathy) are 
affected by T2D phenotype (classical/insulinopenic/hyperinsulinemic or SIDD/SIRD/MOD/MARD) 
4. METHODS 
4.1. Study Design 
Cross sectional study. 
4.2. Study population 
The study strives to include a total of 300 subjects with T2D divided into three well-defined groups:  
• 
T2D F-/N-: Subjects with T2D and no previous history of any fractures or diabetic neuropathy (n=160) 
• 
T2D F+: Subjects with T2D with a previous history of a fracture(s) (any fracture, major osteoporotic 
fracture (MOF) and peripheral) (n=100) 
• 
T2D N+: Subjects with T2D matched by age and sex with severe peripheral (vibration perception 
threshold (VPT) > 50) or a history of autonomic neuropathy (n=40). 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
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Figure 2: Study design  
Inclusion criteria 
1. Men and women with minimum 40 years of age.  
2. Diagnosis of T2D. At least one of the following criteria must be met at diagnosis:  
a. HbA1c ≥ 48 mmol/mol (6,5 %) 
b. Plasma glucose ≥ 11,1 mmol/l 
c. Fasting plasma glucose ≥7,0 mmol/l 
Clinical effect of oral antidiabetic medication strengthens the diagnosis.  
3. Diagnosis of diabetes at least one year prior to inclusion of the study.  
4. A history of fracture(s) (confirmed by radiographs analyzed by radiologist) following the diabetes 
diagnosis (T2D F+ group) 
5. Diagnosed with severe peripheral (VPT ≥ 50) or autonomic neuropathy defined by cardiac autonomic 
reflex tests or severe abnormalities in orthostatic blood pressure (T2D N+ group) 
6. Signed the informed consent.   
7. Not defined by the exclusion criteria.  
 
Exclusion criteria 
1. 
Severe 
decreased 
liver 
function (Alanin 
amino-transaminase 
(ALAT) 
>250 
U/l, 
Gamma-
Glutamyltransferase (GGT) >150 U/l). 
2. 
Moderate to severe kidney dysfunction, estimated Glomerular Filtration Rate (eGFR) <15 
mmol/L/1,73m2.  
3. 
Pregnancy or breast feeding. 
4. 
Active malignancy or terminal ill.   

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
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5. 
Previous chemotherapy or immunomodulating treatment 
6. 
Known severe vitamin deficiency 
7. 
Current or previous alcohol- or drug abuse. 
8. 
Not being able to understand Danish written and/or verbally. 
9. 
Terms according to investigators judgement that makes subjects unsuitable to participate including lack 
of ability to understand and comply with instructions and/or reduced physical ability, limiting the ability 
to participate in the examinations.  
10. Participating in other clinical studies utilizing experimental treatment or medication. 
11. Subjects with pathologic fractures (defined as fractures due to local tumors, tumor-like lesions, or focal 
demineralization as visualized on radiographs). 
12. Primary hyperparathyroidism, Paget’s disease and other metabolic bone diseases, uncontrolled 
thyrotoxicosis, celiac disease not controlled by diet, known hypogonadism, severe COPD, 
hypopituitarism, Cushing’s disease. 
13. Fracture < 6 month ago  
14. Initiation of antiresorptive or bone anabolic drugs <12 months ago to ensure stable bone turnover 
markers.  
15. History of fractures following the diagnosis of diabetes (T2D F-/N- and T2D N+ groups). 
16. History of peripheral or autonomic neuropathy defined by cardiac autonomic reflex tests or severe 
abnormalities in orthostatic blood pressure (T2D F-/N- group). 
17. Suspected peripheral or autonomic neuropathy of other causes than diabetes (e.g., concomitant 
central nervous system pathologies). 
18. Known peripheral arterial disease (PAD) defined as toe pressure <40mmHg, no palpable foot pulses or 
clinical claudicatio intermittens. 
19. Active diabetic foot ulcers at time of inclusion. 
20. Previous or expected amputations in lower extremities, of any cause, during the study. 
21. Known allergy to lidocaine.  
 
4.3. Assessment of resources 
The study will be conducted in collaboration with the department of Endocrinology and Steno Diabetes 
Center North Jutland, which has access to subjects with diabetes and can provide the necessary premises.  
 
The department of Endocrinology has a large laboratory with 7 Bio. Med. Lab. Technologists dedicated for 
research. We have extensive experience with DXA technology and highly skilled technicians with years of 
experience. An extensive International Quality Control (IQC) program according to the guidelines of the 
Society for Clinical Densitometry is in place for the DXA scanners.   
The laboratory offers blood sampling facilities as well as facilities for blood testing including state of the art 
validated bone turnover markers. A collaboration with the department of Clinical Chemistry also exists.  
 
