EVALUATION OF INFLAMMATORY-OXIDATIVE PARAMETERS AND CAROTID ARTERY INTIMA-MEDIA THICKNESS IN OVERWEIGHT/OBESE SUBJECTS

Introduction: Obesity is a rapidly growing health problem, conferring substantial excess risk for morbidity and mortality, especially for type 2 diabetes and atherosclerotic cardiovascular disease (CVD). Obesity is frequently associated with various metabolic disorders defined altogether as Metabolic Syndrome (MS), whose incidence is high and increasing; MS refers to a constellation of disturbances including glucose intolerance, central obesity, dyslipidemia (hypertriglyceridemia, decreased high-density lipoprotein (HDL cholesterol) and hypertension. It can present in several forms, according to the combination of the different components of the syndrome, and it is well established that it increases the risk for the development of cardiovascular disease and type 2 diabetes. Obesity and MS are characterized by oxidative stress (OS), defined as unbalance between Reactive Oxygen Species (ROS) and Total Antioxidant Capacity (TAC), and by subclinical inflammation, both responsible for the development of atherosclerosis. In addition, in the diabetic condition, oxidative stress impairs glucose uptake in muscle and adipose tissue and decreases insulin secretion from pancreatic β cells. Elevated levels of proinflammatory proteins are found in blood and metabolically relevant tissues of obese individuals. In fact adipose tissue, which is considered as an active endocrine and paracrine organ, releases a variety of biologically active molecules collectively known as adipocytokines or adipokines, that influence body weight homeostasis and induce changes in cardiovascular structure and function, glucose metabolism, blood pressure, lipid metabolism, coagulation, fibrinolysis and inflammation; these changes lead to endothelial dysfunction and atherosclerosis. Aims of the study: The first aim of this study was to evaluate, in overweight/obese subjects, the classical risk factors (such as dyslipidemia) and less conventional mechanisms, such as oxidative stress, involved in the development of endothelial dysfunction, increasing cardiovascular risk. Several inflammatory markers (C Reactive Protein, fibrinogen), associated with abdominal obesity, were also assessed. Secondly, because carotid artery intima-media thickness (IMT), which is significantly associated with prevalent and incident carotid plaques, is a convenient marker of atherosclerosis, we studied the association between IMT and oxidative stress, inflammatory markers (cytokines) and others biochemical parameters in a subgroup of our overweight/obese population. Subjects, Material and Methods: Three-hundred and sixty consecutive overweight-obese subjects (99 males and 261 females; mean age 48.912.1 years; mean BMI 34.25.6 Kg/m2; mean waist circumference 99.7±12.4 cm for females and 111.5 ±10.3 cm for males) were enrolled at the ‘‘Obesity and Work’’ outpatient clinic of the Clinica del Lavoro L. Devoto, Fondazione IRCCS, Cà Granda Ospedale Maggiore Policlinico Milano. Each participant signed informed consent and was interviewed about the general health, life-style and smoking habit. Exclusion criteria were: Body Mass Index (BMI)<26 Kg/m2, any previous cardiovascular and pregnancy. Each subject was evaluated by standard routine examination including Complete Blood Count (CBC), lipid panel (Triglycerides, TG; Total Cholesterol, T-Chol; LDL Cholesterol, LDL-Chol; HDL Cholesterol, HDL-Chol), glycemic status (glucose concentration; insulin and glycated haemoglobin, HbA1c), creatinine, high sensitive C Reactive Protein (hsCRP), fibrinogen, uric acid. Oxidative status was evaluated by measuring Serum Reactive Oxygen Species (ROS) concentrations and Total Antioxidant Capacity (TAC) with spectrophotometric method, using commercial kits (dROMs test and OXY-Adsorbent test respectively; Diacron International, Grosseto, Italy) on F.R.E.E. analyzer (Diacron) as previously described. Serum levels of oxidized LDL (oxLDL) were measured by a commercial Enzyme-Linked-Immunoassorbent Assay (Oxidized LDL ELISA, Mercodia, Uppsala, Sweden) on EASIA reader (Medgenix Diagnostics, Fleurus, Belgio). Data of oxidative status analytes were compared to relevant reference values used in our laboratory. Out of the study population, one-hundred and fifty-seven subjects (36 males and 121 females; mean age 52.112.6 years; mean BMI 33.66.0 Kg/m2; mean waist circumference 97.3±11.5 cm for females and 111.0±10.3 cm for males), underwent a carotid B-mode ultrasound imaging and Doppler ultrasound to measure IMT bilaterally at the level of the common carotid artery and its bifurcation and to detect the presence of any plaques (focal