Correlation of Resistin levels with risk factors in type 2 diabetes mellitus patients with ischemic stroke

 

 

Abstract               Background: Ischemic stroke in diabetic individuals is complex in nature with contributions from both environmental and genetic factors. Resistin links the metabolic dysregulation to atherosclerosis through selective inhibition of PKB insulin signalling and activation of MAPK pathway. Aim: To find the correlation of resistin levels with risk factors in type 2 diabetes mellitus patients with ischemic stroke. Material and Methods: Study population was grouped as Group 1: 60 unrelated Type 2 diabetic adults diagnosed with ischemic stroke and Group 2: 60 unrelated Type 2 diabetic adults without ischemic stroke and controls including 60 individuals not fitted in either group.Results: There was a significant weak positive correlation between serum resistin levels and age, waist circumference, fasting plasma glucose, lipid profile except for HDL-c levels. Significant weak negative correlation was found between serum resistin levels and HDL-c. Conclusion: Among patients with diabetes several risk factors play a role together to promote the development of ischemic stroke. Significant correlation found between serum resistin levels and age, waist circumference, fasting plasma glucose, lipid profile except for HDL-c levels.

Key Word: Type 2 diabetes mellitus, ischemic stroke, resistin level, risk factors

 

 

INTRODUCTION

Type 2 Diabetes mellitus affects large number of people worldwide. It is related with cerebrovascular and cardiovascular complications. Diabetes is continuing as global epidemic and it is particularly concentrated in Indian subcontinent¹ and that there is also a greater propensity of Indian population developingstroke.²A cerebrovascular complication that is more commonly associated with diabetes is ischemic stroke.³ Diabetes is an independent risk factor for ischemic stroke. Diabetes mellitusis associated with 2 to 5 fold increased risk for the first or recurrent ischemic stroke.^4,5 Diabetes as an independent factor affects the stroke in age of onset, in its types, also in prognosis. In India, it has been found that 80% of stroke are due to ischemia.⁶ Identifying the factors that predisposes the diabetic patients to macroangiopathy will be helpful in monitoring the disease progression. Ischemic stroke in diabetic individuals is complex in nature with contributions from both environmental and genetic factors. Ischemic stroke is a complicated vascular disease and it is affected by many factors. Conventional risk factors can explain only half of the ischemic stroke incidence.⁷Resistin, is a11.4 kDa protein rich in cysteine residues. This is expressed mainly in monocytes and macrophages. Though it was discovered from the adipose tissue, later it is reported to be secreted from macrophages infiltrating the adipose tissue and preadipocytes. It is involved in inflammatory and metabolic signals.⁸ Thus resistin could be a link between diabetes and ischemic stroke. Resistin links the metabolic dysregulation to atherosclerosis through selective inhibition ofPKB insulin signalling and activation of MAPK pathway. The gene, RETN at chromosome 19p13.2 codes for human resistin.⁹ Thus, this study was conducted with an aim to find the correlation of resistin levels with risk factors in type 2 diabetes mellitus patients with ischemic stroke.

 

MATERIAL AND METHODS

This case control study was approved by ethical committee. All the participating individuals and their relatives were informed about the study.

Inclusion criteria: The study population was categorised into two groups:

Group 1: included 60 unrelated Type 2 diabetic adults diagnosed with ischemic stroke of age around 40-70 years (32 males and 28 females). This study population was selected among the atherothrombotic stroke patients admitted in the neurology and medicine wards.

Group 2: Included 60 unrelated Type 2 diabetic adults without ischemic stroke of age around 40-70 years (33 males and 27 females). These individuals were selected from diabetology outpatient department. They were diagnosed diabetic patients without any history and symptoms of stroke.

Exclusion criteria: Type 2 diabetics with other chronic illness such as renal or hepatic failure, autoimmune diseases, neoplasms, coagulopathies, myocardial infarction, family history of autoimmune or chronic inflammatory diseases and those on thiazolidones group of hypoglycemicdrugs were excluded from both the groups 1 and 2. Diabetics with less than 5 years of duration of diabetes were also excluded from both the goups. Ischemic stroke patients with known embolic source were excluded from group 2.

