IS THERE A DIFFERENCE IN THE PHTHALATE EXPOSURE BETWEEN ADULTS WITH METABOLIC DISORDERS AND HEALTHY ONES? DA LI POSTOJI RAZLIKA U IZLOŽENOSTI FTALATIMA IZMEĐU ODRASLIH OSOBA SA METABOLIĈKIM POREMEĆAJIMA I ZDRAVIH OSOBA?

INTRODUCTION: Phthalates are recognized as endocrine disrupting compounds and are extensively present in variety of every day products. The chronic exposure to phthalates is suspected to be associated with range of health disorders. The study aim was to examine the abundance of phthalate metabolites in the urine samples among adults in the Province of Vojvodina, Serbia, and to determine the prevalence of phthalate metabolites in healthy individuals and those with metabolic disorders such as obesity and newly diagnosed type 2 diabetes mellitus. MATERIAL AND METHODS: The first morning urine sample of 308 participants was screened for the presence of 10 phthalate metabolites: mono-ethyl phthalate (MEP), mono-(2-ethylhexyl) phthalate (MEHP), mono-n-butyl phthalate (MBP), mono-iso-allyl phthalate (MiAP), mono-n-allyl phthalate (MnAP), mono-cyclohexyl phthalate (MCHP), mono-benzyl phthalate (MBzP), mono-n-octyl phthalate (MOP), mono-n-propyl phthalate (MPP) and mono-methyl phthalate (MMP). RESULTS: At least one phthalate metabolite was detected in the first morning urine sample in 50.32% of the examined population. The most frequently detected phthalate metabolites were MEP and MEHP. Out of all phthalate positive participants, 38.3% of them had one, 10.7% had two, while 1.3% of participants had three phthalate metabolites in the first morning urine sample. Significant difference ( p <0.05) between groups was observed on MEP and MMP frequency, while borderline significant difference ( p <0.1) between groups was observed on MEHP and MCHP frequency. CONCLUSION: In Vojvodina region, both healthy adults and those with metabolic disorders such as obesity and newly diagnosed type 2 diabetes mellitus are predominantly exposed to Di-ethyl and Di-(2-ethylhexyl)phthalate metabolites. Further research is required in order to provide more details of the phthalates influence on the adverse health effects.


INTRODUCTION
Phthalates represent a large group of omnipresent industrial chemicals ordinarily used as plasticizers and can make up to 40-50% of the polyvinyl chloride plastic products weight.
They are known to act as endocrine-disrupting chemicals (EDCs) 1 . Phthalates can be found in food packaging, furniture, toys, and many other household products, but also in medical devices, such as tubing and intravenous bags. Phthalates are also popular in the cosmetic industry. Since they are not covalently bound to the plastic, phthalates can leach and transfer to the air, food, and water, and thus become inhaled, ingested or absorbed through the skin 2 . After being absorbed in the circulation, phthalates are metabolized in two phases: hydrolysis (monoester phthalates are produced), and conjugation. Phthalates are mainly excreted through urine, but they can be also detected in various fluids like blood (serum and plasma), breast milk, saliva, feces, etc. 3 .
Monoester phthalates have a fairly short half-life in humans. Despite this fact, numerous scientific evidence imply that phthalate diesters and monoesters can lead to health disorders such as mental retardation, body composition problems, as well as endocrine, pulmonary and cardiovascular diseases 4 .
Phthalates can activate different pathways, but nuclear receptors (NR) are recognized as a primary target 5 . Acting as a partial or complete agonist or antagonist, phthalates can alter NR signalling that is involved in the regulation of the metabolism and energy homeostasis.
By binding to the different components of the PPARs involved in the regulation of adipose tissue and lipid homeostasis, phthalates affect the fat distribution and alter the lipid status 8,9 . Moreover, through the PPAR-γ receptor component, phthalates could induce insulin resistance and impair glucose homeostasis. Besides genetic inheritance and lifestyle, chronic exposure to environmental pollutants, including chronic phthalate exposure, may attribute to the global epidemics of obesity and type 2 diabetes mellitus (T2DM) 10 .
The study goal was to examine the presence of phthalate metabolites among adults in the Province of Vojvodina (both healthy and with metabolic disorders) in order to find the most abundant metabolites. An additional aim was to determine the prevalence of phthalate metabolites in the control group, obese and group of participants with newly diagnosed T2DM.

