Biomarkers of Metabolic Obesity.

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.

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.

Eligibility Criteria:
Inclusion Criteria:

* women and men aged 20-55
* primary obesity
* certificate from the dentist confirming the absence of periodontitis and inflammation in the oral cavity.

Exclusion Criteria:

* secondary obesity,
* type I and II diabetes,
* exacerbated coronary artery disease,
* endocrine disorders,
* eating disorders,
* pregnancy and lactation period,
* use of hormonal contraception or hormone replacement therapy, steroid therapy, antiretroviral therapy,
* surgical or pharmacological history obesity treatment,
* pacemaker