An assessment of correlation between serum asymmetric dimethylarginine and glycated haemoglobin in patients with type 2 diabetes mellitus

Studies that investigated an association between asymmetric dimethylarginine (ADMA) and glycated haemoglobin (HbAc) in type  diabetes mellitus (TDM) have given discordant results. Th e aim of this study was to determine and compare serum ADMA concentration in patients with TDM and healthy controls, and to assess correlation between ADMA and HbAc in patients with TDM. Serum ADMA concentration was determined by ELISA method with the use of ADMA ® ELISA kit (DLD Diagnostics, Hamburg, Germany) and HbAc levels were determined by an immunoturbidimetric method in  patients with TDM and  healthy individuals matched for age and sex. Results have shown that mean serum ADMA concentration was signifi cantly higher in TDM patients (.±. μmol/L) compared to mean serum ADMA concentration (.±. μmol/L; p<.) in healthy subjects. A signifi cant, positive, correlation between serum ADMA concentration and HbAc levels was observed (r=.; p<.) in TDM patients. Our results suggest that there is an association between endothelial dysfunction and glycaemic control in type  diabetes mellitus. Possible explanation for obtained results may be oxidative stress that is increased in conditions of hyperglycaemia and it also promotes endothelial dysfunction. Larger, longitudinal studies are required that will evaluate relation between metabolic abnormalities and increased ADMA levels in patients with type  diabetes mellitus. ©  Association of Basic Medical Sciences of FB&H. All rights reserved


INTRODUCTION
Asymmetric dimethylarginine (ADMA) is a novel risk factor of endothelial dysfunction [].Its role in the development and progression of type  diabetes mellitus (TDM) is not fully known.ADMA is an amino acid discovered in  and presents non-invasive marker of endothelial dysfunction, as well as novel cardiovascular risk factor [].It circulates in plasma, and is present in different tissues and cells.Determined plasma values of ADMA using ADMA ELISA techniques in  healthy white individuals  to  years old were in a range from . to . μmol/L [].Studies have shown that ADMA increases systemic vascular resistance and mean arterial blood pressure, and decreases cardiac output [].It is established that infusion of exogenous ADMA decreases heart rate, and increases pulmonary vascular resistance.Administration of ADMA disturbs renal blood flow, sodium reabsorbtion and decreases cerebral perfusion [].One of the most prominent features of ADMA is that it represents inhibitor of nitric oxide (NO) synthesis [, ].Major mechanisms that lead to increased ADMA values in diff erent diseases include shear stress, oxidative stress, hyperhomocysteinaemia, and high concentration of glucose in blood [, ].Studies that investigated an association between ADMA and glycated haemoglobin (HbA c ) in patients with TDM are scarce and have given discordant results [-].The aim of the present study was to investigate whether serum ADMA is increased in patients with TDM compared to healthy individuals.Also, we aimed to assess whether there is a correlation between serum ADMA concentration and HbA c levels in TDM patients.

Subjects
We recruited  patients with type  diabetes mellitus ( male,  female), as defi ned by American Diabetes Association [], who regularly attend the Out-Patient Family Medicine Clinic "Višnjik" in Sarajevo.All patients were receiving antidiabetic and antihypertensive therapies and some were receiving antilipidemic drugs and/or aspirin for at least the previous  months.Exclusion criteria were the presence of sustained acute and chronic infections, malignancy, hepatic or renal disease, diabetic retinopathy and nephropathy, and other endocrine dysfunctions.Th e control group consisted of  apparently healthy subjects ( male,  female) with no history of TDM, other endocrine dysfunctions, hyperlipidemia, hypertension, or coronary heart diseases.None of the control subjects had received any medication (hormone replacement therapy, corticosteroids, vitamin supplements, antioxidant formulations and thiazolidinediones) which may have aff ected insulin resistance and/or endothelial function and none of the subjects were current smokers and consumers of alcohol.

Blood sampling
Blood was collected in the morning after an overnight fast after a -min rest in a semi-recumbent position.Sampling was done without stasis, using the vacutainer technique.

Blood chemistry analysis
Serum ADMA concentration was determined by ELISA method (machine STAT FAX , USA) at the Department of Physiology and Department of Biochemistry, Faculty of Medicine in Sarajevo.ADMA ® -ELISA kit (DLD Diagnostika GmbH, Germany) was used as reagent.ADMA standards range from . to . μmol/L and the detection limit for ADMA with the use of this method is . μmol/L.HbA c was measured by an immunoturbidimetric method (TINIA; Siemens Healthcare Diagnostics Ltd, Camberley, United Kingdom).Normal range was .-..Th e total cholesterol (TC), HDL-cholesterol (HDL-C), and triglyceride levels were measured with the use of standard enzymatic methods.Th e LDL-cholesterol (LDL-C) levels were calculated using the Friedewald formula.All biochemical tests, except for ADMA determination, were performed at Institute for clinical chemistry and biochemistry University of Sarajevo Clinics Centre.The study was approved by the Ethics Committee of the Medical Faculty University of Sarajevo.Written informed consent was obtained from all subjects.Investigations were carried out in accordance with the Declaration of Helsinki as revised in .

