Oxidative, inflammatory and cardiometabolic biomarkers of clinical relevance in patients with metabolic syndrome

Universidade do Oeste de Santa Catarina (UNOESC), Santa Catarina, Brazil

Endereço para correspondência

Eduardo Ottobelle Chielle
Rua Oiapoc, 211; Agostini;
CEP: 89900-000; São Miguel do Oeste-SC, Brasil
Phone: +55 (49) 3631-1072
e-mail: eduardochielle@yahoo.com.br

INTRODUCTION

Once obesity is present in the metabolic syndrome (MetS) the release and production of pro-inflammatory adipokines and free fatty acids (FFA) occurs. This action is associated with chronic cases of low-grade inflammation and infiltration of additional products of macrophages in the tissue, providing a decrease of insulin receptors⁽¹⁾. When this happens, the extracellular concentration of adenosine increases progressively⁽²⁾, interfering with the biosynthesis of proinflammatory cytokines and decreasing the action of neutrophils⁽³⁾. In a counterregulatory response to this process, adenosine deaminase (ADA) is released in greater amounts to catalyze adenosine into isonine, regulating the intracellular and extracellular concentrations of this nucleoside. Thus, ADA is suggested as a key enzyme for the modulation of insulin bioavailability, once it regulates several aspects of adipose tissue function, including lipolysis and increased insulin sensitivity (IS) in adipocytes⁽⁴⁾.

Recent research suggests that dipeptidil peptidase-IV (DPP-IV) is also associated with the hability to induce insulin resistance in adipocytes and muscle cells in obese individuals^{(5, 6)}. DPP-IV induces the modification and/or inactivation of N-terminal peptides and is, therefore, a strong inhibitor of the antilipolytic activity of the neuropeptide Y^{(7, 8)}. It has been proposed that the association between ADA and DPP-IV on the surface of the T cell determines costimulation of the T cell receptor against the antigen⁽⁹⁾, in this context, DPP-IV activity together with ADA may be necessary for the cascade of T cell activation, playing a key role in the development of immune responses^{(6, 10)}.

In addition to the abnormal production of adipocytokines and deregulated proinflammatory responses, oxidative stress is another mechanism associated with the onset of MetS^(11-13). The oxidative stress occurs when there is a significant increase of free radicals in the tissues, exceeding the neutralizing capacity of the antioxidants, observed especially by the increase of the thiobarbituric acid reactive substances (TBARS)⁽¹⁴⁾. The increase of oxidative stress is involved in the pathogenicity of hypertension, atherosclerosis and contributes to cardiometabolic disorders⁽⁵⁾. Therefore, a large number of body’s cells have adequate defensive action to avoid harmful oxidative events. This action includes the presence of antioxidant enzymes, such as peroxides and non-enzymatic antioxidants, including uric acid, sulfhydryl (SH) groups and total ferric antioxidant power (FRAP) (for determination of total antioxidant capacity)⁽¹⁵⁾. It is also observed that the levels of TBARS increase progressively according to the increase of body weight, unlike SH groups and FRAP that decrease according to body weight⁽¹⁶⁾.

OBJECTIVE

Since MetS is associated with a chronic inflammatory response, characterized by abnormal adipokine production and the activation of several proinflammatory and oxidative signaling pathways resulting in the increase of several biomarkers, the objective of this study was to evaluate the levels of adiponectin, inflammatory markers with ultra-sensitive C-reactive protein (us-CRP), interleukin-6 (IL-6), ADA, DPP-IV and oxidative markers (TBARS), SH groups, FRAP and vitamin C, and to clarify, in adults, a profile of biomarkers that influence the metabolic risk of development of MetS.

METHODS

Study design and population

This was a cross-sectional study in which measurements and analyzes were performed in a single moment. The participants were recruited from January to May 2017 in the city of São Miguel do Oeste, in the state of Santa Catarina, Brazil. The patients were from basic health units. The Ethics Committee of the Universidade do Oeste de Santa Catarina (UNOESC) approved the study protocol no. 219091, and a written informed consent document was provided to all participants. The study groups included 84 adults between 22 and 58 years of age.