4.4. Recruitment 
Recruitment will be targeted at subjects with T2D in the Region of North Denmark and can include both T2D 
patients in general and participants in the DD2 study (https://dd2.dk/). Recruitment of participants will be 
based on advertisements in media, e.g., on www.forsoegsperson.dk, social medias as Facebook and 

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
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Instagram, local medias and papers. Patients will also be recruited from the outpatient clinics of the 
department of Endocrinology, Aalborg University Hospital in Aalborg and Farsø as well as North Denmark 
Regional Hospital in Hjørring. Lists of patients from the BI (Business intelligence) Unit at the Region of North 
Denmark will be used for direct mailing via secure mail pending approval from the competent bodies at the 
Region of North Denmark. An existing database where participants have given written consent (within the 
last year) to be contacted regarding research projects may also be used. This database is created and 
administrated by the North Denmark Region and approved by its legal staff. Potential participants are 
required to use a secure access (MitID), and if they do not actively renew their consent within a year, it is 
automatically withdrawn. We will hang posters and hand out flyers at general practitioners, podiatrists, 
supermarkets and training centers at places flyers are allowed to hang. 
The healthcare professionals at Aalborg University Hospital, regional hospitals and home-care services in the 
North Jutland Region will be contacted and asked to be involved in the patient recruitment.  
 
4.5. Study procedures  
General health data from medical records and interview 
To enable the best possible adjustment of confounding factors in later analysis, general health information 
for each subject will be assessed through medical records and interviews. These include assessment of 
weekly alcohol consumption, daily tobacco use, daily activity level (at work and at home), comorbidities, 
signs and symptoms of neuropathy, familiar disposition to osteoporosis, risk factors/comorbidities associated 
with osteoporosis in accordance with the National treatment guideline in Endocrinology (NBV), prior 
biochemistry, duration of diabetes, prior fractures (localization, high/and medication use (including hormone 
therapy such as oral contraceptives etc.) for at least one month priory to baseline measurements. 
Laboratory assessments 
Individual laboratory values will be compared to their relevant reference range (when existing) and flagged 
as being below or above the range. The blood samples will be taken and handled as usual by the Research 
bio. Med. Lab. technologists at Aalborg University Hospital.  
If laboratory samples need to be re-done, due to missing results (e.g., hemolysis, sample leaked, inconclusive, 
lost in transit etc.), the subject should be called in for resampling. Final laboratory reports must be reviewed, 
dated, and signed by the investigator on the day of evaluation. It must be specified by the investigator 
whether out of range results are clinically significant. The subjects are allowed to receive or betray results 
(See informed consent form).  
 
Biochemistry:  
The following samples will be taken at the screening visit (total 4 ml): 
• 
Creatinine, eGFR 
• 
ALAT 
• 
Gamma-Glutamyltransferase 
• 
TSH 
• 
25-hydroxy D-vitamin 
• 
Cobalamin  

Deep Phenotyping of Bone Disease in Type 2 Diabetes and Relations to Diabetic Neuropathy 
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The following samples will be taken at the study day (total 84 ml including samples for the research 
biobank): 
• 
HbA1c 
• 
Fasting-C-peptide and fasting P-glucose (for HOMA2-%B and -IR) 
• 
CRP 
• 
Lipids (LDL, HDL, total cholesterol, triglycerides) 
• 
PTH 
• 
Calcium 
• 
Phosphate 
• 
Magnesium 
• 
Carbamide 
• 
Sodium 
• 
Potassium 
• 
Alkaline phosphatase 
• 
Bilirubin 
• 
Albumin 
• 
M-component  
• 
 
• 
Hematology 
o Hemoglobin  
o Erythrocytes  
o Reticulocytes  
o Hematocrit  
o Middle Cell Volume  
o Middle Cell Hemoglobin Concentration   
o Leukocytes and diff. counting  
o Thrombocytes  
o Folate  
o Ferritin  
o Transferrin  
• 
hsCRP* 
• 
FGF23* 
• 
Sclerostin* 
• 
Osteoglycin* 
• 
Osteocalcin* 
• 
Undercarboxylated osteocalcin (ucOC)* 
• 
Osteopontin* 
• 
P1NP* 
• 
CTX* 
• 
EsRAGE* 
• 
GIPR* 
• 
Testosterone/estradiol * 

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• 
FSH*  
• 
LH*  
• 
Adiponectin* 
• 
Leptin* 
• 
IGF-1* 
 
*: The sample will be analyzed after study completion from blood stored in the research biobank 
(seesection 7.2) 
Estimated time: 10 min 
24-hour urine collection:  
• 
Creatinine 
• 
Carbamide  
• 
Sodium 
• 
Potassium 
• 
Volume  
• 
Protein  
• 
Albumine 
• 
Ratios  
Assessment of vital signs and body measurement   
Cardiovascular parameters including heart rate, systolic and diastolic blood pressure. This will be assessed 
using an Electronic Blood Pressure Device (Omron M6 Comfort IT Intelligence) after 5 min of quiet sitting. 
Blood pressure will be measured three time and the mean value will be calculated of the last two 
measurements. If the subject is using antihypertensive medication to control blood pressure, then the 
medication should be taken as usual prior to assessing vital signs.  
Height: (Without shoes) will be measured and rounded to the nearest half centimeter.  
Body weight: Should be measured in kilograms (kg) without overcoat and shoes and wearing only light 
clothing. Body weight will be recorded to one decimal place.  
BMI is calculated.   
 
