A STUDY ON THE CORRELATION OF ADENOSINE DEAMINASE AND GLYCATED HAEMOGLOBIN IN THE PATIENTS OF TYPE 2 DIABETES MELLITUS

Background & Objectives: Adenosine modulates insulin action on various tissues and its concentration in tissues is affected by Adenosine Deaminase (ADA) levels. ADA is an enzyme involved in purine metabolism and is considered to be a marker of T cell activation. Immunological disturbances in type 2 diabetic individuals have an association with cell mediated responses and inappropriate T-lymphocyte function. Hence, the study was undertaken to determine the levels of Serum ADA activity in patients of type 2 DM and its correlation with parameters of glycemic profile such as Fasting blood sugar (FBS) and Glycated Haemoglobin. Material and Methods: A total of 100 patients diagnosed for type 2 DM visiting the Outpatient Department of General Medicine and Endocrinology at Mahatma Gandhi Medical College & Hospital, Jaipur were enrolled for the study based on predefined inclusion and exclusion criteria. Blood samples were collected for all enrolled patients and analysed for the investigations like Serum BSF, HbA1c and Serum ADA. Results: In the study, BSF, mean HbA1c and serum ADA level was significantly higher in diabetic group in comparison to control group (p=0.000). The diabetic group was subdivided on the basis of HbA1c levels, HbA1c ≤ 8% as good glycemic control and HbA1c > 8% as poor glycemic control. BSF, mean HbA1c and serum ADA levels were observed to be significantly higher in poor glycemic control group as compared to that of good glycemic control. A significant positive correlation between S. ADA and HbA1c activity was also seen (r= 0.388). Conclusion: Increased ADA level can be used to determine the glycemic status in the patients of type 2 DM and serve as a marker for insulin resistance. Hence, by analysing ADA levels in diabetes, glycemic control and insulin resistance can be assessed. Raised ADA levels can be an early indicator of progressive diabetic change and help to take preventive measures for the development of diabetic complication and thereby improving the outcome of the disease.


Introduction
The worldwide prevalence of diabetes has continued to increase dramatically. [1] World Health Organization (WHO), shows that lndia is going to face a big challenge posed by the rising prevalence of diabetes and its complications, unless steps are taken to implement the primary and secondary prevention measures in diabetes. [2] According to the WHO in 2016, about 422 million people worldwide were living with diabetes [3] and International Diabetes Federation (IDF) estimated that the number of people worldwide affected with diabetes is expected to be around 438 million by 2030 and 642 million by 2040. [4] Indian Council of medical research estimated that the diabetes prevalence in individuals above 14 years was 2.1% in urban areas and 1.5% in rural areas while in those above 40 years of age, the prevalence was 5 per cent in urban and 2.8 per cent in rural areas in 1972-1975. [5] Type 2 DM is a metabolic disorder characterized by the presence of chronic hyperglycemia with disturbances in the metabolism of carbohydrates, fats, and proteins. [1] Long term hyperglycemia predisposes to long term micro and macro vascular complications. [5] The American Diabetes Association (ADA) includes the following criteria to be the investigative benchmarks: [7] • A fasting plasma glucose (FPG) level of 126 mg/dL (7.0 mmol/L) or more, or • A 2-hour plasma glucose level of 200 mg/dL (11.1 mmol/L) or more during a 75-g Oral glucose tolerance test (OGTT), or • Random plasma glucose of 200 mg/dL (11.1 mmol/L) or more in a patient with typical signs of hyperglycemia and hyperglycaemic emergency. Serum Adenosine deaminase activity (ADA), an enzyme present in red cells and vessel wall catalyses the irreversible hydrolytic deamination of Adenosine to inosine and 2'deoxyadenosine to 2'-deoxyinosine which is then further converted to hypoxanthine, xanthine and finally to uric acid (UA). [7] ADA activity is widely distributed in most organs like heart, skeletal muscle, liver, fatty tissues etc. [8] In addition to this, Adenosine is responsible for increasing glucose uptake into cells. [9] Thus, higher ADA activity in insulin sensitive tissue will decrease adenosine levels which in turn would decrease glucose uptake into cells. [10] Serum ADA plays an important role in maturation and activation of lymphocyte. ADA is associated with Tlymphocyte activity and high lymphocyte ADA activities were found in diseases with cell mediated immune response. [11] Its blood levels may help in predicting immunological dysfunction associated with Type 2 DM. [12] Chronic hyperglycemia leads to increased oxidative stress by forming enediol radicals and superoxide ions by NADPH oxidase system and increases ADA levels, both leading to insulin resistance. [14] Glycated haemoglobin (HbA1c) is a routinely used marker for long-term glycemic control. It is an indicator for the mean blood glucose level and predicts the risk of development of complications in diabetes patients. Apart from classical risk factors like dyslipidaemia, elevated HbA1c has now been regarded as an independent risk factor for cardiovascular disease in subjects with or without diabetes. [15] Materials & Methods: The study was conducted in Department of Biochemistry in collaboration with the Department of General Medicine of Mahatma Gandhi Medical College& Hospital, Jaipur. Patients diagnosed with type 2 DM visiting the Outpatient Department of General Medicine & Endocrinology were enrolled for the study. The study was conducted after seeking approval from the Institutional Ethics Committee (IEC) informed consent was taken before enrolling the patients for the study.
The study subjects were divided into two groups. • Control group A control group comprising 50 healthy non-diabetic subjects of comparable age and sex distribution were enrolled for comparative study. Detailed history, clinical examination and relevant investigations were conducted to exclude controls suffering from any such disease which is likely to affect serum ADA and blood glucose level.