Group 3 (Control): 60 individuals of age around 40-60 years (29 males and 31 females) were selected as controls. Exclusion criteria same as above is applied to control group. In addition, they should not be diabetics and there should not be history of stroke. Age, sex and other confounding factors like smoking and alcoholism were matched. The definition of stroke was set as neurological deficit of abrupt onset due to vascular originlasting longer than 24 hours. Ischemic stroke patients with history of diabetes were diagnosed with clinical history, neurological examination and brain computer tomography (CT) scan to rule out hemorrhagic stroke and then included in the study. This was followed by echocardiography and carotid doppler to rule out the embolic source.

Characteristics of study population

• Participant's baseline information and anthropometric measurements were recorded.
• Assessment of vascular risk factors were also done.

 In this study some of the risk factors were defined as:

• Hypertension defined as previous diagnosis of hypertension by physician and/or treatment with antihypertensive drugs.
• A positive smoking and alcohol consumption history were recorded if the individuals had smoked or consumed alcohol regularly at any time in their lives respectively.

Sample collection: Blood was collected after an overnight fasting of 8-12 hours. About 4 ml of blood was collected from the antecubital vein of the subjects by aseptic procedures.2 mL was transferred into a EDTA tubes and another2 mL was transferred in to plain non additive tubes. Blood was collected from the stroke patients after 7-10days of the onset of stroke to avoid the transient early changes of biochemical markersin the blood. The plain non additive tubes were allowed to clotfor 30 minutes and then centrifuged for 15 minutes at 3000 rpm. Serum was separated, collected in 2 ml microcentrifuge tubes and stored at -80c for the estimation of resistin.

Estimation of serum resistin: Serum resistin was measured by Sandwich Enzyme Linked Immunosorbent Assay by using Quantikine- R and D Systems. This assay reagent is highly specific detects only recombinant and human resistin. No significant cross reactivity was observed with analytes such as leptin and RELMβ. The intra assay precision is 4.7% and the inter assay precision is 8.4%.

Resistin standard preparation: All the reagents were in room temperature. 1mL of distilled water is added to reconstitute the resistin standard. The stock solution of resistin standard of 100ng/mL was prepared. The following serial dilution of stock standard solution(100ng/mL) was done with the calibrator diluent.

Sample preparation: Serum samples were brought to room temperature. 5-fold dilution of sample is done as 50µL of sample + 200µL of calibrator diluent.

Assay procedure: 100µL of assay diluent was added to each well of ELISA microplate. Then 100µL of resistin standards S1 to S8 were added to the wells.100µL of samples were added into the remaining wells. The plate was covered with adhesive strips and then incubated for 2 hours at room temperature. Standards and samples added were recorded in the plate layout. In the meantime20 mL of wash buffer concentrate was diluted to 500mL by adding distilled water. The program was set in the automated ELIS Aplate washer with soaking time as 30 seconds and repeating the wash three times.

After 2 hours of incubation, the ELISA microplate was placed in the ELISA washer. After the last wash, the plate was inverted and blotted against the clean paper to remove the remaining wash buffer. 200µL of resistin conjugate was then added to each well. The plate was covered with adhesive strip and incubated for 2 hours at room temperature. The plate was washed with the same program in the automated ELISA washer. Substrate solution was prepared by mixing the Colour Reagent A and B and protected from light by covering with aluminium foil.200µL of Substrate solution is added to each well. The plate is then incubated for 30 minutes and covered with adhesive strip to protect from light.. 50µL of Stop solution was added to each well. On adding the Stop solution, blue colour in the wells changed to yellow colour. The program was set in the ELISA microplate reader to determine the optical density of each well at 450nm.