Estimated time: 10 min.   
Orthostatic blood pressure 
After the normal blood pressure procedure, the subjects will be asked to stand for three minutes and 
afterwards a new blood pressure will be measured. 
Orthostatic hypotension is defined as a decrease in systolic blood pressure of 20 mmHg or a decrease in 
diastolic blood pressure of 10 mmHg within three minutes of quiet standing when compared with blood 
pressure from the sitting position. 
 
Estimated time: 5 min.  
 

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12 lead-Electrocardiogram  
An ECG-12 lead will be performed in a lying position and the subject will have bare chest. The ECG must be 
interpreted by the investigator and documented.  
The evaluation must follow the categories:  
- 
Normal  
- 
Anormal  
o Clinically significant? (Yes/no)  
ECG findings will be summarized descriptively. If an ECG-12 lead has already been performed within three 
weeks before entering the trial and if the results are available, the procedure does not need to be repeated. 
However, if clinical warranted as judged by the investigator the ECG-12 lead should be repeated.  
 
Estimated time: 10 min.  
 
DXA scan 
Will provide BMD at the hip and spine, TBS (trabecular bone score) and body composition measures (lean 
body mass, fat mass and distribution, muscle mass). 
Overview  
DXA will be used to assess overall skeletal changes that often occur with age by measuring bone mineral 
content (BMC) and bone mineral density (BMD). In addition, total body fat and lean muscle mass 
measurements can give insight into the influence of age, sex, and race/ethnicity on the skeleton relative to 
these measures. DXA measurements can be used to determine the prevalence of osteopenia and 
osteoporosis. DXA technology has evolved from pencil beam to fan beam, allowing short acquisition time 
and improved image quality. In clinical practice, ‘areal’ bone mineral density (aBMD; g/cm2) assessment of 
lumbar spine (L1–L4), proximal femur (femoral neck and total hip) and forearm (distal) is made by central 
DXA. Interpretation of aBMD measurements is based on the World Health Organization (WHO) 
recommendations. Osteoporosis can be diagnosed if the value of aBMD is 2.5 or more standard deviations 
(SD) below the mean value of a young reference population (T score ≤−2.5). Central DXA can also provide 
whole-body imaging for total and regional aBMD, body composition (lean muscle and fat mass) and Vertebral 
Fracture Assessment (VFA). Using device-specific thresholds peripheral DXA may play a role in identifying 
those at risk of osteoporotic fracture, especially when there is limited or no access to central DXA75.  
The greatest increase in the DXA usage has been observed in the use of central densitometry76. As with other 
X-ray-based imaging methods, radiation dose from bone densitometry techniques that use ionizing radiation 
(DXA) must be kept as low as reasonably achievable.  
 
Radiation dose  
Various radiation dose parameters are used in diagnostic radiology, the most commonly being absorbed dose 
and effective dose. Absorbed dose, expressed in Grays (Gy), is a measure of the energy per unit of mass 
deposited in the tissue and organs of the body. Radiation dose from ionizing radiation is frequently quantified 
in terms of the effective dose. The effective dose, expressed in Sieverts (Sv), is calculated from information 
about absorbed doses to the organ or tissue exposed to X-rays and the relative radiation risk assigned to each 
of these organs or tissues.  The effective dose is a useful quantity for comparison among different sources of 

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ionizing radiation, such as that from DXA and natural background radiation. The worldwide average effective 
dose from natural background radiation is 2.4 mSv/year.  
Whole-body DXA is an established procedure for the assessment of skeletal mineral status of the whole body 
and the measurement of body composition. Effective doses for whole-body DXA examinations varies and 
were found to be between 0.001 and 0.01 mSv. Comparable with chest-x ray 0.01mSv and dental x-ray 
0,005mSv a low dose.  
We plan to perform Whole Body and central DXA scan to all subjects. Pregnancy status will be assessed on 
all fertile females. If the result of the pregnancy test is positive, the sampled participant will be excluded from 
the entire study.  Participants will receive a maximum of three DXA scans (whole body DXA scan, AP spine 
scan and femur scan). Under no circumstances should a whole-body scan be repeated. If the subject is 
recently scanned at Aalborg University Hospital within 6 months, there is no need for additional scan.  
 
Personnel  
Health technologists who are certified Bio. Med. Lab. Technologists will conduct all DXA scans.  
 
Estimated time: 45 min.  
 
HRpQCT 
Bone structure is an integral determinant of bone strength. The availability of HR-pQCT has made it possible 
to measure three-dimensional bone microarchitecture and volumetric bone mineral density in cortical and 
trabecular compartments of the distal radius and distal tibia, with accuracy previously unachievable and with 
relatively low-dose radiation.   
Analogously to DXA, radiation exposure from HRpQCT is low (0.01 mSv), although slightly higher if compared 
with DXA77.  
 
