On this page we will introduce methodology used in our studies.
How do we do it?
Oral glucose tolerance test (OGTT) is performed mainly to find out whether an individual has developed diabetes or pre-diabetes (elevated fasting glucose or impaired glucose tolerance).
Study participant drinks rapidly 75g of liquid glucose (sugar). Blood samples will be drawn before and 30, 60 (90) and 120 minutes after drinking, to measure glucose and insulin concentrations.
In a normal situation, when glucose absorbs into blood circulation and blood glucose starts to elevate, pancreas secretes insulin into blood circulation. Insulin is a hormone that helps the body to take glucose into cells and use it as an energy.
Insulin deficiency: Without insulin, blood sugar elevates rapidly and remains at high level.
Impaired insulin sensitivity (insulin resistance): Pancreas produces insulin and secretes it into blood circulation but the efficacy of insulin is poor (impaired insulin sensitivity), and although the insulin level is high, the blood glucose elevates.
OGTT= Oral glucose tolerance test
Body mass index (BMI) describes the ratio of weight and height in humans. BMI can be calculated with a simple formula [weight in kg/ (height x height in metres)] or it can be checked from a table. The unit of BMI is kg/m2.
The interpretation of BMI: 20-25 (normal), 25-30 (overweight), >30 (obesity)
In most cases, high BMI indicates obesity, but large muscle mass (for example body builders) can also lead to high BMI.
Waist circumference reflects the fat accumulation in stomach and around internal organs (abdominal obesity). If the waist circumference exceeds 100cm in men and 90cm in women, it indicates abdominal obesity.
By measuring also the hip circumference, hip-waist ratio can be calculated (waist-to-hip), which reflects the distribution of fat. World Health Organization (WHO) has defined that hip-waist ratio over 0.95 in men and over 0.85 in women indicates abdominal obesity.
The body composition (how large portion of the weight is fat) can be measured with two different methods. In the older method Bioimpedans, electrodes are connected to wrist and ankle. In the more recent method, person stands on a scale, a mild electric current goes through the body and the scale measures the resistance of the current. Based on these resistance measurement, the fat percentage can be estimated when persons gender, age and weight is known.
GAD-antibodies: antibodies against Glutamate decarboxylase enzyme.
In Type 1 diabetes, the immune system of the body attacks against the beta cells in pancreas that produce insulin. As a result, some of the beta cells die, which can lead to insulin deficiency. The immune system’s war against its own tissues is called autoimmunity.
You cannot measure the amount of beta cells and it is not known why the immune system attacks against them. The presence of antibodies in the blood circulation is considered to be a sign of the process (“war”). 85-90% of the children that are diagnosed with Type 1 diabetes, have different pancreas-antibodies in their blood circulation. On the other hand, adults with Type 1 diabetes have often only GAD antibodies. About 10% of the adults with Type 2 diabetes have GAD antibodies – these patients are called LADA (Latent Autoimmune Diabetes in Adults). Half of the LADA patients later develop significant insulin deficiency and a need for insulin treatment.
What is the meaning of GAD antibodies for people who do not have diabetes? GAD antibodies increase the risk of getting diabetes. The degree of risk depends on the level of the antibodies. Not all the people that have GAD antibodies will eventually get diabetes.
Diabetes leads to changes in both small (fundus=retinopathy; kidneys=nephropathy), and large blood vessels (coronary artery disease, heart infarction, stroke and blood circulation disturbances in legs). In addition, especially the function of the long nerves can be damaged (neuropathy).
Retinopathy is examined with the help of fundus photography. Nephropathy can be detected by measuring the albumin secretion in urine, which is divided in microalbuminuria (night urine 20-200ug/min) and macroalbuminuria (night urine over 20-200ug/min). Neuropathy can be detected with a clinical examination, when the activity of the nerves in the lower limbs are examined by either a neuropathic meter, a sound iron (vibration), a monofilament (touch sensation), or more complex methods such as ENMG (electronuromyography).
Data collection on the complications of diabetics participating in the study, have been done in collaboration with the health centers involved in the study. During the study visit, samples are taken to evaluate known risk factors (blood lipid levels, night urine albumin secretion, HbA1c). In addition, blood pressure, EKG and waist circumference will be measured and with the help of questionnaires, exercise habits, alcohol consumption and smoking habits will be monitored. The goal is to find out the factors that are connected to the elevated risk of getting complications. The special interest of Botnia study has been to examine the genetic and environmental factors predisposing to diabetic complications.
The illnesses of the study participants are also followed through the Hospital- and Primary Health Care Removal Register (HILMO and AvoHilmo) maintained by the Institute of Health and Welfare (THL). In addition, the causes of death are monitored through the mortality register maintained by Statistics Finland.
We also investigate the prevalence of diabetic complications in different subgroups, such as in MODY, LADA and in families that have both Type 1 and Type 2 diabetes among family members (MIX study).
The UKK walk test measures the physical fitness of an individual. Participants walk 2km distance as fast as possible and at the finish line, the duration of the walk and pulse are registered. The duration of the walk and pulse together with BMI, age and gender it is possible to estimate individuals oxygen uptake (unit ml/kg*min).
Fitness index is measured with the help of the same variables, which are adjusted to gender, age and weight. Normal fitness index is 90-100, lower value indicates poor physical fitness and higher value better fitness than average.