The control group consisted of 48 subjects Without MetS, healthy volunteers of both sexes (28 women and 20 men) and a test group composed of 36 subjects clinically characterized by the laboratory With MetS (21 women and 15 men). Participants were non-smokers and did not use any medication continuously, and did not report metabolic diseases or events, such as coronary diseases, strokes, neoplasia and other diseases or disorders that could influence the biomarkers studied.

Indexes and ratings

MetS was verified from a careful definition, taking into account the presence of at least three of the following risk factors: 1) waist circumference ≥ 90 cm for men or ≥ 85 cm for women [using cuts established by the World Health Organization (WHO)]; 2) serum high-density lipoprotein cholesterol (HDL-C) level < 40 mg/dl for men or < 50 mg/dl for women; 3) serum triglyceride level ≥ 150 mg/dl; 4) systolic blood pressure ≥ 130 mmHg, or diastolic blood pressure ≥ 85 mmHg, or treatment with antihypertensive; and 5) fasting blood glucose level ≥ 100 mg/dl⁽¹⁷⁾.

Anthropometric measurements

All measures were taken in the Anthropometry Laboratory at UNOESC. Standing height (H, cm) using a wall mounted stadiometer (Charder, model HM-210D). Weight (W, kg) was measured using a calibrated electronic scale (Toledo, model 2124). Body mass index (BMI) was calculated as W/H2 (kg/m²). Waist circumference (WC), neck circumference (NC) and hip circumference (HC) were measured in centimeters with a flexible tape. For WC the tape was applied above the iliac crest with the subject standing upright with abdomen relaxed, arms at the sides and feet together (feet close in the same position and facing forward fully supported on the platform). For NC measurement, the participant remained in the same position and tape was placed around the half of the neck on the hyoid bone. The percentages of fat and fat weight were determined by bioimpedance (Biodynamics Model 450). All measurements were taken on the left side of the body, according to standardized procedures by Lourie and Weiner (1981)⁽¹⁸⁾. During the anthropometric measurements, all participants were barefoot and clothed appropriately.

Laboratory measurements

Blood samples containing ethylenediamine tetraacetic acid (EDTA) and serum samples were obtained from blood samples collected from participants after an overnight fast of at least 12 h. Total blood cholesterol, HDL-C, triglyceride, creatinine, urea, glucose, gamma-glutamyl transferase (GGT), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), amylase, estimated average blood glucose (GMe), insulin sensitivity (IS) and uric acid were measured enzymatically using a commercial assay kit (Labtest Diagnostics^® – Brazil). Low-density lipoprotein cholesterol (LDL-C) was subsequently calculated using the Friedewald formula⁽¹⁹⁾.

The IS and the high-sensitive C-reactive protein (hs-CRP) were determined by electrochemiluminescence immunoassay using an Elecsys 2010 analyzer (Roche diagnostics^®). Insulin resistance index was calculated by homeostasis model assessment of insulin resistance (HOMA-IR) as (fasting insulin mIU/l) × (fasting glucose mg/dl)/22.5, and evaluation of insulin sensitivity the quantitative insulin sensitivity check index (QUICKI) was used. Glycated haemoglobin (HbA1C) was measured by highperformance liquid chromatography and expressed as %.

The serum adiponectin concentration were measured in duplicate using an enzyme-linked immunosorbent assay (ELISA), according to manufacturer (EMD Millipore Corporation, Billerica, MA, EUA) in the Luminex 100 IS Analyzer System (Luminex Corp, Austin, TX, USA). Adiponectin showed a sensitivity of 1.5 ng/ml, accuracy of 92%-102%, inter-assay precision was 2.4%-8.4%, intraassay 1%-7.4% and the curve range: 1.5-100 ng/ml. Serum IL-6 levels were determined by ELISA using commercial kits from R&D Systems (Minneapolis, MN, USA) according to the manufacturer’s instructions. The detection limits of the assays were: 0.09 pg/ml, the sensitivity of 2 pg/ml and the curve range: 23.3 to 2560 pg/ml.

