Evaluation of the Relationship between Insulin Resistance and 8-Iso Prostaglandin Levels in Obesity Children

Evaluation of the relationship between insulin resistance and 8-iso


INTRODUCTION
Obesity is defined as an increase in energy intake over energy expenditure and an increase in fat tissue in the body. Excessive energy intake causes the accumulation of adipose tissue in the body. Today, obesity is considered among the diseases that carry the most important risk of mortality and morbidity, together with the complications it brings. The World Health Organization (WHO) has ranked obesity in first place among the most important diseases. [1] It is known that obesity causes a number of diseases, such as cardiovascular diseases, insulin resistance, oxidative stress, and dyslipidemia. An increased body mass index (BMI) poses a serious risk to body health, and those with a BMI above 30 are considered obese. Obesity directly leads to insulin resistance, increasing insulin levels in the blood. [2] The need for more insulin to produce a normal biological response is called insulin resistance. Insulin resistance has been demonstrated in almost all type 2 diabetes patients. [3] Obesity has become a major health concern for both children and adults as a result of factors such as improved economic conditions, increased consumption of readymade foods in recent years, children spending more time at home due to technological advancements, shrinking playgrounds, and climate change. [4] According to some reports, one-third of obese children grow up to be obese adults. Furthermore, different disorders are observed in these people at an adult age. One of the issues that the health community is most concerned about is the fact that childhood obesity will rise in the twenty-first century. [5] Obesity has become more prevalent in Western societies, according to studies conducted on children aged 6 to 19. [6] While the prevalence of being overweight is 13.9% on average in the children of our country, it has been reported that the average prevalence of obesity is 4.8%. [7] However, a study on children in Turkey found that obesity was increasing in both sexes. [8] Childhood obesity can be said to be attributed to genetic and familial factors, psychological factors, a sedentary lifestyle, and poor eating habits. [9] Fatigue, difficulty in breathing, and extremity pain are observed in very few of obese children. It is observed that they have a high appetite, they eat carbohydrate and fat-based diets, and they are reluctant to consume fruit and vegetables. [10] Reactive oxygen species (ROS) play a role in the pathophysiology of many diseases, including cardiovascular diseases, obesity, type 2 diabetes, and atherogenic mechanisms. It is stated that oxidative stress is associated with the formation of adipose tissue, which contributes to the development of obesity and metabolic syndrome. [10] The level of antioxidant capacity decreases with obesity and the increase in adipose tissue. [11] In obesity, mechanical work force and myocardial mechanism increase and oxygen consumption accelerates. Increasing oxygen consumption accelerates the formation of ROS. [12] Isoprostanes are products formed by the nonenzymatic peroxidation of polyunsaturated fatty acids such as ara-chidonic acid induced by free radicals. Isoprostane and prostaglandins are called 8-iso prostaglandin F 2α because they are isomers of prostaglandin F 2α formed by cyclooxygenase. [13] 8-iso-PGF 2α has been measured as an indicator of lipid peroxidation in various body fluids such as urine, blood, bile, pericardial fluid, cerebrospinal fluid, and in tissues such as the brain and liver. [14] 8-iso-PGF 2α , an indicator of oxidative stress and lipid peroxidation, has been studied in cardiovascular diseases, lung diseases and neurodegenerative diseases. [15]

AIM
The study aimed to evaluate the relationship between insulin resistance and the oxidative stress biomarker 8-iso-PG-F 2α levels in obese children in our country where parent-child association is high.

MATERIALS AND METHODS
The study included 44 children, 21 boys and 23 girls, between the ages of 6 and 15, with obesity-related diagnoses who attended the Faculty of Medicine Pediatric Endocrinology Department at Kahramanmaras Sutcu Imam University between December 2020 and June 2021. The control group consisted of 40 children (20 boys and 20 girls) who were free of any systemic diseases.

Collection and storage of samples
Venous blood samples taken from the children in the patient and control groups in the morning after 8-12 hours of fasting were placed in tubes that did not contain anticoagulant substances. Then, the blood samples were centrifuged at 4000 rpm for ten minutes and their serum was separated with a Hettich centrifuge device. Glucose, triglyceride, total cholesterol, HDL cholesterol, and insulin analyses were performed. Furthermore, a sufficient amount of serum samples were separated for 8-iso PGF 2α analysis and stored in a deep freezer at −80°C until analysis.