The scanner’s gantry is relatively narrow and shallow (rear physical stop) only allowing the distal peripheral 
skeleton to be accommodated. The limb being scanned is immobilized in a carbon fiber shell. A scout view, 
essentially a two-dimensional x-ray scan, is obtained so that the operator can identify a precise region for 
the three-dimensional measurement. Because HR-pQCT uses a polychromatic X-ray source it is subject to 
beam hardening as well as scatter artefacts, which can significantly impact geometric and densitometric 
measures. Once the images have been acquired, a default patient evaluation protocol is used to analyze the 
scans over the entire 9.02 mm three-dimensional region to assess a wide range of standard and optional 
structural and density parameters.  
HR-pQCT has been demonstrated to be able to discern between women and men with and without fractures. 
The inherent strength of HR-pQCT is its ability to assess a large slice of bone in three-dimensions for 
differences in cortical and trabecular characteristics and to identify those characteristics that are most 
associated with bone weakness, by FEA bone strength modelling. When compared with DXA aBMD, HR-pQCT 
measures have a better discriminatory ability to discern between women with and without fractures  
 
Estimated time: 20-25 min.  
 

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Microindentation 
Using OsteoProbe®, we will by microindentation measure the material strength index (BMSi), a measure of 
the hardness and stiffness of the cortical bone in the tibia. The participant will be positioned in  supine 
position with the leg to be measured rotated to orient the flat surface of the medial tibia diaphysis. The mid 
distance between the medial malleolus and the medial border of the tibia plateau are marked. Following a 
disinfection of the area using chlorhexidine,  Lidocaine is administered by inserting a syringe both 
subcutaneously and in the periosteal surface. A sterile probe is then inserted at the marked mid-diaphysis, 
penetrating the skin and periosteum until reaching the bone cortex. While maintaining probe contact with 
the bone surface, as well as orienting the probe perpendicularly to the tibia surface, the outer housing of the 
device is slid forward to the subject’s leg to initiate a measurement. After the first measurement, the probe 
is moved to a new location, at least 2 mm from the prior measurement, to obtain another measurement. At 
least 8 and maximum 18 indentations are performed on each subject. 
Estimated time: 20 min. 
Skin AGE Reader  
All subjects will be tested with the AGE Reader (Diagnoptics®, Groningen, The Netherlands), which is a non-
invasive device that uses ultra-violet light to excite autofluorescence in human tissue. The skin 
autofluorescence (SAF) is strongly correlated with AGEs. AGEs bind to type 1 collagen in both skin and bone. 
A study showed that levels of SAF was inversely associated with bone material strength, as confirmed by 
microindentation31. Thus, measuring AGE levels by means of SAF might be a reflection of bone AGEs and a 
potential biomarker of bone strength and fracture risk. The AGE Reader is validated in subjects with UV 
reflection on >6% corresponding to Fitzpatrick skin phototypes I-IV. 
Estimated time: 5 min. 
Assessment of Neuropathy 
Perception Threshold Tracking (PTT) 
Perception Threshold Tracking is a potential new method for early detection of neuropathy. It is a further 
development of conventional threshold tracking that excels in describing membrane potential of not only 
large, but also small nerve fibers. The trial is conducted using two types of surface electrodes placed on the 
dorsum of the foot: patch and pin electrodes for stimulation of large and small nerve fibers respectively. The 
perception threshold is then estimated by slowly increasing the intensity of stimuli until the subject presses 
a button indicating that the stimuli is perceived. As this happens, the intensity is initially heighted by 20 % 
and then lowered until the subject relieves the button indicating that the stimulus is no longer felt. The 
intensity is then lowered by 20% and then increased until perception is indicated by pressing the button. This 
is repeated three to five times to increase precision. The perception threshold is taken as the average of the 
six to ten times the subject pressed the button. This procedure will be repeated several times using different 
electrodes. The subject will feel no significant pain, as the stimuli given is just around the individual 
perception threshold, which is a huge benefit of this method compared to other available neural tests. There 
will be no risk of burns or injury to the skin, as the stimulator is limited to supplying electricity well below any 
dangerous level.  
Estimated time: 10-15 min. 

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NC-stat DPNCheck  
Gel is applied to the apparatus and the one-time electrode is connected. The device is then placed following 
the sural nerve from just behind the malleus and up the back of the leg. Using a short electrical stimulation, 
the device records the nerve conduction velocity and amplitude.   
Estimated time: 5 min.  
Skin biopsy 
Skin biopsy will be performed on the 40 subjects in the T2D N+ group, 40 subjects in the T2D F+ group and 
40 subjects in the T2D F-/N- group. After ensuring sufficient blood flow the skin is anesthetized using 0,5-1 
ml Lidocaine. After 3-5 minutes it is ensured that the skin is indeed numb. After confirming this, two punch 
biopsies are taken just above the ankle. The biopsies will be taken using a disposable 3-mm punch under 
usual sterile conditions. Potential bleeding is stopped by compression and the wound is bandaged as per 
normal clinical procedure. Before the subject leaves, it is ensured that the bleeding has stopped. Participants 
are informed to keep the wound dry for 2 days and to change the bandages if needed. Skin biopsies will not 
be taken if the participant has skin diseases in the biopsy area or has any known hematologic disorders 
resulting in markedly reduced ability to stop small bleedings.  
 