RESEARCH DESIGN AND METHODS
In total 308 volunteers aged 18-50 years from Vojvodina region, Serbia, were enrolled in a cross-sectional study. The participants were divided into 3 groups: 103 in the control, 104 in the obese (body mass index (BMI) >30kg/m 2 ) and 101 in the group of volunteers with newly diagnosed T2DM (fasting plasma glucose value >7.0mmol/l), without medical treatment.
Participants with a history of chronic diseases such as dyslipidaemia, autoimmune disease, chronic infections, malignant disease, or those with possible or proven pregnancy or lactation were not involved in the study. Volunteers who were treated with any kind of medication that could affect the lipid status or the body composition (such as hypolipidaemia, glucocorticoids, oral contraceptives or immune-suppressive drugs) were not included in the study.
The study participants provided written informed consent and the study protocol was approved by the Ethics Board of the Faculty of Medicine, University of Novi Sad, Serbia.
All subjects who decided to withdraw their informed consent were excluded from the study.
Firstly, all participants were surveyed and asked specific questions about their medical and personal history. Afterward, anthropometric values such as weight, height, waist circumference were taken and BMI was calculated using the following formulaweight/height 2 (kg/m 2 ). Waist circumference was measured in the middle of the line joining anterior superior iliac spine and rib arc.
The first morning urine sample of the volunteers who participated in this study was screened for the presence of 10 phthalate metabolites: mono-ethyl phthalate (MEP), mono- After enzymatically treatment of collected urine samples, methyl-tert-butyl-ether was used as a solvent for the extraction of phthalate metabolites. The samples were prepared and analyzed by the previously developed method accurately described by Milošević et al. 11 Gas chromatography coupled to mass spectrometric detection (Agilent GC 7890A, 5975C VLMSD) equipped with a fused silica capillary column (30 m, 0.25 mm i.d. and 0.25μm film thickness; J&WScientific, Folsom, CA, USA) was used for the determination of phthalates residues in urine. The limit of detection (LOD) for 10 phthalate metabolites was 0.25 ng/ml. Separate groups were designed for each phthalate metabolite dividing them between phthalate-free and phthalate positive samples (binary distribution), as the span of positive values was too wide so that standard deviations would exclude valuable patients.

Statistical analysis
The data were analyzed using the Paerson's Chi-squared test (in cases of low number of positive phthalate values coefficient of contingence was used) with the significant results being recorded at p<0.05 and p<0.1. The statistical analyses and graphical representation were done using SPSS 23.0 (SPSS Inc., Chicago, Illinois, USA) and MS Excel Package.

General characteristics of the studied group
General characteristics of the analysed population such as male to female ratio, sex, height, age, body weight, waist circumference and BMI values are shown in Table 1.

Statistically significant differences in age, body weight, waist circumference and BMI
were observed between studied groups.  The frequency of the detection for the analyzed phthalate metabolites in each observed group was compared, respectively, and the results are presented in Fig. 1.  had p values less than 0.1 and therefore were considered as significant and will be further discussed.

MEP
In the examined population of 308 volunteers, a significant difference was seen on MEP frequency between control group and T2DM volunteers (χ²= 5.058, df= 1, p=0.025).

DISCUSSION
Many chemicals which presence in nature has been increased after the industrial revolution can act as endocrine disruptors by interfering with endogenous hormonal pathways.
Epidemiological studies 12-14 have shown the link between exposure to these chemicals and the development of common disorders and diseases such as obesity and T2DM. Taking into consideration that pathogenesis of these disorders depends on the combination of lifestyle habits and genetics, it is lately hypothesized that exposure to endocrine disruptors during or after pregnancy can play a significant role in the onset of some diseases 15 . Phthalates are usually found in large quantities in daily products. The number of publications that investigate the positive linkage between phthalates exposure and adipogenesis and T2DM, some of the largest epidemics of the modern world, increases continuously. Although chemical industry representatives assert that levels of phthalates found in the human body are well below the "safe" concentrations by some regulatory agencies, endocrinologists consider that phthalates exposure even at low doses, during vulnerable periods, can lead to adverse health effects 16 . Although it is estimated that the average level of human exposure to DEHP is around 0.0024 mg/kg per body weight per day, much below the current DEHP "No Observed Adverse Effect Level" (NOAEL) by the European Food Safety Authority, the chronic exposure even at low doses could be more harmful than single acute exposure to high dose 17 . Natural hormones are active at pico to nanomolar range. Hence, phthalates as EDCs might ameliorate hormone homeostasis and cause biological impact at low doses 18 . According to literature data 12,19 , fetuses, newborns and adolescents are vulnerable groups and particularly susceptible to phthalate exposure, which is explained by the high levels of cell activity in those age groups.
The obtained results showed that the urine sample of 50.32% participants was positive for the presence of at least one phthalate metabolite.
The ubiquitous presence of phthalates in human urine samples is documented in the study published by Zota et al. 20 . Eleven phthalate metabolites were analysed in the urine sample of more than 11000 adults and children, and data from five cycles of NHANES (National  28 . The precise mechanism by which phthalates influence these PPARmediated actions is expected to be explained with further experiments.

Limitations and advantages of this study
This study focused only on a middle-aged cluster of white (Caucasian) volunteers, hence, the results cannot be extrapolated to other ethnic and other age groups. Being conducted as a cross-sectional study, this research has a risk of bias of selection. Thereby, further studies are needed to confirm the present data. Further studies are also needed for clustering the geographical, age, ethnic, sexual, and other characteristics.
In the current study, urine was used as a matrix for measurements of phthalate metabolites.
The advantage of urinary measurements is that, apart from low cost and non-invasive methods of obtaining samples, usually higher levels are found compared with serum and thereby more phthalate metabolites could be measured above the lower detection limit.

Conclusion
Approximately half of the examined group (50.32%) had at least one phthalate metabolite in their urine sample. Our study showed that the most abundant phthalate metabolite present in the group of obese participants was MEP while MEHP was the most common phthalate metabolite in T2DM group. Group of healthy individuals had the highest percentage of presence of MEP amongst examined phthalate metabolites. The obtained results indicate that in Vojvodina region, both healthy adults and those with metabolic disorders such as obesity and newly diagnosed type 2 diabetes mellitus are predominantly exposed to widespread DEHP and DEP phthalates.
Further research that will provide more detailed insight into phthalate interference with glucose and lipid metabolism and their influence on the endocrinological balance is needed.