Statistical analysis
Th e Kolmogorov-Smirnov test of normality was used to test the distribution of variables.An unpaired Student t-test was used to compare the diff erence between two groups.Since tested variables were normally distributed, correlations were assessed by Pearson's test.A p value of less then . was considered statistically significant.The software used was SPSS for Windows (version .; SPSS, Chicago, IL, USA).

RESULTS
Th e baseline characteristics of study participants are reported in Table .Age and body mass index (BMI) did not diff er between control group of subjects and patients with TDM.Th e mean duration of diabetes was .±.years.Statistically signifi cant diff erence was established in values of systolic and diastolic blood pressure between control group of subjects and patients with TDM.As it could be expected

).
As presented in Table , there was no statistically significant correlation between levels of lipid profi le indices and ADMA concentration both in the control and TDM group.

DISCUSSION
Results of our study clearly showed that serum ADMA concentration in patients with TDM was signifi cantly higher compared to age and sex -matched healthy subjects.Obtained results are in accordance with majority of studies that tested concentration of ADMA in patients with TDM [,].However, Paiva et al. [] consider that long-term hyperglycaemia may result in decreased ADMA values in patients with TDM.Our results are not consistent with the findings of mentioned authors.Contrary to their opinion, we believe that precisely hyperglycaemia may be behind ADMA increase determined in TDM patients.Th is metabolic disturbance may induce decreased NO formation and lead to endothelial dysfunction [].Furthermore, even though mechanisms that are responsible for ADMA increase in patients with TDM are still not fully elucidated, it is possible that increased serum ADMA concentration may be a consequence of decreased expression or activity of dimethylarginine dimethylaminohydrolase, which is an enzyme responsible for ADMA degradation [].
Results of the studies that investigated correlation between ADMA and HbA c in patients with TDM have given discordant results.Paiva et al. [] determined statistically significant negative correlation between ADMA and HbA c in patients with TDM.Our results have established highly significant positive correlation between ADMA and HbA c in our study sample.Results of Anburajan et al. [] have also observed signifi cant positive correlation between ADMA and HbA c in patients with TDM.
Results of our study suggest that there is an association between endothelial dysfunction and glycemic control in patients with TDM.However, since some authors have determined negative, and others positive correlation between ADMA and HbA c in the conditions of TDM, we are of the opinion that future studies should give an answer to the question whether good glycemic control is followed by decrease in ADMA concentration or hyperglycaemia per se conditions decrease in ADMA values by promotion of NO synthesis.
Although it has been demonstrated that hypercholesterolemia is associated with endothelial cell dysfunction [, ], results of our study did not show significant correlation between lipid profi le indices and ADMA levels in both control subjects and patients with TDM.Obtained results are consistent with observation of Krzyzanowska et al. [] that also did not find significant association between serum lipids and ADMA in morbidly obese women.Conversely, Yamamoto et al. [] reported significant negative correlation between serum ADMA and HDLcholesterol in patients with TDM.Furthermore, findings of this study have shown that HDL-cholesterol is  independent determinant of serum ADMA in TDM patients.However, our results do not support these findings which might be due to limited size of our study sample.

CONCLUSION
In a conclusion it can be stated that serum ADMA concentration is increased in patients with type  diabetes mellitus.Among many, one of the possible explanations for the observed ADMA increase may be an axis that consists from hyperglycaemia that induces increased secretion of proinflammatory cytokines such as tumour necrosis factor -alpha (TNF-α), that in turn leads to increased ADMA values in the conditions of type  diabetes mellitus.Furthermore, positive correlation between ADMA and HbA c in patients with type  diabetes mellitus is determined.
Although in our study indices of oxidative stress were not measured, we fi nd it plausible that possible explanation for the established association between ADMA as a marker of endothelial dysfunction and HbA c as an index of glycemic control might be this disturbance of oxidant/antioxidant balance since it is known to be increased in conditions of hyperglycaemia and it also promotes endothelial dysfunction.Finally, larger longitudinal studies are required that will further evaluate observed relation between metabolic abnormalities and increased ADMA levels in patients with type  diabetes mellitus

DECLARATION OF INTEREST
Th e authors declare no confl ict of interest.

TABLE 1 .
Baseline characteristics of the control subjects and patients with type 2 diabetes.

TABLE 2 .
Pearson's correlation analysis between levels of serum total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol levels and serum asymmetric dimethylarginine concentration in control subjects and patients with type 2 diabetes mellitus.FIGURE 2. Pearson's correlation analysis of serum glycated haemoglobin values and asymmetric dimethylarginine concentrations in patients with type 2 diabetes mellitus.