Skin biopsies will be analyzed at the Danish Pain Research Center, Aarhus University. Sections will be stained 
using the free-floating protocol, and IENF quantified following available guidelines. Additionally, 
regenerative-related antibodies will be used to identify potential biomarkers of early onset neuropathy. The 
antibodies used will include but are not limited toPGP and GAP-43. 
Estimated time: 15-20 min.  
Quantitative Sensory Testing  
Quantitative Sensory Testing (QST) is a way of assessing large and small sensory nerve fiber function using 
several different stimuli. The method has been verified using different protocols, and will be conducted in 
accordance with the original protocol by German Research Network on Neuropathic Pain (DFNS)78. All 
measurements are made on the dorsum of the foot. The protocol consists of seven tests measuring 13 
parameters including, but in this study, we will only assess thermal detection. The tests will be performed 
using a thermal sensory testing device. Cold detection threshold (CDT) and warm detection threshold (WDT) 
are measured and calculated as a mean of three consecutive measurements. The thresholds are to be 
obtained with ramped stimuli increasing by 1 degree Celsius per second, terminated as the subject presses a 
button. Cut-off temperatures are set at 14 and 50 degrees Celsius, and the baseline temperature is set at 32 
degrees Celsius, as this represents the mean value of skin temperature.   
Estimated time: 10 min  
Vagus Test Procedure 
A commercially available handheld device (VagusTM, Medicus Engineering Aps, Aarhus N, Denmark) will be 
used to test the autonomic nervous system and hereby the autonomic neuropathy. The participant will hold 
the device during four steps, where the device will 1) measure the heart rate at rest, 2) measure the heart 
rate response from laying position to standing position, 3) measure the relationship between heart rate 
during expiration and inspiration, and 4) measure the heart rate conditions during exhalation with a 

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resistance of 40mmHg and at rest. The latter will not be performed if the subject has known proliferative 
diabetic retinopathy or other known diseases with risk of retinal bleeding. The test is defined as abnormal if 
Cardiac Autonomic Neuropathy (CAN)-Score is ≥2. 
Estimated time: 15-20 min.  
COMPASS-31 
A questionnaire about symptoms of autonomic neuropathy (attached). Gives us the opportunity to measure 
non-invasive and stratify people with and without neuropathy. Will be filled on iPadby the participants, and 
entries are transferred to REDCap. 
Estimated time: 10 min. 
Michigan Neuropathy Screening Instrument (MNSI) 
MNSI (attached) will be used as a part of the screening process for diabetic neuropathy. MNSI includes two 
separate assessments: a 15-item self-administered questionnaire (filled in REDCap by the participants) and 
a lower extremity examination that includes inspection and assessment of vibratory sensation and ankle 
reflexes. Estimated time: 15 min. 
Assessment of muscle strength 
Isometric handgrip strength will be assessed by a hand dynamometer (NC70144, Procare.dk, Denmark). Each 
subject is instructed to exert maximal contraction force with their dominant hand (defined as their “writing 
hand”), when standing in an upright position and the hand tested pointing downwards, parallel to the body, 
and unsupported. Each subject repeats the test three times. The maximal contraction level of the three trials 
is selected for further analysis. 
Isometric measurement of knee extension strength will be measured with hand-held dynamometer. Similar 
procedures as for the arms.  
Estimated time: 5 min. 
Test for postural control  
Subjects will be tested standing on a force platform (Plux Biosignals S.A, Arruda dos Vinhos, Portugal) during 
quiet bipedal stance in four different sensory conditions each of 35 seconds: i) eyes open, standing on a firm 
surface, ii) eyes closed on firm surface, iii) eyes open on soft surface, iv) eyes closed on soft surface. The trial 
including four sensory conditions will be recorded in one sequence and repeated two times separated by 
short breaks (30-60 s) in between. Subjects are asked to stand in the middle of the force platform as quiet as 
possible. All tests are performed barefoot, and the feet position is marked on the platform itself and on the 
soft foam pillow, during soft surface conditions. Soft foam pillow characteristics are: 48x40x6 cm (length, 
width, height), density 87 kg*m-3 (O'live Balance pad, Denmark). If subjects cannot hold their feet close 
together during the test, the distance between the widest spacing of the inner feet is measured and verified 
before all trials. Subjects are allowed a maximum of three attempts for each sensory condition. However, if 
they fail to complete the full 35 seconds of recordings in either of these attempts, this particular condition is 
skipped and not included in the analysis. 
The vertical forces are extracted from the force platform (sampling rate at 1 kHz, Open Signals v. 1.2.8). The 
Center of Pressure (CoP) position versus time is extracted The Math Works, Inc., Natick, Massachusetts, 