Anthropometric measurements
Systolic and diastolic blood pressure measurements were made with an Oncomed brand sphygmomanometer on the right arm in a sitting position after 10 minutes of rest. Using height and weight measurements, percentile values of the control group and obese children were calculated according to sex and age. Homeostasis model assessment (HOMA) was used to determine insulin resistance. [16] Fasting insulin (U/ml) × Fasting glucose (mmol/L) HOMA-IR = 22.5

Biochemical measurements
For the analysis of serum 8-iso-PGF 2α level,

RESULTS
Considering the descriptive values, when the obese child and control groups were compared, it was seen that there was no difference between the two groups in terms of sex distribution and age. It was found that the height and weight of the obese children were significantly higher than those in the control group (Table 1). Significant differences were observed between the two groups in terms of age, body mass index, and systolic and diastolic blood pressures (p<0.05). The systolic and diastolic blood pressure of the obesity and control groups are presented in Table 7. The lipid profiles of obese children and control groups were compared and TG, TC, and LDL values were found to be significantly higher in obese children compared to the control group, while HDL values were found to be significantly lower than the respective values in the control group (Table 2).
Obese children and control groups were compared in terms of 8-iso-PGF 2α levels which were found to be significantly higher in obese children than in the control group (Table 3).
A moderate correlation was found between HOMA and 8-iso-PGF 2α levels in obese children (Fig. 1).  When obese children and control groups were compared in terms of BMI and 8-iso-PGF 2α levels, BMI and 8-iso-PGF 2α values of obese children (32.03±4.53) were found to be significantly higher (p>0.001) compared to those in the control group (17.10±2.32) (Fig. 2).
Relationships between descriptive values, routine, and biochemical parameters were also examined in all children, and accordingly, a significant positive correlation was found with children's TG, HOMAR, BMI, glucose, LDL, TC, and 8-iso-PGF 2α levels. HDL and insulin levels were found to  be significantly negatively correlated. The Spearman correlation analysis between groups is shown in Table 4.
Since p<0.05, it was determined that the correlation coefficient was significant. Table 5 shows that 8-iso-PG-F 2α levels are highly positively correlated with the control group and obesity.
Our ROC analysis showed that the measurement of 8-iso-PGF 2α showed a high level of accuracy (AUC=0.966) and a sensitivity of 100%, making it appropriate to trust it (Fig. 3, Table 6).

