Microcirculation and Bone Metabolism in Patients With Type 2 Diabetes Mellitus and Charcot Foot - A Pilot Study
Condition: Diabetic Angiopathies; Bone Diseases, Metabolic
MeSH terms: Bone Diseases; Bone Diseases, Metabolic; Diabetic Angiopathies; Diabetes Mellitus; Diabetes Mellitus, Type 2; Metabolic Diseases
Study type: Observational

Summary: 
      This study is part of a research project for a University MD Program. This is an
      observational study aimed at comparing the differences in bone metabolism and
      microcirculation in patients with type 2 diabetes mellitus (with and without diabetic
      neuropathy and Charcot foot) with healthy subjects.

      Diabetes is gradually becoming a global epidemic along with its associated complications.
      Diabetes can affect several systems in our body particularly the eyes, nerves and the
      kidneys. The damaging effects occur at the level of the small blood vessels
      (microcirculation) that supply these vital structures. Normally, the inner lining of these
      blood vessels (endothelium) plays a very important role in maintaining adequate blood flow.
      The endothelium releases a chemical substance called nitric oxide, which relaxes these small
      blood vessels thereby ensuring sufficient blood supply to these key structures. Nitric oxide
      also prevents blockage of these vessels. Any form of metabolic stress like hyperglycaemia
      (raised blood sugar as seen in diabetes) can cause abnormal changes in the normal behaviour
      of the endothelium (endothelial dysfunction). Therefore hyperglycaemia promotes endothelial
      dysfunction by lowering nitric oxide levels, which may lead to diabetic complications like
      diabetic retinopathy (eye damage), nephropathy (kidney damage) or neuropathy (nerve damage).

      In addition, patients with diabetes also suffer from osteoporosis (thinning of bones).
      Osteoporosis is a bone disorder characterised by a reduction in bone mineral content leading
      to an increased risk of developing fractures. The increased risk of fractures in patients
      with type 2 diabetes is attributed to poor bone quality resulting from the harmful effects of
      high blood glucose. Studies have also shown that nitric oxide has a bone protective effect as
      demonstrated by its ability to prevent bone fragmentation and improve bone strength.

      Study of markers of endothelial function and bone metabolism will facilitate a better
      understanding about the origin of diabetic complications. This will aid in the development of
      novel therapeutic agents that target the harmful triggers in diabetes and eventually may
      prevent and retard the onset of the debilitating diabetic complications.
    

Detailed description: 
      This is an observational study aimed at observing and comparing differences in bone
      metabolism and microcirculation in patients with type 2 diabetes and healthy controls.

      In this study, 50 participants will be recruited and then categorised into the following
      groups:

        1. Control group: 10 healthy volunteers (Hospital staff and spouses/ partners of patients
           will volunteer as healthy subjects)

        2. Type 2 diabetic patients without neuropathy: 10

        3. Type 2 diabetic patients with neuropathy:

             -  10 patients with painless neuropathy

             -  10 patients with painful neuropathy

        4. Type 2 diabetic patients with unilateral Charcot foot: 10

      Visit Schedule The estimated time for enrollment of patients will be within 3 months. The
      duration of the study is 5 months.

      Screening (-2 Weeks): To assess for eligibility. This will include informed consent process,
      medical history and physical examination, screening for neuropathy and routine bloods.
      Laboratory work-up done at screening will be considered for data analysis.

      Visit 1 - Baseline visit

        -  Anthropometric measures: Height, weight, BMI, waist circumference

        -  Vital parameters: supine and standing blood pressure, pulse, respiratory rate and
           temperature

        -  Laboratory assessment that includes markers of endothelial activation, inflammation and
           bone metabolism.

             -  Markers of endothelial activation will be investigated using systemic protein
                suspension array technology. For e.g. adhesion molecules like ICAM, VCAM and
                inflammatory molecules.