lesion). These subjects were also evaluated for serum concentrations of soluble cytokines and adhesion molecules by cytofluorimetric method using Human Flow Cytomix-Kit (Bender MedSystems, Austria) on FACScan Flow Cytometry Becton Dickinson (BD Biosciences, USA). This method is a bead-based immunoassay for the flow cytometer, designed for quantification of different cytokines, in particular we evaluated simultaneously in the same sample: serum soluble Intercellular Adhesion Molecule-1 (sICAM-1), serum Chemokine Monocyte Chemoattractant Protein 1 (MCP-1) and serum Leptin. Statistical analysis: Continuous variables are reported as means±standard deviation or as median and interquartile range[IQR] to properly account for the skewness of most data distributions. Statistical significance was assigned as a p value <0.05. Statistical analyses were performed using R statistical software (Revolution Analytics, Palo Alto, CA). Results: All the subjects had a normal haematological status based on the results of a standard CBC panel (data not shown). Males showed a similar BMI as females (males 34.1±5.1 Kg/m2 vs females 33.3±5.8 Kg/m2, p value NS). The large majority of obese subjects (87.5%) showed an unbalanced oxidative status due to elevated ROS concentrations (380.8592.9 UCarr, cut-off <300 UCarr) in the presence of adequate TAC in about 70% of cases (383.057.9 µmolHClO/ml, cut-off >350 µmolHClO/ml); oxLDL concentrations (83.629.6, cut-off <70 U/L) were higher than cut-off in 63% of subjects As regards inflammatory parameters, fibrinogen (342.1178.26 mg/dL, ref. int. 200-400 mg/dL) and hsCRP concentrations (0.581.18 mg/dl, cut-off<0.5 mg/dL) were altered in 18% and 30% of cases, respectively. ROS concentration strongly correlated with fibrinogen (r=0.3, p=0.0002) and with hsCRP (r=0.3, p=0.0001). Oxidized LDL concentrations correlated significantly with waist circumference (r=0.22; p<0.0001), with the levels of lipid panel parameters (total-Cholesterol, r=0.5, p<0.0001; HDL-Cholesterol, r=-0.25 for males, p=0.01 and r=-0.20, p=0.001 for females; LDL-Cholesterol, r=0.52, p<0.0001; triglycerides, r=0.3, p<0.0001), with fibrinogen (r=0.19; p=0.0005) and with C Reactive Protein even if not significantly (r=0.08; p=0.1). Interestingly Uric Acid concentrations correlated positively with oxLDL (r=0.22, p<0.0001) and TAC (r=0.30, p<0.0001) while negatively with ROS concentrations (r=- 0.24, p=0.001). A strong correlation was found between BMI and hsCRP (r=0.2, p=0.0005) and between BMI and Fibrinogen (r=0.34, p<0.0001). The whole population was also stratified on the basis of glycated haemoglobin percentage according to American Diabetes Association (ADA) Consensus Statement in order to evaluate pre-diabetes and diabetes condition. In our study we found an impaired glucose metabolism in about 55% of subjects; 44.7 % of our subjects presented normal value of HbA1c (5.31.2%, cut-off<5.7%) while 43% and 12.3% of cases showed a pre-diabetes (5.90.2%, ref. int. 5.7%≤HbA1c≤6.4%) and diabetes condition (7.41.2%, cut-off>6.4 %), respectively. According to the Adult Treatment Panel (ATP) III criteria, 34% of our subjects (mean age 52.811.4 years; 40.5% of males and 59.5% of females, p=0.0004) showed a MS condition. We found a strong correlation between this cluster of disorders and oxidative stress condition, particularly with oxidized LDL and Uric acid (p<0.0001); no association between ROS and MS was highlighted. Regarding inflammatory indexes, a significant correlation was found between MS and fibrinogen (p=0.028) and hsCRP (p=0.012). Our results showed also a correlation between MS and HbA1c (p=0.0002). The analysis of IMT in 157 subjects of our study population (121 females, 36 males; mean age 52.112.6 years; mean BMI 33.66.0 Kg/m2), detected the presence of plaques in 42 subjects (35 females, 7 males; mean age 61.39.5 years; mean BMI 33.55.6 Kg/m2). The analysis of oxidative status in this subgroup showed no difference in ROS and oxLDL concentrations between obese with plaques (OP) and obese without plaques (ONP) while uric acid concentrations were significantly higher in OP than in ONP (p=0.0085). No association between inflammatory markers (hsCRP and fibrinogen) and the presence of plaques was found. The analysis of cytokines results showed no significantly differences both in leptin concentrations (OP: 719 [511-1071] ng/mL; ONP: 565.5 [352-898] ng/mL) and sICAM-1 concentrations (OP: 534.5 [435-666.5] ng/mL; ONP: 475 [376-604] ng/mL) while MCP-1 levels were significantly (p=0.02) higher in OP (678 [507.5-932.5] pg/mL) than in ONP (587 [455.7-726.7] pg/mL). In our population serum leptin correlated significantly with oxLDL (r=0.17, p=0.08), anthropometric indexes (BMI and waist