Integrated Haematological Profiles of Redox Status, Lipid, and Inflammatory Protein Biomarkers in Benign Obesity and Unhealthy Obesity with Metabolic Syndrome

1. Introduction

The constant growth of obesity pandemic has fostered the research of reliable biomarkers for routine diagnosis, able to assess the severity of the underlying metabolic impairment, to predict risk of the wide array of multiorgan complications, and to provide solid molecular basis for innovative therapeutic approaches [1–3]. Distinctive ties between metabolic dysfunction and immune system alterations have also produced relevant achievements, leading to the classification of the adipose tissue as an effective immunoendocrinological organ [4–6]. Metabolic syndrome (MetS) is the most distinctive condition featuring “nonbenign obesity” (morbid obesity) [7, 8], which encompasses the health-impairing forms of obesity [9–11]. The different criteria are adopted worldwide, also on ethnic basis, to diagnose MetS; all include the key parameters of fasting plasma glycemia, total and HDL cholesterol levels, fasting triglyceride levels, blood pressure, waist circumference, body mass index (BMI), and insulin resistance [7, 12–15].

Various combinations of these altered parameters have been unequivocally related to the individual higher risk of endocrinological [16, 17], hepatic [18, 19], cardiovascular [20, 21], neurological [22, 23], and ultimately oncological [24] outcomes. More recently the immunological state, namely, the altered transcription levels and/or the altered concentrations levels of several systemic proinflammatory cytokines, chemokines, and growth factors, as well as adipose-tissue-specific adipokines, have gained focus as key parameters driving mere overweight into a MetS [25]. Their alterations have been directly correlated with lipid metabolism, adipogenesis and fat distribution, adipocyte metabolism and function, glucose uptake abnormalities, insulin secretion and resistance, appetite and satiety, energy expenditure, endothelial function, hemostasis, blood pressure, energy metabolism in insulin sensitive tissues, and immune cell migration into adipose tissue [26–30].

The role of systemic redox state and of antioxidant (AO) imbalance has also been studied in obesity in terms of molecular mechanisms of disease (for recent review see [31]) and of nutritional intervention protocols aimed at weight control and normalization of metabolic parameters (for review see [32]). In particular, the etiological relevance of excessive reactive-species-mediated oxidation of lipids, carbohydrates, and proteins in the different organs targeted by MetS has been extensively described [33–35]. The current diagnostic guidelines of MetS and morbid obesity are essentially based on type II diabetes protocols and traditionally employ combined approaches, that is, the anthropometric profile (height, weight, waist/hip/arm circumference, etc.), mainly reported as BMI index [36], coupled with the assessment of clinical laboratory parameters connected with glucose and lipid metabolism [7, 12–15], hormone status [12], routine protein markers of inflammation [37], bone, hepatic, and renal markers [38, 39]. The reliability of BMI and waist circumference parameters is currently being discussed, as unable to correlate with the individual levels of lipid and glucose dysmetabolism or of chronic inflammation [40, 41]. Overcoming biopsy invasiveness, technology-assisted noninvasive evaluation methods of fat and lean mass, of liver or cardiac fat, and so forth have been introduced as additional clinical parameters correlating with severity of obesity [39, 42]. Conversely, the laboratory blood test protocols for MetS and obesity have not been updated with clinical research outcomes. Thus far, neither the proinflammatory cytokines and adipokines panels, in effect requiring elevated costs, nor the redox parameters, more easily measurable on a routine basis, have been examined extensively in the different obese types.

In the attempt to contribute to the ongoing efforts for the identification of new pathologically relevant molecular markers of obese morbidity, we attempted here to identify possible significative alterations of a panel of 55 metabolic blood markers of redox status, nutritional and endogenous AO/free radical-detoxifying defenses, and unsaturated lipid quality and oxidation grade, in a group of 112 Italian Caucasian obese patients, of which 67 with obese state aggravated by MetS, as compared with a control group of 90 healthy subjects. In addition, the concurrent analysis of a panel of 37 serum cytokines and adipokines enabled a comparative evaluation of the chronic proinflammatory levels in the two obesity subgroups. Ultimately, the study protocol aimed at identifying possible specific redox-related metabolic and immunologic patterns marking the transition from a state of “healthy” obesity to morbid obesity with MetS and increased type II diabetes risk. The selection of a specific panel of reliable molecular determinants for this transition bears promising implications for diagnosis, risk prevention, and follow-up of obese subjects with different degrees of impaired metabolism and possibly will provide new hints for future treatment innovation.

2. Materials and Methods

3. Results

4. Discussion

The primary goal of the present clinical laboratory trial was to evaluate the patterns of oxidative stress, adipokines, and inflammatory factors in two groups of obese people with similar clinical features of obesity but with different metabolic status. One group was clinically and biochemically defined as having a MetS, and the other did not have as yet all metabolic derangements characteristic for “unhealthy” obesity (Table 1). The table shows that patients of both groups were overweighted (BMI), had central type of obesity with fat accumulation around the waist (Circumference), and similar highly increased versus normal ratios of fat/lean tissue mass. Nevertheless, the obese groups under investigation differed substantially in the widely accepted metabolic markers of MetS, such as dyslipidemia, insulin resistance, and borderline fasting glucose levels attributed to prediabetes [12, 15, 16, 21]. Majority of the OB+MetS group patients suffered from arterial hypertension (data not shown). The large female sex prevalence is in fact a common occurrence in obesity facilities in Italy. The group of males (12 out of 112 obese subjects included in the study) did not show peculiar differences in the metabolic and immunologic parameters with the females and therefore was included in the general evaluation.

