Oxidative stress parameters as biomarkers of risk factor for diabetic foot among the patients with type 2 diabetes

Oxidative stress parameters as biomarkers of risk factor for diabetic foot among the patients with type 2 diabetes

Ana Carla Pozzi Oliveira^I; Caio Jordão Teixeira^II; Talitha Fernandes Stefanello^I; Marcia Aparecida Carrara^II; Roberto Barbosa Bazotte^{II, *}; Anacharis Babeto Sá-Nakanishi^III; Jurandir Fernando Comar^III; Marcia Regina Batista^I

^IDepartamento de Análises Clínicas e Biomedicina; Universidade Estadual de Maringá; Maringá - PR - Brasil

^IIDepartamento de Farmacologia e Terapêutica; Universidade Estadual de Maringá; Maringá - PR - Brasil

^IIIDepartamento de Bioquímica; Universidade Estadual de Maringá; Maringá - PR - Brasil

ABSTRACT

The aim of this study was to determine whether plasma levels of carbonylated proteins, total antioxidant capacity (TAC) and reduced protein thiols could be suitable biomarkers of risk factors for diabetic foot. Individuals with type 2 diabetes with normal protective sensation (normal foot group) vs. loss of protective sensation and/or signs of peripheral arterial disease and/or foot deformities and/or history of ulcers and/or neuropathic fractures and/or amputation (diabetic foot group) were compared. The diabetic foot group showed higher carbonylated protein levels (P = 0.0457) and lower levels of TAC (P = 0.0148) and reduced protein thiols (P = 0.0088), compared with the normal foot group. In general, several other parameters of risk of diabetes complication (blood levels of glycated hemoglobin, glucose and cholesterol, duration of diabetes, body mass index and waist circumference) showed a tendency of higher values in the diabetic foot group. The results suggest that the plasma levels of carbonylated proteins, TAC and reduced protein thiols could furnish information about the risk of diabetic foot, considering that the changes in these biomarkers were associated with the loss of sensitivity and foot ulcerations.

Key words: Diabetic foot, oxidative stress, total antioxidant capacity, reduced protein thiols, protein carbonyls

INTRODUCTION

Combined peripheral neuropathy and ischemia result in a higher risk of foot ulcers in type 1 and type 2 diabetic patients (Singh et al. 2005). The risk of patients with diabetes developing foot ulcers in their lifetime could be as high as 25% (Singh et al. 2005). For this reason, diabetes is the leading cause of amputation worldwide. For example, a global study of lower extremity amputation estimated that 25–90% of all the amputations were associated with diabetes (Global Lower Extremity Amputation Study 2000). Regardless of the high incidence of foot ulcers and amputations associated with diabetes, the characterization of risk factors that could prevent foot ulcers and amputations is not well established (Sun et al. 2012). Therefore, the determination of biomarkers for early detection not only of foot ulcers but also nerve damage, infection and gangrene should be investigated.

Because several studies have described the role of oxidative stress in causing diabetic foot ulcers (Bolajoko et al. 2008), the evaluation of blood parameters of oxidative stress as biomarkers of diabetic foot risk must be considered. Considering that 20-40% and 5-7% of patients with type 2 diabetes have neuropathy and foot ulcers, respectively, (Sun et al. 2012) and that type 2 diabetes represents 95% of diagnosed patients, this study focused on the patients with type 2 diabetes. Accordingly, the blood levels of total antioxidant capacity (TAC), reduced protein thiols and carbonylated proteins in the patients with type 2 diabetes with normal foot vs. diabetic foot were compared.

MATERIALS AND METHODS

Eligibility criteria were confirmed diagnosis of type 2 diabetes and age over 40 years. Exclusion criteria were pregnancy, gestational diabetes, type 1 diabetes and other specific types of diabetes. The study followed the guidelines as described in the Declaration of Helsinki and written consent of the participants was obtained. The study was approved by the Ethics Committee of the State University of Maringá, PR, Brazil (COPEP - CAAE 381/2010). During the consultation, the patients were interviewed using a structured questionnaire, and information about the socio-demographic and disease factors (age, sex, medical history, educational level, marital status, duration of diabetes, diabetes-related disorders, etc.), pharmacotherapeutic profile and lifestyle were obtained. After the interview, the measurements of body mass index (BMI) and waist circumference and foot examination were performed.

The foot examination was based on the National Hansen's Disease Program (NHPD) developed by the University of Baton Rouge, LA, USA, which identified those patients who had lost protective sensation (Tan 2010). This diabetic foot screen used a 5.07 monofilament, which delivered 10 g of force on 12 places of application to identify the patients with the risk of developing diabetic foot. The results obtained from this test permitted the classification of the foot into four categories: a) normal protective sensation; b) loss of protective sensation; c) loss of protective sensation plus signs of peripheral arterial disease and/or foot deformities; d) history of ulcers and/or neuropathic fractures and/or amputation. The patients with normal protective sensation were included in the normal foot group, and the other patients were included in the diabetic foot group.

Just before finishing the consultation, the patients received instructions for blood collection. In general, the instructions and procedures for blood collection were similar to those adopted in a previous study investigating the risk factors of coronary heart disease in the population of northwestern Paraná (Silva et al. 2004). The biochemical parameters investigated were blood glycated hemoglobin A1c (Metus et al. 1999) and serum glucose (Bergmeyer and Bernt 1974), cholesterol (Allain et al. 1974), triacylglycerol (Bucolo and David 1973), creatinine (Bartels et al. 1972), reduced protein thiols (Faure and Lafond 1995), TAC (Erel 2004) and carbonylated proteins (Levine et al. 1990). Part of the results was presented as a percentage (%) and part of the results as mean ± standard deviation (SD). Comparison between the normal foot group and diabetic foot group was carried out using the unpaired Student t-test. P< 0.05 was considered statistically significant.