DPP-IV activity was determined by spectroscopic quantification of glycyl-prolyl-p-nitroanilide hydrolysis⁽²⁰⁾. Results were expressed as the specific activity (U/l). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). ADA activity was determined by commercial kit (Ebram Products Laboratory Ltda^®, SP, Brazil) according to the enzymatic deamination of adenosine to inosine by kineticmanner. The values were expressed in U/l.

Lipid peroxidation was estimated by TBARS measuring according to the method of Lapenna, et al. (2001)⁽²¹⁾. The determination of sulfhydryl group levels was based on Boyne and Ellman (1972)⁽²²⁾. FRAP levels was estimated according to Singh et al. (2012)⁽²³⁾. All were determined by spectroscopic quantification. Vitamin C was measured by the Enzyme Linked Immunosorbent Assay (ABCAM – Ascorbic Acid Assay Kit^® and Rac Beta – Tocopherol Assay Kit^®), expressed as nmol/µl.

Statistical analysis

The data were analyzed using Statistica 6.0 software (StatSoft, Tulsa, OK, USA). Data are expressed as means ± standard deviation (SD) or median (interquartile ranges). The Kolmogorov-Smirnov test was used to examine the distribution of variables. Comparisons of baseline data between groups were performed using the unidirectional variance analysis (Anova) followed by the Tukey’s test or the Kruskal-Wallis test followed by the Dunn Multiple Comparison Test to determine the statistical differences between groups. A p < 0.05 value was considered statistically significant.

RESULTS

General characteristics of the study population

The general characteristics of the study participants are described in Table 1. As expected, the weight, BMI, body fat percentage, diastolic and systolic blood pressure, and hip, waist and abdomen circumferences showed a significant increase in the With MetS group (p < 0.001) when compared to the Without MetS group.

Biochemical analyzes

The concentrations of biochemical, inflammatory and oxidative parameters are presented in Table 2. The MetS group had a significant increase in insulin, triglycerides, cholesterol, low-density lipoprotein cholesterol (LDL-C), GPT and uric acid (p < 0.001), as well as GGT, GOT, HbA1C, HOMA-IR, glucose, IL-6, FRAP, SH groups and TBARS (p < 0.05) when compared to the Without MetS group. MetS group presented a significant reduction in IS, (p < 0.001) HDL-C and vitamin C (p < 0.05) when compared to the Without MetS group. No significant differences were observed in creatinine, urea, GMe and amylase activity between groups. No statistical differences were observed between genders.

DISCUSSION

Multiple mechanisms may contribute to the development of MetS and its comorbidities, including abnormal cytokine production, aberrant oxidative stress, and dysregulated pro-inflammatory response in tissues, such as muscle and liver. As expected, the results of the research showed that the anthropometric characteristics of the With MetS group were significantly higher than the Without MetS group (Table 1).

It was observed a significant increase in HbA1C, HOMA-IR, glucose, IL-6, hs-CRP in the MetS group, and a decrease in IS. It is noteworthy that the reduction of insulin receptor 1 (IRS-1) and glucose transporter type 4 (GLUT-4) in liver and muscle tissues is associated with increased IL-6 levels under high BMI⁽²⁴⁾. These changes lead to IR and stimulate the production of hs-CRP⁽²⁵⁾. In fact, the action of IL-6 during insulin signaling in adipocytes and hepatocytes causes an increase in free fatty acids by increased lipolysis^{(26, 27)}, directly interferes with glucose metabolism⁽²⁸⁾, besides inducing reduction secretion of adiponectin⁽²⁹⁾.

The laboratory analyzes revealed a significant increase in insulin, triglycerides, total cholesterol, LDL-C in the MetS group when compared to the Without MetS group, associated with a significant reduction in adiponectin (Table 2). These results are associated with a simultaneous increase in BMI. Adiponectin levels are closely associated with the amount of body fat and its resistance and or decreased stimulation during adipogenesis may modulate several steps during the insulin-signaling pathway leading to IR⁽³⁰⁾. Studies suggest that the reduction of adiponectin may induce release of glycerol and fatty acids, which, in excess, are associated with RI^{(31, 32)} and may affect lipid metabolism in adults by stimulating the production of LDL-C in liver cells, as well as, increasing the degradation of LDL-C receptors in the liver⁽³³⁾. The effect on lipid concentrations in triglycerides blood, on the other hand, can be mediated through its effect on the metabolism of liver fatty acids, which regulate the expression of genes involved in lipid metabolism⁽³⁴⁾.