Methodology
Detailed history and physical examination of the patients were done. Patients were asked to provide a detailed history and were subjected to a physical examination. An informed consent was taken before the collection of the sample from cases and controls. The control subjects had the same exclusion criteria as the cases and were not on any drug regimens which could influence the study. The study was conducted after approval from the institutional Ethics committee. Blood samples after overnight fasting were collected by standard aseptic techniques.
Plasma blood sugar in fasting sample, HbA1c, Serum ADA were estimated by colorimetric method on fully automated analyser VITROS 4600.
Following Parameters to be estimated 1) Blood Sugar Fasting (BSF) 2) Glycosylated haemoglobin (HbA1c) 3) Serum Adenosine Deaminase (ADA) Estimation of Blood Glucose Quantitative determination of Serum glucose was done by colorimetric -Glucose Oxidase Peroxidase method.

Principles
The VITROS GLU Slide method is performed using the VITROS GLU Slides and the VITROS Chemistry Products Calibrator Kit 1 on VITROS 250/350/950/5, 1 FS and 4600 Chemistry Systems and the VITROS 5600 Integrated System. The VITROS GLU Slide is a multilayered, analytical element coated on a polyester support. A drop of patient sample is deposited on the slide and is evenly distributed by the spreading layer to the underlying layers. The oxidation of sample glucose is catalyzed by glucose oxidase to form hydrogen peroxide and gluconate. This reaction is followed by an oxidative coupling catalyzed by peroxidase in the presence of dye precursors to produce a dye. The intensity of the dye is measured by reflected light. The ADA assay consists of four steps: The ADA assay is based on the enzymatic deamination of adenosine to inosine which is converted to hypoxanthine by purine nucleoside phosphorylase (PNP). Hypoxanthine is then converted to UA and hydrogen peroxide (H 2 O 2 ) by xanthine oxidase (XOD). H 2 O 2 is further reacted with N-Ethyl-N-(2-hydroxy-3-sulphopropyl)-3-methylaniline (EHSPT) and 4-aminoantipyrine (4-AA) in the presence of peroxidase (POD) to generate quinone dye which is monitored in a kinetic manner.

Statistical Analysis
All results obtained were presented as mean ± SD in the patients as well as control group. The diabetic group was subdivided on the basis of HbA1c levels, HbA1c ≤ 8% as good glycemic control and HbA1c > 8% as poor glycemic control. The results were compared by applying Student's t-test. The correlation of Serum ADA with BSF and HbA1c also calculated by applying Pearson's correlation. P-value of ≤ 0.05 was considered as statistically significant.

Discussion:
ADA plays a crucial role in lymphocyte proliferation and differentiation and its highest activity is seen in T-lymphocytes. High ADA activity might be due to abnormal T-lymphocyte responses or proliferation. [14] In 1995, Reddy M et al. reported in their study that ADA activity in patients with Type 2 DM was higher as compared to the control group. [16] Kurtul N. et al., 2004 reported in their study that serum ADA activity was higher in type 2 DM patients and correlated with HbA1c levels. They also suggested that Adenosine modulates the action of insulin on various tissues and its concentration in tissues is affected by ADA levels.
ADA plays important role in insulin effect and glycemic control. Thus, depletion of adenosine due to increased adenosine deaminase activity would lead to insulin resistance in the body & subsequent hyperglycemia, which is a hallmark feature of diabetes mellitus. [17] [21] Serum ADA level had a significant positive correlation with FBS and HbA1c in type 2 diabetes mellitus patients. [22] In accordance with our findings, Anjali C. Warrier et al., in 1995 showed in their study that increased serum ADA activity were correlated with Glycated hemoglobin and lipid peroxidation in DM patients. They suggested that decreased tissue adenosine levels is due to increase in ADA activity and is related to the severity of hyperglycemia and lipid peroxidation in diabetes mellitus. [23] The enzyme ADA has a major role in purine metabolism and has also been identified as a reliable marker of cell mediated immune responses. However, its diagnostic importance has not been explored much. The present study reported a significant rise in serum ADA levels in type 2 DM patients and demonstrated a significant association of ADA with glycemic index (measured by HbA1c). This association suggests that estimation of serum ADA levels may have an important role as risk marker of CVD and other associated complications. The study, therefore, recommends further research on the importance of serum ADA estimation in type 2 DM and its association with other independent markers like lipid profile, CRP, homocysteine.

Conclusion:
The study was undertaken to determine the levels of Serum ADA activity in patients of type 2 DM and its correlation with parameters of glycemic profile such as FBS and Glycated Hemoglobin. This association suggests that estimation of serum ADA levels may have an important role as risk marker of CVD and other associated complications. Increased ADA level can be used to determine the glycemic status in the patients of type 2 DM and serve as a marker for insulin resistance. Raised ADA levels can be an early indicator of progressive diabetic change and help to take preventive measures for the development of diabetic complication and thereby improving the outcome of the disease. The study recommends further research on the diagnostic importance of Serum ADA and its association with HbA1c & insulin resistance in larger cohort studies.