Biomarkers of Metabolic Obesity.
Condition: Obesity
MeSH terms: Obesity
Study type: Observational

Summary: 
      The main aims of the work: Analysis of the concentration of selected pro-inflammatory
      parameters in blood serum and saliva of people with different distribution of subcutaneous
      and visceral adipose tissue. Analysis of metabolic obesity biomarkers in saliva and blood
      serum. Evaluation of new diagnostic methods for the early determination of the risk of
      metabolic obesity and its complications.

      Based on the inclusion and exclusion criteria the observational study was conducted among 119
      people (79 women and 41 men). Study group included 79 people (48 women and 31 men with
      obesity), and control group included 41 people (31 women and 10 men with normal body weight).

      The study consisted of three visits. The first included qualification for the study, body
      composition measurements, ankle-brachial index test and completing the questionnaires and
      3-day nutrition diaries. At the second visit blood was collected to conduct the morphological
      test. At the third visit saliva was collected to determine the proinflammatory cytokine and
      adipokine concentration.
    

Detailed description: 
      The main aims of the work:

        1. Analysis of the concentration of selected pro-inflammatory parameters in blood serum and
           saliva of people with different distribution of subcutaneous and visceral adipose
           tissue.

        2. Analysis of metabolic obesity biomarkers in saliva and blood serum.

        3. Evaluation of new diagnostic methods for the early determination of the risk of
           metabolic obesity and its complications.

      The study protocol was approved by the Bioethics Committee of the Medical University, no.
      R-I-002/647/2019 and APK.002.39.2021. All participants gave informed written consent to
      participate in the study before its commencement. The study began in January 2020 and ended
      in May 2022.

      The research was carried out at the Department of Dietetics and Clinical Nutrition (Faculty
      of Health Sciences of the Medical University of Bialystok), Biochemical Diagnostics
      Department (University Clinical Hospital in Bialystok), as well as at the Department of
      Biotechnology (Faculty of Pharmacy with the Division of Laboratory Medicine of the Medical
      University of Bialystok).

      The first visit took place in the Department of Dietetics and Clinical Nutrition at the
      Medical University of Bialystok. It included qualification for the study based on the
      inclusion and exclusion criteria, discussion of the purpose of the study and signing the
      written consent for the study by the participants, as well as information about the
      protection of their personal data and full anonymity. 200 people volunteered for the study.
      Based on the inclusion and exclusion criteria the observational study was conducted among 119
      people (79 women and 41 men). Each participant received an individual code (e.g. A01, A02,
      B01, B02 etc.), which was used to identify the participants in further stages of the study.

      All participants completed: The World Health Organization Quality of Life (WHOQOL- BREF)
      survey, International Physical Activity Questionnaire - IPAQ, Beliefs and Eating Habits
      Questionnaire created by Behavioral Conditions of Nutrition Team, Committee of Human
      Nutrition Science, Polish Academy of Science, and 3-day Nutrition Diary (participants
      described meals consumed for the next three days, including two working days and one day
      off).

      The participants of the study had measurements of anthropometric parameters (body weight,
      height, waist and hip circumference) and the BMI (Body Mass Index) was calculated. On its
      basis the participants were divided into the study and control group. Moreover, the WHR ratio
      (Waist to Hip Ratio), WHtR ratio (Waist to Height Ratio) and RFM ratio (Relative Fat Mass)
      were calculated.

      In order to determine the body composition of the study participants, an electrical
      bioimpedance body composition analysis was carried out using the devices: BioScan 920-2
      (Great Britain, Essex) and InBody 270 (South Korea, Seoul). The examination was performed in
      the morning, fasting, without any intense physical exertion.

      An ankle-brachial index test (MESI, Cracow, Poland) compares the blood pressure measured at
      the ankle with the blood pressure measured at the arm was also used in the study. This
      analysis was performed to assess the risk of lower limb atherosclerosis. Moreover blood
      pressure and resting heart rate using an electric sphygmomanometer was measured for all the
      study participants.