             -  Nitric oxide will be analysed using Griess assay

             -  Bone turnover markers [e.g., P1NP (Procollagen type 1 amino terminal propeptide),
                CTX (C-terminal cross-linked telopeptide of type-I collagen), Sclerostin, RANKL
                (receptor activator for nuclear factor (NF)-kB (RANK) ligand), OPG
                (osteoprotegerin), OPN (osteopontin), OCN (osteocalcin), BMP4 (Bone morphogenetic
                protein 4) and TGF-1β(Transforming growth factor-1β) - using ELISA (enzyme-linked
                immunosorbent assay).

             -  IL-6 - Interleukin-6, an inflammatory cytokine implicated in osteoporosis (using
                ELISA)

        -  Assessment of skin microcirculation with Laser Doppler Iontophoresis

        -  Assessment of calcaneal bone mineral density (BMD) with Bone Sonometer (ultrasound)

        -  Advanced glycation end-products will be assessed by an AGE reader which uses the
           technique of skin autofluorescence

      Clinical Procedures

      Assessment of the microcirculation with Laser Doppler Iontophoresis:

      A standard measurement of microcirculation is laser Doppler iontophoresis, which is used by
      several research institutes. In this trial the skin microcirculation will be measured on the
      dorsum of the foot using a Perimed Laser Doppler imager and iontophoresis system.

      Endothelial-mediated vasodilation will be measured by the iontophoresis of acetylcholine,
      while sodium nitroprusside will be used to measure endothelium-independent vasodilation. The
      iontophoresis system consists of an ION chamber (iontophoresis delivery vehicle device) that
      sticks firmly to the skin and a reference electrode. The response in blood flow will be
      imaged and quantified using the Perimed Laser Doppler Imager (PeriScan PIMII; Perimed,
      Sweden).

      Analysis of AGE:

      An AGE reader, which utilizes the principle of skin autofluorescence, will measure the
      accumulation of AGE in the skin. This validated device provides a real-time, non-invasive
      assessment of cardiovascular risk for chronic diseases like Diabetes, renal failure,
      cardiovascular disease, etc. AGE-modified proteins have autofluorescent properties and when
      excited by ultraviolet (UV) light they emit fluorescence in particular wavelengths. The
      levels of AGE in skin correlate with AGE levels in blood.

      The volar aspect of the forearm will be placed on the AGE reader, which is equipped with a UV
      light source. The UV light triggers AGE in skin, after penetrating 1mm of the dermis, to emit
      autofluorescence that is then detected by the AGE reader. The intensity of fluorescence
      correlates with the quantity of AGE in the skin.

      Assessment of calcaneal BMD:

      This is a simple and convenient method to assess peripheral BMD and assess fracture risk. The
      device used is a quantitative ultrasound called Sahara Clinical Bone Sonometer (Sahara
      Clinical Bone Sonometer; Hologic, Waltham, MA). The calcaneus is the preferred peripheral
      site to assess fracture risk. This device uses ultrasound waves to determine the BMD of the
      calcaneus. In this procedure, once the bare heel is placed in the device, the BMD is
      calculated within 30 seconds and the results are then generated on paper by the device.
    

Intervention name(s): Baseline comparison of microcirculation and bone metabolism
Intervention type(s): Other
Intervention description(s): Baseline comparison of microcirculation and bone metabolism among the 5 different groups
Observational model: Other
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        40 patients with type 2 diabetes mellitus will be considered eligible for the study.
        Patients with a previous diagnosis of type 2 diabetes mellitus and newly diagnosed type 2
        DM seen at Tameside Hospital will be recruited into the study. In addition, eligible
        patients will be identified and referred from University Hospital of South Manchester
        (UHSM) and appropriate GP practices (from Oldham, Tameside and Wythenshawe).

        Source of healthy controls: Hospital staff and spouses/ partners of patients will volunteer
        as healthy subjects.
      
Biospecimen description: 
      Analysis of serum samples for markers of endothelial function, inflammation and bone
      metabolism