A second objective of this study was to explore oxidative biomarkers and, therefore, a reduction of SH groups and vitamin C was observed in the volunteers with MetS, with a significant increase of TBARS, evidencing the decrease in the capacity of the organism with MetS to neutralize free radicals, leading to an increase in reactive oxygen species (ROS) and favoring lipid peroxidation. As vitamin C is a water-soluble vitamin, with the increase of body weight and the concomitant reduction of the ratio between lean mass and fat mass, there is a reduction of the aqueous phase to the lipid phase in the body and, therefore, a decrease in vitamin C concentration, exposing the cells to deleterious effects of oxidative stress⁽³²⁾. These data are highlighted because the increase of oxidative stress in vascular walls is involved in atherosclerosis, hypertension and induces damage to cellular structures including membranes, proteins and deoxyrribonucleic acid (DNA)⁽³⁵⁾.

Although it was observed an increase in the serum concentration of FRAP, we believe that this data does not represent an important and reliable antioxidant defense in MetS patients, since the increase of uric acid can interfere significantly in this result, since uric acid can chelate ions metals, such as iron, contributing to total antioxidant capacity⁽³⁶⁾.

The results of this study reinforce the growing evidence of ADA and DPP-IV increase in patients with MetS when compared with patients without MetS. The increased ADA activity found in this study may be a contributing factor to insulin sensitivity in individuals with MetS, since a reduction in ADA levels reduces glucose transport in adipocytes and interferes with lipid hydrolysis⁽³⁷⁾. At the same time, increased DPP-IV activity in individuals with MetS may substantially increase lipolytic activity in adipocytes. For this reason, the increase in serum DPP-IV and ADA activity in these patients could be related to the hyperinsulinemia present in MetS. As previously shown, insulin, glucose, HbA1C and HOMA-IR levels were significantly higher in individuals with MetS, suggesting an important role of ADA and DPP-IV activity in the development of IR in these individuals. In fact, studies have found an association between HbA1C and DPP-IV activity in diabetes mellitus type 2 (DM2)⁽³⁸⁾ and in obese individuals⁽³⁹⁾.

These results may integrate new knowledge about possible interactions of inflammatory mediators and MetS helping on prevention of future chronic diseases and aggravation of MetS. The results presented here are of relevant clinical importance because, as demonstrated, the evaluation of the several components of MetS (adiposity, dyslipidemia and hypertension) and the biomarkers studied may have beneficial effects in the prevention of DM2, cardiovascular diseases and in the improvement of insulin sensitivity, since they reinforce the need to reduce weight and practice physical activity, confirming the need to develop and strengthen public health policies to prevent early-onset MetS and reduce its effects. However, further longitudinal studies that include assessment of lifestyle, ethnicity, and genetic characteristics of volunteers are needed to promote understanding of this disease and its associations in other populations.

This study emphasized that MetS may predispose significant changes in adipokines, inflammatory and oxidative markers. The combination of IL-6, us-CRP, ADA, DPP-IV, increased TBARS with reduction of vitamin C, SH groups and adiponenctin, favors the infiltration and activation of macrophages in adipose tissue, promote inflammation and compromise IS, therefore, it presents a critical role in the pathogenesis of MetS, development of RI, dyslipidemia and atherosclerosis.

These findings are particularly significant because they may assist in monitoring clinical changes and preventing future cardiometabolic events in individuals with MetS. The results may help determine the pathways involved in inflammation related to this condition and prevent the future development of DM2. These results may also be useful in the identification of inflammatory and oxidative markers that assist in monitoring and especially for obese and overweight individuals, thus preventing the development of MetS or helping the follow-up of patients who already have this disease.