DISCUSSION
Obesity is a chronic, progressive disease characterized by psychological issues that limit physical activity. The prevalence of obesity is increasing rapidly in both developing and  follow-up. [19] BMI values vary according to age and sex in children. Therefore, BMI percentile curves prepared for Turkish children were taken into account when determining obese and control children during the study. [20] In the study, those with 95th percentile values and above were determined as the obese group, and children between the 5th and 85th percentiles were determined as the control group. Therefore, the BMI values of the obese children were found to be higher than those of the control group. Candido et al. and Mantovani et al. obtained similar study results. We think that the BMI values of obese children are an indicator of increased adipose tissue in their bodies. [21,22] When obese children and control groups were compared in terms of glucose and insulin levels, there were significant differences seen in the study. In our study, glucose levels were found to be higher in the obese children group than those in the control group, while insulin levels were lower in the obesity group than in the control group. There are studies in the literature in which both insulin and glucose levels are higher in the obesity group than in the control group. [22,23] In addition, there are studies similar to ours that evaluated significant differences between glucose levels and insulin levels. [24] Insulin sensitivity is best assessed using the clamp technique. However, the invasive nature of the clamp technique makes it difficult to use it in practice. Instead of this technique, insulin sensitivity is evaluated according to the fasting insulin values and the insulin values measured during the oral glucose tolerance test. For this purpose, the insulin sensitivity index and the HOMA index are frequently used, and these methods are reported to be correlated with the hyperglycemic clamp technique. [24] There are studies showing that HOMA can be used to evaluate the sensitivity of insulin resistance and can be applied precisely and easily. [25] Similarly, the HOMA index was used for IR evaluation in the study. It can be said that the increase in fat mass is a factor in the high HOMA values in obese children in the study. In addition, the lack of sufficient number of receptors despite the increase in insulin requirement can be considered as another factor.
There are studies which evaluated different plasma lipid levels in obese children. In general, studies on obese children have shown that HDL levels decrease as the degree of obesity increases. On the contrary, increases are observed in triglyceride and LDL levels. [26] Likewise, Reinehr et al. developed countries. This disease affects children as well as adults negatively day by day. In our country, the problem of obesity has not been emphasized much; moreover, many families have adopted the view that an overweight child is healthy. Today, it is known that there is a relationship between obesity and cardiovascular diseases, type 2 diabetes, lung diseases, and neurodegenerative diseases. In addition, it is well known that obesity begins in childhood in most obese adults. [17] Lifestyle, feeding frequency and type, child's birth weight are the factors that should be focused on the mechanism of childhood obesity. Children born to obese parents are more likely to be obese. While the concept of obesity is taken into account in adults, it is observed that there are deficiencies in childhood obesity. [17][18] Considering that obesity is a common health problem in the society, a cost-effective and easily applicable method should be used in diagnosis and stated in their study that there were decreases in the serum HDL levels in obese children. [27] In our research, we found that the triglyceride, total cholesterol, and LDL levels were significantly higher in obese children than those values in the control group. In addition, HDL levels were observed at lower levels compared to the control group. Our research result parameters are similarly supported by the study of Boyd et al. [26] In addition, correlations between parameters were made in children and it was shown that there is a positive relationship between the triglyceride, total cholesterol, and LDL levels.
A study found that plasma 8-iso-PGF2 levels, which are used as an indicator of oxidative stress, were significantly higher in obese children than in the healthy control group. It is seen that there is a direct correlation between plasma 8-iso-PGF 2α level and visceral adipose tissue. In addition, there was no significant correlation between subcutaneous adipose tissue and plasma 8-iso-PGF 2α levels in nonobese children. It is stated that 8-iso-PGF 2α is a strong and independent determinant of visceral adipose accumulation. [33] In a study including 58 patients, Kelly et al. found that the plasma 8-iso-PGF 2α levels of obese children were higher than those in normal children. High levels of plasma 8-iso-PGF 2α can be interpreted as the possibility of a higher incidence of type 2 diabetes and cardiovascular diseases in obese children in the future. [28] When the plasma 8-iso-PGF 2α values are examined, it is seen that results consistent with the studies in the literature are obtained. [29][30][31] Many researchers have reported an increase in plasma 8-iso-PGF 2α levels in patients with insulin resistance. [30,31] In another study evaluating the relationship of oxidative stress with diabetes, obesity and atherosclerosis, 8-iso-PGF 2α levels were found to be significantly higher in obese adults. In a different study investigating the relationship of oxidative stress with obesity and insulin resistance only in adult males, it was reported that 8-iso-PGF 2α levels were higher in obese males than in normal males. [32] In the literature, it is seen that the obesity 8-iso-PGF 2α correlation is mostly evaluated in adults. This study investigated the correlation between obesity in children and 8-iso-PGF 2α .
The blood pressure values obtained in the study show that there is a correlation between 8-iso-PGF 2α levels and obesity. While the mean systolic blood pressure was 140.18±26.78 mmHg in the obesity group, the mean systolic blood pressure was 118.26±26.78 mmHg in the control group. While the diastolic blood pressure was 88.16±14.78 mmHg in the children with obesity, it was 74.32±10.48 mmHg in the control group. It is known that 8-iso-PGF 2α , which is administered endogenously into the vein, increases blood pressure. Therefore, increased 8-iso-PGF 2α level in obese children is thought to be effective in increasing blood pressure.

CONCLUSIONS
Oxidative stress forms the infrastructure of many diseases. Today, exposure to oxidative stress due to various factors has decreased to very young ages. It has been proven by various studies that oxidative stress can disrupt the normal working principles of the energy production mechanisms in the body and affect the general working principles of the cell. In this context, understanding the metabolic and biochemical background of oxidant mechanisms in the development of diseases, interpreting their measurable results and identifying biomarkers will contribute to early diagnosis and treatment. There are many biomarkers used as indicators in oxidative stress studies. In the present study, 8-iso-PGF 2α levels were based on as a biomarker of oxidative stress, and obese children were our area of interest. Our results showed that 8-iso-PGF 2α concentrations were higher in obese children than in the control group. We can say that elevated 8-iso-PGF 2α levels are related to high blood pressure and visceral adiposity. In this respect, we can say that increased levels of 8-iso-PGF 2α , which we consider as a biomarker of oxidative stress, may be a factor in the formation of cardiovascular diseases in obese children. Furthermore, we can say that the diagnosis of the disease can be made with 99% sensitivity of 8-iso-PGF 2α levels in obese children. As insulin resistance increased, there was an increase in the 8-iso-PGF 2α levels suggesting that this biomarker is important in the diagnosis of obesity.

Compliance with the ethical standards
All human studies have been approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. All persons gave their informed consent prior to their inclusion in the study.