circumference: r=0.4, p<0.0001; r=0.28, p=0.006, respectively), inflammatory parameters (uric acid: r=0.27, p=0.006, fibrinogen: r=0.4, p<0.0001; hsCRP: r=0.5, p=0.0001) and lipid panel parameters (t-Chol: r=0.26, p=0.008, LDL-Chol: r=0.24, p=0.01, triglycerides: r=0.25, p=0.001). Serum sICAM-1 correlated positively with uric acid (r=0.23, p=0.02), MCP-1 (r=0.28, p=0.004). Patients with MS showed sICAM-1 median concentrations significantly higher than patients without MS (571[487-727] vs 452[363-587] ng/mL; p=0.0006). We found glycaemic status significantly altered in OP compared to ONP: serum fating glucose concentrations (105.826.5 vs 97.024 mg/dL; p=0.01) and serum post prandial glucose concentrations (119.436.6 vs 102.823.2 mg/dL; p=0.001). Moreover glycated haemoglobin percentage was higher in OP than in ONP (6.20.9 vs 5.70.5 %; p=0.01). Notably, even if we didn’t find correlations between the presence/absence of plaques and each Metabolic Syndrome parameter, Fisher’s exact test highlighted an association between the presence of plaques and MS (p=0.00067). Discussion: Obesity and Metabolic Syndrome are characterized by an altered oxidative status and by subclinical inflammation, both responsible for the development of atherosclerosis; in fact some indicators of oxidative stress and inflammation are elevated in obese subjects, especially in those exhibiting MS according to ATP III criteria. Reactive Oxygen Species are produced by multiple pathways within the cell and are essential for many cellular functions. ROS production is buffered by enzymatic and non-enzymatic antioxidant systems. The perturbation of the pro-oxidant/antioxidant balance can lead to oxidative stress. The most of our overweight-obese subjects showed an oxidative stress condition due to elevated ROS concentration despite a TAC which, in contrast with other authors, was adequate in the large majority of cases (70%). Our findings highlighted that oxidative stress seems to be an independent risk factor in obese subjects; ROS values correlated neither with BMI nor with lipid panel parameters. Increased concentrations of ROS (particularly superoxide and hydrogen peroxide) lead to enhanced oxidation of low-density lipoprotein (LDL), inactivation of endothelium-derived nitric oxide and vascular dysfunction. Previous studies reported that elevated oxLDL concentrations can predict myocardial infarction in well-functioning elderly people, even after adjusting for age, gender, race, smoking and metabolic syndrome. In the present study, elevated oxLDL concentrations in most of subjects (63%) indicated lipid peroxidation, more advanced in the cases of serious dyslipidemia and significantly higher in subjects with MS. Recent studies indicated that uric acid, despite being a major circulating antioxidant, both correlates and predicts development of obesity, hypertension, and cardiovascular disease, conditions associated with oxidative stress. Our results seemed to confirm the double role of uric acid. The positive correlation with TAC, together with the negative correlation with ROS, confirmed this molecule as an important oxygen radical scavenger. On the other hand, uric acid was associated with lipid peroxidation, evaluated by oxLDL assay. In our study, we can hypothesize that uric acid may function either as an antioxidant, primarily in plasma, or pro-oxidant, primarily within the cell. As reported by others, hyperuricemia increases the risk of MS. Our results are in agreement with this findings; infact we found a strong positive relation between MS condition and uric acid concentrations. Inflammation plays a central role in atherosclerosis and hsCRP and fibrinogen are important biochemical markers for low-grade vascular inflammation and endothelial dysfunction that characterize the earliest events in the pathogenesis of atherosclerosis. The altered levels of fibrinogen and hsCRP in the 18% and 30% of cases respectively, and the correlation between BMI and hsCRP and BMI and fibrinogen in these subjects confirmed the low-grade chronic inflammation state associated with obesity. The inflammatory state accompanying MS shows a quite peculiar presentation, as it is not associated with infection or sign of autoimmunity and no massive tissue injury seems to have taken place. According to Monteiro R, we found that fibrinogen and hsCRP concentrations were significantly higher in subjects with MS than in others. Inflammation and oxidative stress are tightly interrelated since inflammation causes ROS generation and ROS promote the synthesis of pro–inflammatory cytokines. The linkage of both systems is also evident in the present study where we found a strong correlation between ROS and hsCRP concentration