ACKNOWLEDGMENTS

The authors are grateful to the UNOESC (SC), Brazil and to the Programa de Bolsas Universitárias de Santa Catarina (UNIEDU) for their support in this study. In addition, we thank all the volunteers who participated in this study.

CONFLICT OF INTERESTS

There are no conflicts of interest.

REFERENCES

1. Karalis KP, Giannogonas P, Kodela E, Koutmani Y, Zoumakis M, Teli T. Mechanisms of obesity and related pathology: linking immune responses to metabolic stress. FEBS J. 2009; 276: 5747-54.

2. Kuno M, Seki N, Tsujimoto S, et al. Anti-inflammatory activity of nonnucleoside adenosine deaminase inhibitor FR234938. Eur J Pharmacol. 2006; 534: 241-9.

3. Haskó G, Cronstein BN. Adenosine: na endogenous regulator of in nate immunity. Trends Immunol. 2004; 25: 33-9.

4. Khemka VK, Bagchi D, Ghosh A, et al. Raised serum adenosine deaminase level in non obese type 2 diabetes mellitus. ScientificWorldJournal. 2013; 2013: 404320.

5. Grundy SM, Brewer HB Jr, Cleeman JI, Smith SC Jr, Lenfant C; National Heart, Lung, and Blood Institute; American Heart Association. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Arterioscler Thromb Vasc Biol. 2004; 24(2): e13-8.

6. Yazbeck R, Howarth GS, Abbott CA. Dipeptidyl peptidase inhibitors, an emerging drug class for inflammatory disease? Trends Pharmacol Sci. 2009; 30: 600-7.

7. Mentlein R. Dipeptidyl-peptidase IV (CD26) – role in the inactivation of regulatory peptides. Regul Pept. 1999; 85: 9-24.

8. Matteucci E, Giampietro O. Dipeptidyl peptidase-4 (CD26): knowing the function before inhibiting the enzyme. Curr Med Chem Rev. 2009; 16: 2943-51.

9. Stone JD, Chervin AS, Kranz DM. T-cell receptor binding affinities and kinetics: impact on T-cell activity and specificity. Immunology. 2009 Feb; 126(2): 165-76.

10. Deacon CF, Carr RD, Holst JJ. DPP-4 inhibitortherapy: new directions in the treatment of type 2 diabetes. Front Biosci. 2008; 13: 1780-94.

11. Espeland MA, Rejeski WJ, West DS; Action for Health in Diabetes Research Group. Intensive weight loss intervention in older individuals: results from the action for health in diabetes type 2 diabetes mellitus trial. J Am Geriatr Soc. 2013; 61: 912-22.

12. Furukawa S, Fujita T, Shimabukuro M, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004; 114: 1752-61.

13. Hee Park K, Zaichenko L, Brinkoetter M, et al. Circulating irisin in relation to insulin resistance and the metabolic syndrome. J Clin Endocrinol Metab. 2013; 98: 4899-4907.

14. Higdon JV, Frei B. Obesity and oxidative stress: a direct link to CVD? Arterioscler Thromb Vasc Biol. 2003; 23: 365-7.

15. Gallagher D, Heymsfield SB, Heo M, Jebb SA, Murgatroyd PR, Sakamoto Y. Healthy percentage body fat ranges: an approach for developing guidelines based on body mass index. Am J Clin Nutr. 2000; 72(3): 694-701.

16. Chielle EO, Bonfanti G, de Bona KS, Moresco RN, Moretto MB. Adenosine deaminase, dipeptidyl peptidase-IV activities and lipidperoxidation are increased in the saliva of obese Young adult. Clin Chem Lab Med. 2015; 53(7): 1041-7.

17. Kang DR, Yadav D, Koh SB, Kim JY, Ahn SV. Impact of serum leptin to adiponectin ratio on regression of metabolic syndrome in high-risk individuals: the ARIRANG study. Yonsei Med J. 2017; 58(2): 339-46.

18. Lourie JA, Weiner JS. Practical human biology. London: Academic Press; 1981.

19. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972; 499-502.

20. Nagatsu T, Hino H, Fuyamada H, Hayakawa T, Sakakibara S. New chromogenic substrates for X prolyldipeptidyl-aminopeptidase. Anal Biochem. 1976; 74(2): 466-76.