There is a continuous debate over the causes and possible mechanisms of transition from benign obesity to obesity aggravated by serious metabolic disorders (MetS) bearing high incidence of cardiovascular complications and type II diabetes [9–11, 16, 19–25, 57]. Here, we attempted to elucidate an impact of redox and adipokine homeostasis players and definite inflammatory factors to the effective metabolic differences in benign obesity and unhealthy obesity complicated with MetS. It was not surprising for us that a redox imbalance in favour of oxidative stress with suppressed glutathione and GST (Figure 1), higher-than-normal levels of 4-HNE protein adducts, and luminol-dependent chemiluminescence (Figure 2) was observed in both groups of obese people independently on the presence of metabolic abnormalities. In accordance with recent literature data, 4-HNE has been found in greater-than-normal amounts in obese people [58, 59]. The aldehyde is a final product of lipid peroxidation with diabetogenic potential through induction of insulin resistance and capacity to disrupt adipogenic functions [58–60]. GST protein is sensitive to 4-HNE adduct formation and therefore may be partially inactivated by 4-HNE excess [61] in the obese. In a loop, enzyme inactivation could sustain the increased levels of 4-HNE observed, since GST enzyme together with glutathione inactivates aldehydes [60, 62, 63]. Luminol-dependent chemiluminescence (CL) reflects the capacity of ROS production by circulating granulocytes and monocytes [64]. Since these circulating phagocytes are primed by oxidized low-density lipoproteins (oxLDL) [65–67] to release large amounts of ROS, it appears to be logic that ROS production in obese subjects in general is increased. A vicious cycle is formed when LDL are oxidized by ROS, while oxLDL induce ROS overproduction.

The combination of low level vitamin E, activated SOD, and inhibited CAT in the group with uncomplicated obesity (Figures 1 and 2) allowed us to hypothesize that the high levels of hydrogen peroxide formed in the reaction of superoxide anion-radicals dismutation by SOD (2O₂ ⁻ + 2H⁺ + 2H₂O → 2H₂O₂) persist due to inadequate activity of CAT, a hydrogen peroxide decomposing enzyme (2H₂O₂ → 2H₂O + O₂). Catalase activity is usually inhibited by excess H₂O₂, as well as by 4-HNE, which forms aldehyde-protein adducts with the enzyme, thus inactivating its active centre [61, 68, 69]. Hydrogen peroxide belongs to stable reactive oxygen species which, in the presence of transition metals, could be easily converted into hydroxyl radicals (H₂O₂ + Fe⁺ⁿ → 2OH^∙), classical initiators of lipid peroxidation chain reaction. Under these circumstances, α-tocopherol plays a classical role of sacrificing antioxidant being consumed in the process of interruption of the chain reaction in lipid compartments [70–72].

Of great importance, GPX is hyperactivated in the group with MetS exclusively (Figure 1). PUFA in the membrane phospholipids or in circulating lipoproteins are subjected to intense enzymatic (12/15 lipoxygenase) or nonenzymatic free radical-driven lipid peroxidation, under certain pathological conditions such as inflammation, obesity, or atherosclerosis [73]. Both types of lipid oxidation result in the formation of highly reactive lipid hydroperoxides (LOOH), proinflammatory leukotrienes among them, which are neutralised by GPX (mainly, by isoform Gpx4) [74], GST [75], peroxireductase VI, and aldo-keto-reductases [73]. LOOH [73, 76] and small end-products of lipid peroxidation such as 4-HNE and other aldehydes (malonyldialdehyde, acrolein, etc.) transported by activated granulocytes/monocytes may facilitate and maintain generalised inflammation [77–79]. Of interest, successful anti-inflammatory therapy of psoriasis patients with TNF-α antibodies led to suppression of GPX activity, while in nonresponders to the therapy, GPX activity was further stimulated [80].

In addition to the possible pathogenic role(s) of oxidative processes in lipids, a significant elevation of monounsaturated fatty acids (MUFA) in erythrocyte membranes assessed by the decreased ratio of saturated FA/MUFA was shown for both experimental obese groups, while increased content of ω-6 versus decreased ω-3 membrane-bound FA was found in uncomplicated obesity group only (Figure 3). Membrane-bound MUFA are considered as a strong proinflammatory factor in obesity because their biosynthesis is associated with hyperactivated enzyme stearoyl-delta9-desaturase [81, 82]. The combination of two factors, highly increased MUFA and PAI-1 (Figure 4), observed in the Ob+MetS group is associated with the increased risk of cardiovascular diseases, high blood pressure [83], and the elevated coagulation rates in MetS [84].