      The second visit took place at the Biochemical Diagnostics Department (University Clinical
      Hospital in Bialystok). 10 ml of venous blood were collected from all participants in the
      study. Oral glucose tolerance test (OGTT) was performed, and on its basis, the concentration
      of glucose and insulin was determined (fasting, and after 60 and 120 minutes of the test).
      Concentrations of total cholesterol, LDL cholesterol, HDL cholesterol, triglyceride,
      C-reactive protein, glucose, insulin, serum urea, serum creatinine, serum uric acid, alanine
      aminotransferase (ALT) and aspartate aminotransferase (AST) was assessed on ALINITY ci,
      Abbott.

      Blood samples were taken patients and frozen at -80 °C. Serum levels of total adiponectin
      (Quantikine ELISA Human Total Adiponectin/Acrp30 Immunoassay, R&D Systems, Abingdon, UK),
      resistin (Quantikine ELISA Human Resistin Immunoassay, R&D Systems, Abingdon, UK), IL-6
      (Quantikine ELISA Human IL-6 Immunoassay, R&D Systems, Abingdon, UK), TNFα (Quantikine ELISA
      Human TNFα Immunoassay, R&D Systems, Abingdon, UK), IL-1β (Quantikine ELISA Human
      IL-1β/IL-1F2 Immunoassay, R&D Systems, Abingdon, UK), IL-23 (Quantikine ELISA Human IL-23
      Immunoassay, R&D Systems, Abingdon, UK), MMP-9 (Quantikine ELISA Human MMP-9 Immunoassay, R&D
      Systems, Abingdon, UK) and total MMP-2 (Quantikine ELISA Total MMP-2 Immunoassay, R&D
      Systems, Abingdon, UK) were assessed with the enzyme-linked immunosorbent assay (ELISA)
      according to the manufactures' instructions.

      The third visit took place at the Department of Biotechnology (Faculty of Pharmacy with the
      Division of Laboratory Medicine of the Medical University of Bialystok).

      Saliva was collected using a standard method. Samples from the subjects were collected
      between 9:00 and 11:00 a.m. All subjects abstained from eating and drinking for 2 h. The
      subjects rinsed their mouths with deionized water and were sitting in a comfortable position
      with their eyes open and head titled slightly forward. Unstimulated whole saliva was
      collected for 10 min by spitting described by Navazesh. Saliva samples were homogenized and
      clarified by centrifugation at 1200 RPMI for 15 min at 4°C. The aliquots of clarified
      supernatants were stored at -70 °C for the ELISA measurements.

      Highly sensitive assay kits (R&D Systems, Minneapolis, USA) were used to determine the
      concentrations of IL-6, IL-1β, IL-23, resistin, MMP-9, MMP-2 and TNF-α in the salivary
      samples from the study participants. The tests were carried out according to the
      manufacturer's protocols. The microtiter plate provided in kits was pre-coated with
      monoclonal antibody specific for analyzed protein. Standards and samples were added to the
      appropriate microtiter plate wells. After the incubation at room temperature, enzyme-linked
      polyclonal antibody was added. Then, the microplate wells were aspirated and washed four
      times. Next, a substrate solution was added to each well. The enzyme-substrate reaction was
      terminated by the addition of a stop solution and the color change was measured
      spectrophotometrically at 450 nm ±2 nm. The antigen concentration in the samples was
      determined by comparing the O.D. to the standard curve. The antigen concentration in the
      samples was determined by comparing the O.D. to the standard curve.
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Non-Probability Sample
Study population: 
        200 people volunteered for the study. The participants came from the Podlaskie Voivodeship,
        Poland. The first visit took place in the Department of Dietetics and Clinical Nutrition at
        the Medical University of Bialystok. Based on the inclusion and exclusion criteria the
        observational study was conducted among 119 people (79 women and 41 men).
      
Keywords: obesity; saliva; blood serum; BMI; proinflammatory cytokines; proinflammatory adipokines; IL-6; IL-23; IL-1beta; resistin; TNF-alpha; MMP-2; MMP-9