21. Lapenna D, Ciofani G, Pierdomenico SD, Giamberardino MA, Cuccurullo F. Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma. Free Radic Biol Med. 2001; 31: 331-5.

22. Boyne AF, Ellman GL. A methodology for analysis of tissue sulfhydryl componentes. Anal Biochem. 1972; 46: 639-53.

23. Singh N, Bhardwaj P, Pandey RM, Saraya A. Oxidative stress and antioxidant capacity in patients with chronic pancreatitis with and without diabetes mellitus. Indian J Gastroenterol. 2012; 31(5): 226-31.

24. Senn JJ, Klover PJ, Nowak IA, Mooney RA. Interleukin-6 induces cellular insulin resistance in hepatocytes. Diabetes. 2002; 51: 3391-9.

25. Jones SA, Horiuchi S, Topley N, Yamamoto N, Fuller GM. The soluble interleukin 6 receptor: mechanisms of production and implications in disease. FASEB J. 2001; 15: 43-58.

26. Tsigos C, Papanicolaou DA, Kyrou I, Defensor R, Mitsiadis CS, Chrousos GP. Dose-dependent effects of recombinant human interleukin-6 on glucose regulation. J Clin Endocrinol Metab. 1997; 82: 4167-70.

27. Boden G, Shulman GI. Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and betacell dysfunction. Eur J Clin Invest. 2002; 3: 14-23.

28. Petersen EW, Carey AL, Sacchetti M, et al. Acute IL-6 treatment increases fatty acid turnover in elderly humans in vivo and in tissue culture in vitro. Am J Physiol Endocrinol Metab. 2005; 288: E155-62.

29. Fasshauer M, Kralisch S, Klier M, et al., Adiponectin gene expression and secretion is inhibited by interleukin-6 in 3T3-L1 adipocyte. Biochem Biophys Res Commun. 2001; 301: 1045-50.

30. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes. Nature. 2001; 409(6818): 307-12.

31. Ort T, Arjona AA, MacDougall JR, Nelson PJ, Rothenberg ME, Wu F. Recombinant human FIZZ3/resistin stimulates lipolysis in cultured human adipocytes, mouse adipose explants, and normal mice. Endocrinology. 2005; 146: 2200-9.

32. Kok BP, Brindley DN. Myocardial fatty acid metabolism and lipotoxicity in the setting of insulin resistance. Heart Fail Clin. 2012; 8: 643-61.

33. Melone M, Wilsie L, Palyha O, Strack A, Rashid S. Discovery of a new role of human resistin in hepatocyte low-density lipoprotein receptor suppression mediated in part by proprotein convertase subtilisin/kexin type 9. J Am Coll Cardiol. 2012; 59(19): 1697-705.

34. Xia M, Liu Y, Guo H, Wang D, Wang Y, Ling W. Retinol binding protein 4 stimulates hepatic sterol regulatory element-binding protein 1 and increases lipogenesis through the peroxisome proliferator-activated receptor-γ coactivator 1β-dependent pathway. Hepatology. 2013; 58: 564- 75.

35. Grundy SM. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Circulation. 2005; 109: 433-8.

36. Yeum KJ, Russel RM, Krinky NI, Aldini G. Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma. Arch Biochem Bioph. 2004; 430(1): 97-103.

37. Frühbeck G, Gómez-Ambrosi J, Salvador J. Leptin induced lipolysis opposes the tonic inhibition of endogenous adenosine in white adipocytes, FASEB J. 2001; 15(2): 333-40.

38. Bellé LP, Bitencourt PE, de Bona KS, Moresco RN, Moretto MB. Association between HbA1c and dipeptidyl peptidase IV activity in type 2 diabetes mellitus. Letter to the editor. Clin Chem Acta. 2012; 413: 1020-1.

39. Chielle EO, Souza WM, Silva TP, Moresco RN, Moretto MB. Adipocytokines, inflammatory and oxidative stress markers of clinical relevance altered in young overweight/obese subjects. Clin Biochem. 2016; 49(7-8): 548-53.