Adipose tissue was considered as a mere energy storage until the first adipokine, a bioactive product synthesised within and released from adipose tissue, was identified in 1994 [27]. This very first adipokine was leptin, levels of which are highly increased in obese people (Figure 4 and [85, 86]). Adipokines control distinct essential physiological processes, such as appetite and satiety, energy metabolism, endothelial function, hemostasis, blood pressure, insulin secretion and sensitivity, adipogenesis, adipocyte functions, and fat distribution [29]. Among the more than 600 putative adipokines, there are numerous proinflammatory mediators, including interleukins, tumor necrosis factor- (TNF-) alpha, chemokines, and growth factors [87, 88]. A distinct adipokine pattern associated with body weight includes insulin, triglycerides, leptin, PAI-1, chemerin, MCP-1, and retinol-binding-protein-4 (RBP-4) [86]. Some adipokines, such as adiponectin, leptin, chemerin, visfarin, and PAI-1, are produced exclusively by adipose tissue, while others, tumor necrosis factor- (TNF-) alpha, interleukins, chemokines, and growth factors, could be produced by circulating blood leukocytes, tissue macrophages, keratinocytes, and other cells. Therefore, blood levels of these factors reflect not only their release from adipose tissue but also a significant contribution of circulating leukocytes, cells-effectors of inflammatory responses in the organism, and endothelial cells. Out of all pure adipokines, plasma levels of which were measured in the study; exclusively leptin, a marker of obesity, exhibited higher-than-normal values in both obese groups (Figure 4), while visfatin and resistin remained within the normal range (data not shown). Another adipokine PAI-1 produced in visceral and not in subcutaneous adipose tissue was overexpressed in patients with MetS; its high levels are connected with high risk of thrombosis [84]. A clearcut proinflammatory pattern of cytokines (IL-4, IL-6, IL-7, and IL-9), chemokines (IL-8), growth factors (VEGF, PDGF, G-CSF, GV-CSF), and TNF-α was found in obese subjects with MetS. As an adaptive reaction, the expression of anti-inflammatory IL-10 was also increased (Figures 5(a) and 5(b)). These findings corresponded to an assumption that MetS is a pathology characterised by generalised chronic inflammation [2, 4, 31, 89–91].

Of major relevance is the contribution of TNF-α to metabolic syndrome. TNF-α expression was found to be increased in the adipocytes of obese animals, and its neutralisation by a TNF-α-specific soluble antibody led to the improvement of insulin sensitivity [92]. These observations were then confirmed in obese and diabetic humans [93]. Noteworthy, the major source of TNF-α as well as of multiple cytokines and chemokines in obesity are phagocytes, not adipocytes [94]. The therapy with anti-TNF-α antibodies resulted in attenuation of MetS symptoms in general [95] and decrease of GPX activity to neutralise LOOH and toxic aldehydes [80]. All these inflammation-related cytokines could be overproduced by circulating leukocytes primed with LDL and free fatty acids (FFA) [67].

Of note, obesity itself was not connected to abnormal plasma mediators of inflammation with the exception of RANTES, which was remarkably increased in the OB group. We assume that RANTES could be a key molecular target to interrupt an undesired transition to MetS in obese people. Regulation of leukocyte activation and migration into tissues by chemokines like RANTES and similar monocyte chemotactic protein-1 (MCP-1) are recognised as important factors in the induction of acute and chronic inflammation [96]. RANTES has been shown to stimulate T-lymphocyte extravasation as well as monocyte oxidative metabolism and recruitment to lungs, kidney, liver, skin, and other tissues [97]. The inhibition of RANTES and MCP-1 with corresponding antibodies or low molecular weight antagonists has been suggested and successfully applied in many pathologies accompanied by chronic inflammation [98]. RANTES may represent a valuable target for the prevention of MetS setting as well as for diagnostic purposes.

5. Conclusions

On the grounds of the results obtained, we hypothesised that as a primary step of the transition from the benign obesity to obesity complicated with MetS there could be a long-lasting priming of circulating phagocytes by excessive amount of oxLDL. The primed granulocytes and monocytes produce large amount of ROS and redox-dependent proinflammatory cytokines. Primed leukocytes in benign obesity overexpress chemokines like RANTES, thus prompting leukocyte migration into tissues, for example, in adipose tissue, pancreas, muscles, and liver, which are target organs for the initiation and maintenance of metabolic derangements characteristic for MetS (the first loop of transition). Subsequently, ROS-mediated formation of LOOH and toxic end-products of lipid peroxidation occurs. These highly reactive products induce adipogenesis and fat production by adipocytes [65–67, 99–101], which creates a second self-sustained pathological loop of increased fat production and accumulation → increased fatty acid oxidation → chronic proinflammatory pattern formation → redox-dependent derangement of antioxidant and detoxifying systems → redox-dependent derangement of insulin sensitivity → redox-dependent dyslipidemia → MetS. Upregulated GPX could be an early pathogenic marker of MetS setting, as a unique defense against hydrogen peroxides and their metabolites with a pathogenic role for MetS in the obese.

Conflict of Interests

The authors declare that they have no conflict of interests.