and ROS and fibrinogen levels. The second aim of our study was to identify the features of carotid atherosclerosis in our overweight/obese subjects. Carotid intima-media thickness (IMT), a validated marker of subclinical atherosclerosis, is associated with the atherosclerotic process because of its association with known cardiovascular risk factors (hypertension, dyslipidemia, diabetes mellitus, smoking) and less-conventional risk factors such as inflammation and oxidative stress. Our results showed uric acid concentrations significantly higher in OP than in ONP. This finding is in agreement with the hypotesis that uric acid is involved in inflammation because triggers the release of proinflammatory molecules, such as hsCRP; this systemic inflammation contribute to the development of atherosclerosis condition. The adipokine/cytokine networking system is altered in obesity, contributing to low-grade inflammation and impaired adipocyte metabolism. In the majority of obese patients plasma leptin concentration is markedly increased reflecting greater amount of adipose tissue and resistance of hypothalamus to anorectic effect of this hormone. However, hyperleptinemia observed in obesity/MS may contribute to many complications including cardiovascular diseases. According to Bełtowski, in our obese subjects, leptin correlated positively with BMI and waist circumference. Moreover, our study highlighted that leptin concentrations were higher in OP than in ONP, even not significantly. Some authors suggested that leptin activity is impaired by its binding to CRP. In addition, leptin stimulates CRP production by cultured endothelial cells. Thus, elevation of CRP could contribute to leptin resistance associated with chronic hyperleptinemia. Our results, in agreement with these findings, reported a positive and significant correlation between leptin and hsCRP. Another potential mechanism which could impair endothelial leptin signalling is oxidative stress; it has been demonstrated that leptin has lipid peroxidation properties [16]. We confirmed this properties by highlighting a correlation between leptin and oxLDL in our population. We also found a correlation between leptin and uric acid; an increase in uric acid levels may indicate unfavourable changes of factors involved in pre-atherosclerosis. Monocyte recruitment into the vascular wall is the early and important event in atheroma development. Local inflammation is attributed to several molecules produced by leukocytes, including sICAM-1and MCP-1. Our results are in agreement with Mizuno et al.: OP showed significantly increased MCP-1 concentrations compared to ONP. sICAM-1 levels were higher in OP than in ONP. Central obesity may play a role in pathogenesis of diabetes. The hallmark of diabetes is hyperglycaemia and in both type 1 and type 2 diabetes, large prospective clinical studies have shown a strong relationship between time-averaged mean levels of glycaemia, as measured by glycated haemoglobin, and cardiovascular complications. Because preclinical atherosclerosis is common in pre-diabetes we studied the prevalence of this condition in our population and we found a significant relationship between HbA1c values and increased carotid IMT. Notably in the pre-diabetic condition there was a significant higher IMT than in non diabetic condition. Moreover, recent findings had demonstrated that glycaemic “instability”, in terms of “postprandial hyperglycaemia” or “glucose variability” should be considered as an independent risk factor for cardiovascular disease because glucose swings could also be very deleterious for the vasculature. According to Ceriello, in our population, we found a significantly increase of glucose postprandial concentrations in subjects with plaques. Previous studies, performed in different population of apparently healthy participants or in participants without known cardiovascular disease or in young participants, identified MS as a risk factor for an increased carotid IMT; we found a strong correlation between MS and an increased carotid IMT, confirming this association. Summarizing, all the subjects showed a condition of oxidative stress due to elevated ROS concentrations in spite of the generally adequate TAC; this condition was exacerbated by the presence of Metabolic Syndrome. The analysis of carotid artery intima-media thickness showed that glycaemic “instability”, in terms of “postprandial hyperglycaemia” or “glucose variability” seemed to be a more predictable risk factor for cardiovascular disease than oxidative stress. However further investigations regarding plaque progression and biochemical follow up of the subjects will be necessary in order to achieve more conclusive results.