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United States of America. First and last 2.5 seconds are excluded after low pass filtering (15 Hz Butterworth, 
2nd order and zero lag). The CoP range and velocity, Anterior-Posterior (AP) and Medial-Lateral (ML) 
directions, are calculated for each sensory condition. Average parameters from the three trials, for each of 
the four sensory conditions are used for analysis. The coefficient of variation for the four different conditions 
is extracted in a pilot study of seven healthy adults (aged 24-31years) and is <8.1% for both CoP range and 
mean velocity. Both CoP range and mean velocity are extracted in the 30s analysis window. The CoP range is 
extract subtracting the maximum value by the minimum CoP position and the mean velocity is calculated 
dividing the total CoP displacement by it respective time window (30s).  
Estimated time: 10-15 min. 
Assessment of gait function 
The Timed up and Go (TUG) test measures time acquired to walk three meters back and forth as fast as 
possible although running is not allowed. The subject starts from a sitting position in an armchair (seat height 
approximately 43-47 cm) and asked to rise from the chair on an “three, two, one, go” start-signal and walk 
three meters, then turn around at the mark on the floor, and walk back to the chair and sit down again. Time 
is measured from the “go” signal and stopped when the subject again is positioned in the chair using the back 
rest. All subjects are asked to wear supporting shoes and subjects not using such shoes are asked to perform 
the test barefooted.   
The Chair Stand Test (CST) assesses lower body strength and power through the ability to rise from a chair 
and sit back down. Test subjects are required to stand up from a standard chair to a fully extended standing 
position with their arms folded across their chest. From the sitting position, the subject stands completely 
up, then completely back down, and this is repeated for 30 seconds. The total number of complete chair 
stands (up and down equals one stand) is counted. 
Usual gait speed (UGS) takes place over 6 meters and composites of 4 meters of measuring, 1 meter of 
acceleration and 1 meter of deceleration. Subjects must walk at their own pace. Time <0,8 m/s (5 seconds 
on 4 meters), is viewed as cut off. 
Estimated time:  5-10 min. 
Assessments of OCT, Fundus and Visual Examination   
All subjects will be tested with Optical Coherence Tomography (OCT), fundus photo and visual examination. 
Eyes will not be dripped. OCT is a non-invasive scan of the retina to measure the size and layer in combination 
with a fundus photo of the retina to assess the visual nerve and macula. If the subjects recently have been 
scanned at Aalborg University Hospital within 3 months, there is no need of a new one in case of stable eye-
condition. The eye department at Aalborg University Hospital will assess the pictures.   
 
Estimated time: 30 min.   
5. DATA COLLECTION 
Patient data will be collected using the EPJ. Physical data achieved doing the study will be stored in locked 
desks with locked doors. The door will be locked if the room is left empty. Computer equipment is borrowed 

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by the North Jutland Region and is password protected in accordance with current guidelines. E-mails will 
only be sent through encrypted servers and mails, also in accordance with current guidelines.   
6. DATA ANALYSIS 
6.1. Sample size considerations 
The total number of individuals that will be recruited for the study is based on previous cross-sectional or 
exploratory studies on diabetes, skeletal parameters, neuropathy, fall risk and postural control45,46,79. The 
primary concern was to assemble a study population large enough to permit multiple testing. Therefore, by 
applying a risk of 5% for type 1 errors (2α) and a risk of 20% for type 2 errors (β), we estimated that 300 
people with T2D divided into the respective groups (F-/N-, F+ and N+) were acceptable for the procedures 
to avoid standard errors in analyses. 
6.2. Statistical methodology 
Mean and Standard deviation will be used as descriptive statistics for Gaussian distributed variables, 
median and 75 percentiles for skewed distributions. T-tests for two samples and multiple linear regression 
will be used for Gaussian distributed variables or variables who can be log-transformed. For non-Gaussian 
distributed variables non-parametric statistics will be used. 
6.3. Statistical significance 
We use a statistical significance level of α < 5 %.  
6.4. Missing data 
Missing data, interpretation errors, coagulation defects are considered missing values and will not be 
included in the analysis. In this case, each sample requisition is numbered (N). Furthermore, samples that 
cannot be measured in numerical values are categorized as missing values.  
6.5. Procedures in change 
The procedures for change in original study plan are stated at publishing scientific material. Furthermore, the 
local scientific committee and GCP-units are notified as required of these changes.  
7. DATA AND SAFETY MONITORING PLAN 
7.1. Data 
The project will be registered at the North Denmark Region Research Department according to the GDPR 
regulations. All sensitive data will be stored in either REDCap, on the protected server of Aalborg University 
Hospital or locally at DISK´s in DXA and HR-pQCT machines. Data will be stored anonymized after termination 
of the project.  
7.2. Establishing of biobanks 
Research biobank 
Blood will be stored in a research biobank for later analysis. The blood is cooled down to -80 degrees and 
stored. Blood is handled by the research bio. Med. Lab. Technologists to safekeeping of 
EthyleneDiamineTetraAcetic acid (EDTA)-full blood, EDTA plasma, lithium-heparin plasma and serum. 
Subjects will be asked permission for storage in the research biobank.  

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Biobank for future research projects 
Subjects will be asked permission to store an additional blood sample of 24 ml blood as well as an additional 
8 ml urine sample from the 24-hour urine sample in a separate biobank with the perspectives of conducting 
future research projects. Only around 10 % of the tissue from skin biopsies is used for analysis. If the subject 
consents, excess material will be transferred back to Aalborg for storage in the biobank for future research. 
If the subject does not consent, excess material will be destroyed after analysis. Subjects will receive separate 
written information material about the additional blood sample, urine sample and storage of the excess skin 
tissue and will have to sign a separate consent form. Material will be stored in the separate biobank for 15 
years after project termination or until a subject wants it destroyed. Use of material from the separate 
biobank will require permission from the regional ethics committee. Data will be handled in accordance with 
GDPR.  
 