Complete data for 28 patients showed that 11 belonged to normal foot group and 17 to diabetic foot group. Most patients (Table 1) in both the groups (normal, or diabetic foot group) were female (> 70%), were knowledgeable about diabetic foot (> 80%) and had a family history of diabetes (> 75%). The diabetic foot group had a higher age (P = 0.0235) and tendency of longer duration of diabetes, higher BMI and waist circumference and higher percentage of nephropathy. In spite of diabetic foot, 11.8% patients in this group were not receiving any medication (Table 1). On the other hand, the normal foot group showed a higher percentage of patients graduated from high school, engaged in physical activity at least three times a week and using oral antidiabetic drugs. Marital status, smoking and presence of retinopathy were similar in the two groups (Table 1). The diabetic foot group revealed a tendency of higher blood glycated hemoglobin A1c, glucose, total cholesterol, low-density lipoproteins, triacylglycerols and creatinine, compared with the normal foot group. Moreover, a tendency for lower high-density lipoproteins was observed in the diabetic foot group (Table 2).

As shown in Table 3, the diabetic foot group showed higher carbonylated proteins (P = 0.0457) and lower TAC (P = 0.0148) and reduced protein thiols (P = 0.0088), compared with the normal foot group. It must be emphasized that these significant differences were observed in a small number of patients (11 normal foot vs. 17 diabetic foot), while for Hb A1c (Table 2), only a tendency for higher values was observed.

DISCUSSION

Diabetes is not only a disease of the altered metabolism of carbohydrates, lipids and protein, but also of the altered chemistry of carbohydrates, lipids and proteins. However, while short-term insulin deficiency could easily explain the classical changes in carbohydrate, lipid and protein metabolism (Monnier et al. 2012), the altered chemistry of carbohydrates, lipids and proteins as consequence of chronic insulin deficiency is not well established. Poorly controlled diabetes accelerates chemical modification of proteins and function of tissue proteins, precipitating the development of diabetic complications. In this context, there are several hypotheses on the origin of complications, including mitochondrial damage, mitochondrial defect in oxidative phosphorylation, increased oxidative and reductive stress, increased formation of advanced glycation end products (AGES), increased activity of the polyol pathway, hypoxia, altered lipoprotein metabolism, increased protein kinase C activity, altered growth factors and cytokine activities (Baynes and Thorpe 1999; Arya et al. 2011; Papanas and Ziegler 2011).

Increased levels of reactive carbonyl compounds derived from proteins by both oxidative and non-oxidative reactions lead to increased chemical modification of proteins, and then, at a later stage, to oxidative stress and tissue damage (Baynes and Thorpe 1999). Therefore, this study evaluated plasma carbonyl proteins as a biomarker of diabetic foot. In addition, two parameters of defense against oxidative stress were assessed, i.e., plasma reduced protein thiols and TAC. The results showed increased plasma levels of carbonylated proteins (P = 0.0457) in patients with diabetic foot.

Carbonyl stress is the result of a higher level of reactive carbonyl species and may be the consequence of an increased substrate stress and/or a decrease in the efficiency of detoxification of carbonyl compounds, which leads to increased chemical modification of biomolecules and thereby to a series of tissue dysfunction (Noeman et al. 2011). Therefore, the clinical severity of diabetic foot could be related to the development of carbonyl stress. The present results also showed lower plasma levels of reduced protein thiols (P = 0.0088) and TAC (P = 0.0148) in the patients with diabetic foot. In agreement with these findings, other studies (Bolajoko et al. 2008; Yang et al. 2011) have suggested that elevated oxidative stress could be associated with increased risk of diabetic foot.

Interestingly, the diabetic foot group showed a higher percentage of patients not on insulin therapy, or on antihypertensive, or hypolipidemic therapy. In agreement with these observations, the diabetic foot group showed a tendency of higher glycated hemoglobin level and plasma levels of glucose, triacylglycerol, and cholesterol and its fractions. This study did not include the localization of tissue-specific change in oxidative stress in the foot, nor did it establish a clear role for blood levels of altered protein thiols, TAC and carbonyl protein in the pathogenesis of diabetic foot. Despite these limitations, results provided evidence that reduced protein thiols, TAC and carbonyl proteins in plasma could be useful biomarkers in the early detection of diabetic foot in the patients with type 2 diabetes.

CONCLUSION

Results showed a decreased defense against oxidative stress in the patients with diabetic foot. Therefore, the plasma levels of TAC, protein thiols and carbonyl proteins could provide additional information about the risk of diabetic foot, considering that the alteration of these biomarkers was associated with the loss of sensitivity and foot ulcerations.

ACKNOWLEDGMENTS

This study was supported by CNPq grant 563870/2010-9 and PRONEX/CNPq/Fundação Araucária. The authors are grateful to C.E. Oliveira, M. Guilhermetti and S. C. Oliveira for their technical assistance during the experiments and Carlos A. dos Santos for data management. Dr. A. Leyva helped with English editing of the manuscript.

Received: June 18, 2012;

Accepted: July 22, 2013.

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Publication Dates

• Publication in this collection
  19 Nov 2013
• Date of issue
  Apr 2014

History

• Accepted
  22 July 2013
• Received
  18 June 2012