The Botnia clamp includes two separate examinations that measure insulin production and insulin sensitivity and that are performed one after the other during a same day.
First part that measures insulin production, is called Intravenous Glucose Tolerance Test (IVGTT). A plastic cannula (“IV drip”) is placed into the crook of the elbow and glucose is administrated to the vein. When glucose enters the blood circulation, the blood glucose level starts to elevate and pancreas secretes insulin to the circulation. Blood samples are drawn before and 2, 4, 6, 8, 10, 20 after the glucose injection, in order to measure how much insulin is being secreted and how high the glucose level will rise before insulin fixes it.
The difference between glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) is that when you get the glucose orally it affects also the stomach and gut. The peptide hormones in the gut are important for glucose metabolism and the rate of glucose absorption into the gut affects the results of OGTT. IVGTT measures only the ability of the pancreatic cells to produce insulin.
The second part that measures insulin sensitivity is called "euglycemic hyperinsulinemic clamp". It measures the insulin sensitivity, in other words how much glucose the body the body utilizes when insulin is given. During the examination, insulin and glucose are administrated simultaneously to the blood circulation via intravenous drip. Blood glucose level is kept steady, by either adding or reducing the glucose drip. If the individual is insulin sensitive, it is necessary to give a lot of glucose in order to keep the blood glucose steady and if the individual is not sensitive to insulin, hardly any of glucose is needed.
During the Botnia-Clamp, energy consumption is measured through an indirect calorymetry. The examination take about 30 minutes, and during this time the study participant wears a transparent plastic helmet and breathes inside of it. Fresh air current runs through the helmet and the oxygen and carbon dioxide contents of the exhale air are measured. With the help of these measurements, it is possible to calculate the body’s energy consumption.
Likewise Botnia-Clamp, the GITT-test measure both insulin production and insulin sensitivity, but the implementation is simpler.
In the first part (Glucagon-C-peptide-test), glucagon hormone is injected to the vein. Glucagon elevates blood glucose and accelerates insulin production. The blood samples are drawn before and 6 and 20 minutes after the injection to measure blood glucose and C-peptide levels. Actually pancreas produces one bigger pre-hormone called proinsulin that is split into two parts: insulin and C-peptide. C-peptide measures insulin production.
In the second part, insulin is injected to vein and blood glucose levels are measured before and after the injection, in order to find out how fast the blood glucose level goes down. The faster the blood sugar goes down, the more insulin sensitive the person is.
Biopsies can be used to study the changes in the metabolism of tissues and the expression of diabetic genes in the different tissues. All the tissues and cells contain the same genes but they do not operate in all tissues. Some of the genes are operating (expressing) in certain tissues and some genes in other tissues. In addition, the genes can be turned off or be over activated in some conditions such as diabetes (expressing can be decreased or increased).
After local anesthesia, a small slash is made to the skin and sample is taken with a biopsy needle. The sample is taken usually from the quadriceps and/or from the fat tissue of the stomach or the buttock. The wound is sewed with two stitches, which will be taken away after a week. After the biopsies, the examinee has to avoid a heavy muscle exercise until the stitches have been removed.
It is possible to measure energy consumption, by registering body movements and heart rate levels. In our intervention study, we have followed the energy consumption of the study participants using combined accelerometer and heart rate monitor (Actiheart; www.camntech.com), which monitors the physical activity of an individual by measuring movements ("sensitive accelerometer") and heart rate. The device includes two small electrodes that are fastened to the skin.
The monitoring results are uploaded into a computer program that calculates energy consumption and the duration of physical strain in different exertion levels (MET-level).
A test meal stimulates insulin secretion not only through glucose, but also through other nutrients, and is performed for individuals taking part in various studies such as zinc and hypoglycemia study. The test meal used in Botnia Study includes 77 g glucose and 518.7 kcal. A test meal is eaten after overnight fasting during 10 minutes and blood samples including glucose, insulin, glucagon, pro-insulin, c-peptide, free fatty acids, incretin hormones GLP1 and GIP are drawn at fasting state and every 30 minutes until 120-180 minutes after finishing the test meal.
Urine is traditionally used for the diagnostics and monitoring of the diabetic kidney disease (e.g. albumin to creatinine ratio).
The urine research field is currently focused on screening for more precise and specific novel biomarkers (see extracellular vesciles below) that could detect the disease or alert about fast progressing disease earlier than the traditional methods.
Urine analytics has the benefit that urine can be collected non-invasively unlike plasma samples and biopsies.
Albumin-to-creatinine ratio: The concentration of albumin in urine is indicative of the functioning of the kidney i.e. it tells how much protein leaks into the urine through the kidney. High concentration indicates deterioration of the kidney function. The concentration of albumin is typically determined as a ratio to creatinine in order to overcome the differences in urine concentration caused by differential fluid intake.
Extracellular vesicle research: Extracellular vesicles (EVs) including exosomes are small (app. 30-1000 nm) membrane-bound sacs secreted by all cells. EVs act in cell-to-cell communication transferring their rich molecular content, received as building blocks and messages, between cells. The molecular information of EVs has been found to reflect different pathological states. EVs are found in all body fluids including urine, from where they can be isolated using many techniques such as ultracentrifugation. The molecules within the EVs, such as RNA, can then be further purified and studied using global molecular detection techniques (omics). The analytics requires super sensitive methods due to the small size of EVs and therefore small sample amounts.