The collected and stored material will be encrypted after the project has been terminated via a serial number 
which cannot be related to the test subject. This number can however be related to the test subject’s data 
and will be handled confidentially. If the test subject decides so, the material will be destructed after project 
termination. The project is covered by the North Jutland Region joint directory. The material will be stored 
with opportunity of identification in accordance with the North Jutland Region joint directory. This will be 
linked to an identification number if the approval from the North Jutland Region joint directory expires before 
new ways of use have been tested.  
 
The biobank will function as a database, where additional and potential important information can be 
collected. This database will be used in future studies. Reasons are:  
• 
Some biomarkers are in such low concentration that it is uncertain to perform routine analysis. In 
time, technology will improve thus providing an opportunity for new and smarter ways of testing. 
Therefore, this is of great research potential that the samples are kept.  
• 
Some biomarkers are not discovered and therefore not available doing the trial.  
There are found no risks associated with storage of these samples in the biobank.  
8. STUDY LIMITATIONS 
Some limitations must be considered in this study. First, all participants may not be able to complete all test 
procedures due to individual restrictions. Further, it is voluntary if subjects want to participate in skin 
biopsies, which is a limitation.  Both PTT and QST require corporation from the subjects and include subjective 
measures of pain. As the measured thresholds are not purely objective, psychosocial factors might influence 
test results.  
9. ETHICAL CONSIDERATIONS 
The trial will be conducted in compliance with Harmonised Tripartite Guideline for Good Clinical Practice (ICH 
GCP)84 and applicable regulatory requirements, and in accordance with the Helsinki Declaration85 for 
biomedical research involving test subjects. The methods used have been tested and performed in several 
studies both in Denmark and abroad and no long-term side effects have been reported. The risks associated 

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with the project are few as low dose radiation is used and the tests imply limited risks. The potential benefits 
in terms of well-being are large and are estimated to outweigh the potential risks.  
 
9.1. Informed consent 
Subjects interested in participating in the study will receive written information about the project (”Dine 
rettigheder som forsøgsperson i et sundhedsvidenskabelig forskningsprojekt” produced by Den National 
Videnskabsetiske Komité (NVK)) and a folder of the study (“deltagerinformation”)). If the subject is interested 
in participating in the project after reading the written information, they will be asked permission to access 
medical records for screening in accordance with the Danish law. Medical records will be assessed for 
information on comorbidities and diabetes complications, relevant diagnostic imaging, medications, and 
previous biochemistry to ensure the potential participant is suitable for inclusion. If suitable for inclusion 
after screening medical records, they will be invited for a personal meeting, where all details for the study 
will be outlined by the investigator or a delegated assistant. The subject will be informed about the 
opportunity to bring a lay representative in advance of the meeting. The meeting will take place in a closed 
room without any disturbances, e.g. phone calls. Subjects will be invited for an additional visit at our center 
to sign the informed consent (IC) form and further screening. After signing the IC, the subject will have blood 
samples taken for screening. Furthermore, we will assess status of neuropathy with questionnaires and 
physical examination including biothesiometry, NC-stat-DPNCheck, orthostatic blood pressure and Vagus 
test, and the subjects will receive equipment and instruction for the 24-hour urine sample to collect before 
the study day.  
 
The subjects must be fully informed of their rights and responsibilities while participating in the trial as well 
as possible disadvantages of potential risks. The investigator must ensure the subject has ample time to come 
to a decision whether to participate in the trial. A voluntary, signed, and personal dated IC must be obtained 
from the subject before any trial-related activity. The responsibility for seeking IC must remain with the 
investigator, but the task may be delegated by the investigator to a medically qualified person, in accordance 
with local requirements. The IC must be signed and personally dated by the person who seeks the IC before 
any trial-relate activity. Subjects will be informed that information given in relation to the project before 
signing the IC will be passed on to the investigator.  
If information becomes available that may be relevant to the subjects willingness to continue participating 
in the trial, the investigator must inform the subject in a timely manner, and revised IC must be provided and 
a new IC must be obtained.  
Participants have time for reflection and an additional informational meeting (with participating relatives 
etc.) will be scheduled.   
 
The given information will include:  
• 
That this a scientific project investigating the T2D bone phenotype and potential risk factors of 
diabetic bone disease including diabetic neuropathy.  
• 
Information regarding the different tests involved including risks.  
• 
That withdrawal from the project is possible at any time.  
• 
That data collected will be published in an anonymized form, without any traces of personal data  
• 
That data collected will be safely stored in accordance with the rules in force.  

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• 
The IC gives the investigator, the sponsor, and the sponsor’s representatives as well as any 
supervisory authority direct access to obtain information in the patient’s medical record, etc., 
including electronic medical records, in order to see information about the subject’s health 
conditions, which is necessary as part of the implementation of the research project as well as for 
control purposes, including self-control, quality control and monitoring, which they are obliged to 
carry out.  
 