Statistical analysis: Data were analysed using SPSS statistical software version 16 and was considered as statistically significant if the p value is less than 0.05. Age, Waist circumference, fasting plasma glucose and fasting lipid profile were compared between the three study groups by ANOVA. Gender, smoking, alcoholism hypertension were compared between the three study groups by chi-square test. Duration of diabetes, fasting plasma glucose, hypertension and fasting lipid profile were compared between the two study groups by students' independent t-test. Correlation of the parameters namely age, waist circumference, fasting plasma glucose and fasting lipid profile with serum resistin levels were found out by Pearson’s correlation analysis.

RESULTS

There were no significant difference in the distribution of age and sex among the study subjects, showing that this is an age and sex matched case-control study. Also there were no significant difference between the study groups in smoking, alcoholism and hypertension. This shows that these risk factors were also matched among the three groups of this study.We obtained a significant difference between the study groups in waist circumference, concentration of fasting plasma glucose, triglycerides, HDL-cholesterol, TC/HDL-c ratio and NonHDL-cholesterol with the p value <0.001 But there is no significant difference in the concentration of total cholesterol and LDL-cholesterol between study groups. Serum resistin levels were also significantly high in type 2 DM patients with ischemic stroke(39.5±21.2 ng/mL) compared to type 2 DM patients without ischemic stroke (25.9±13.6 ng/mL) and controls (21±9.58 ng/mL) with p value <0.001.

 

Table 2: comparisons of characteristics between diabetics Without ischemic stroke and diabetics with ischemic stroke

* Significant at p<0.05 ** highly significant at p<0.001

able 3 shows the correlation of serum resistin levels with age, waist circumference, fasting plasma glucose and fasting lipid profile of study population. There was a significant weak positive correlation between serum resistin levels and age, waist circumference, fasting plasma glucose, lipid profile except for HDL-c levels. Significant weak negative correlation was found between serum resistin levels and HDL-c.

Table 3: Correlation of resistin levels with other parameters in study groups

DISCUSSION

As diabetes is on increasing incidence, the morbidity and mortality due to diabetic vascular disease is also on parallel rise. Diabetes is found to be an independent risk factor of ischemic stroke. Diabetes mellitus affects the clinical manifestation of ischemic stroke in many aspects like age of onset, type of stroke, prognosis, recurrence and mortality. The reason for the increased risk of stroke in diabetes may be due to the molecular link between the various metabolic, hemodynamic and inflammatory signalling pathways. Resistin is plays a multirole in insulin resistance, inflammation and hence on atherosclerosis. More studies have been done on the development of cardio vascular complication in diabetes mellitus but not so in cerebro vascular disease. Diabetics with same duration of diabetes and glycemia status differs in their susceptibility to ischemic stroke. This points to the role of genetic factors in susceptibility of diabetics to ischemic stroke. Serum resistin levels were positively correlated with age, waist circumference, fasting glucose, total cholesterol, LDL-cholesterol, TC/HDL-C ratio and Non HDL-c. Resistin levels were negatively correlated with HDL-c. But all these correlations had low correlation coefficient value and this may be due to clustered distribution of these parameters in the study population. Even if there is weak correlation, it implies that serum resistin levels increases with age and waist circumference. It also shows that fasting glucose, total cholesterol, LDL-c, TC/HDL-c ratio and Non HDL-c increases with increase in serum resisitn levels.               This similar to the study on Indian females where resistin levels were positively correlated with waist circumference, plasma glucose, serum triglycerides, serum cholesterol, and negatively correlated with HDL-c.¹⁰ Similarly it has been reported by Osawa et al in Japanese population.¹¹ According to Surapon et al.,2010, TC/HDL-c ratio and Non HDL-can be considered as marker of insulin resistance.¹² In this study it has been shown that there was a significant association of resistin levels with HDL-c , TC/HDL-c ratio, non HDL-c and hence with insulin resistance.

 

CONCLUSION

Among patients with diabetes several risk factors play a role together to promote the development of ischemic stroke. In the analysis of these risk factors can be identified diabetes-specific factors such as hyperglycemia and vascular risk factors such as hypertension and dyslipidemia. Significant correlation found between serum resistin levels and age, waist circumference, fasting plasma glucose, lipid profile except for HDL-c levels.

 

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