9.2. Risks and side effects 
As the project is mainly observational and contains no interventions the overall risk for the subject is 
extremely limited. No changes/adjustments in medication will be made and there will be no risk for 
hypo/hyperglycemia. The main issue for the participants is the fact that some time is needed for the 
conduction of the examinations. Furthermore, a small amount of transient pain might be felt during some of 
the examinations; however, no lasting side effects are reported regarding any of the examinations. To 
minimize the risk of side effects the examinations will be conducted by trained personnel, and all equipment 
will be sorely tested before usage on participants.  
Different measurements may possibly reveal pathological conditions among subjects. The participants will 
be informed that the study might find additional medical conditions and they will be given the choice whether 
to be informed or not if this is the case. All findings, which are categorized “abnormal” or outside the normal 
range will be conferred with a medical doctor involved in the study, who will decide the plan and inform the 
subject of this. This might lead to further examinations, which will be offered to the subject. If the subject 
has declined the opportunity to receive any information, this will be respected.  
Listed below are the potential risks and side effects of the different examinations included in the protocol:  
• 
Blood and urine samples: Almost negligible risk of infection, small bleeding, or pain around syringe 
insertion. 88 ml blood will be taken from each participant including screening samples. If subjects consent 
for samples for the biobank for future research projects, additionally 24 ml of blood will be taken, 
bringing the total amount of blood taken to 112 ml. If any blood samples come back with elevated or 
potentially dangerous results, the staff will act accordingly and discuss these findings with a medical 
doctor. No side-effect regarding urine samples. 
• 
DXA and HR-pQCT: The risks of adverse reactions from the DXA or the HR-pQCT scans are minimal due 
to the low dose of radiation applied through the test. In theory this will increase the life-time risk of 
cancer from approximately 25% to less than 25,00002 %. No children or pregnant women are included 
in the project. The DXA scan may show sign of osteoporosis, which is an asymptomatically condition, 
mostly diagnosed after a fracture have occurred. Thus, it will be an advantage for subjects to have this 
diagnose assessed prior to any fracture occurrence. If osteoporosis is diagnosed, they will be referred to 
their general practitioner for further assessment and treatment.  
• 
Microindentation: Participants will experience slight discomfort from microindentation due to localized 
pain in relation to the insertion site and, rarely, hematoma. Complications such as hematoma from 
microindentation are rare. There is a small risk of infection and reaction to local anesthesia such as 
redness, itching and subcutaneous hematomas.PTT: Participants might feel a small amount of transient 
pain during the examination. No lasting pain will occur.   

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• 
Skin biopsy: Participants might feel a small amount of transient pain during and a small bleeding. No 
lasting pain will occur. There is a small risk of infection and reaction to local anesthesia such as redness, 
itching and subcutaneous hematomas. 
• 
OCT, Fundus Photography and visual examination: It is a common procedure performed by trained 
personnel. Eyes will not be dripped. 
• 
Vital signs and body measurements, 12-lead ECG, skin AGE Reader, Vagus™, QST, NC-stat DPNCheck, 
force platform, gait function and muscle strength assessments: No risks or side-effects. 
 
9.3. Benefits to subjects 
The study participants will not necessarily benefit directly from participation in the study. However, they 
will gain information about their general health including bone health, nerve function, balance and 
muscle function, body composition including fat and muscle mass and distribution. Individual 
participants may be diagnosed with illness(es) that may require treatment; in that case, they will be 
referred for relevant treatment. 
Furthermore, the findings might help them later due to a better understanding and prevention/delay of the 
condition. In addition, the study might help their fellow diabetics, as this will emphasis the awareness of 
fractures as a complication to diabetes, which both subjects and the scientific community sorely needs. 
9.4. Costs to subject 
No cost for the subjects is associated with this particular project.   
9.5. Compensation to subjects 
Transport expenses are covered according to current guidelines. No other economical compensation will be 
paid. In case of unforeseen side effects, the subjects will be compensated in accordance with the Danish 
Health Act and “Patienterstatningsordningen”.  
9.6. Provision for vulnerable subjects 
The participants are not expected to be any more vulnerable than the average patient is, and the trial does 
not contain any particularly distressing examinations.  
10. ECONOMY 
The initiative for this project has sorely been made by sponsor and main supervisor Peter Vestergaard 
and the primary investigator Julie LindgårdGraversen.  
The project is funded by the 25 mio. Steno National Collaborative Grant 2020, Closing in on sub-
segmentation in type 2 diabetes in the Danish nationwide DD2 cohort, funded by the Novo Nordic 
Foundation (NNF0063292). Neither of the investigators have any conflicts of interest to declare. 
Furthermore, funding is granted from the Steno Diabetes Center North Jutland who provides support by  Bio. 
Med. Lab. Technologists and support for analysis of all investigations. 

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11. PLAN FOR DISSEMINATION OF FINDINGS 
The study is planned to be submitted to internationally acknowledged scientific magazines. All data are 
published in anonymized form. The results will be published regardless of submission denial or whether the 
results are positive, negative, or inconclusive. Further dissemination might include national and international 
conferences and topical meetings.  The subjects will receive information of the findings at the end of the 
study.  The project will be prospectively registered to www.clinicaltrials.gov.   
12. INSURANCE 
The study is conducted at and covered by Aalborg University Hospitals insurance.  
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14. APPENDICES 
Compass-31 
See separate document. 
Michigan Neuropathy Screening Instrument (MNSI) 
See separate document. 

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Flowchart of study inclusion