Serum adenosine deaminase levels in patients with type 2 diabetes mellitus and its relation with fasting blood glucose and glycated hemoglobin levels

 

P Kiranmai

 

Associate Professor, Department of Biochemistry, Osmania Medical College and Hospital Hyderabad, Telangana, INDIA.

Email: drkiranmai_23@yahoo.co.in

 

Abstract               Background: Insulin resistance and decreased insulin secretion are the main physiological abnormalities of Type 2 Diabetes Mellitus (DM). Cell mediated immune system disturbance and improper T lymphocyte function also contribute to the pathophysiology of Type2 DM. Chronic hyperglycemia leads to increased oxidative stress and increases Adenosine deaminase(ADA) levels, Both these lead to insulin resistance. As there is relationship between cell mediated immunity, ADA and Type 2 DM, the present study was undertaken to determine S.ADA levels in Type 2 DM and highlight its importance in the immunopathogenesis of Type 2 DM. The study also aims to find correlation between S. ADA and Fasting Blood Sugar (FBS), Glycated hemoglobin (HbA1c). Materials and Methods: 100 individuals in the age group 35 to 65 yrs of established Type 2 DM were included in the study group. Study group is further divided into 2 groups Group I includes 50 Diabetics with HbA1c <7 % and Group II includes 50 Diabetics with HbA1C >7 %. A group of 50 Normal healthy age matched individuals served as the control group. In both groups FBS, S.ADA and HbA1C are measured and compared using Student T test and pearsons correlation statistics. Results: The study showed that FBS, S.ADA and HbA1c levels were significantly higher in group I and Group II when compared to control group. Comparison between group I (well controlled Diabetes patients) and Group II (poorly controlled Diabetes Patients) showed significantly increased ADA activity (p<0.001). There was positive correlation between ADA and HbA1c in groups I and II (p< 0.01 and p<0.001 respectively) Conclusions: Increased S.ADA activity may be an important indicator of immunopathogenesis of Type 2 DM and hence can serve as a marker for diagnosis of DM. Positive Correlation between S.ADA and HbA1c suggests that S. ADA levels can be used for assessing the short term and long term Glycemic status in Type 2 Diabetes Mellitus.

Key Words: Type 2 Diabetes Mellitus, Adenosine Deaminase, Fasting Blood Glucose, Glycated haemoglobin.

 

 

 

 

INTRODUCTION

Diabetes Mellitus (DM) is the most important causative factor of mortality in the developing countries affecting more than 170 million persons all over the world.¹ The incidence and prevalence of Type 2 DM are globally increasing according to a study by WHO with prevalence in countries like India and China estimated to cross 228 million^2,3 DM is characterised by Insulin deficiency either in action or secretion associated with hyperglycemia and disturbances of carbohydrate, lipid and protein metabolism⁴. Hyperglycemia causes longterm dysfunction and failure of organs like eyes, kidneys, heart, nerves, and blood vessels.⁵ Insulin resistance and decreased insulin secretion are the main physiological abnormalities of Type 2 DM⁶ Cell mediated immune system disturbance and improper T lymphocyte function also contribute to the pathophysiology of Type2 DM.⁷ Adenosine Deaminase (ADA)catalyses the deamination of adenosine to inosine and 2’ deoxy adenosine⁸. ADA is considered as a good marker of cell mediated immunity⁹. It has a crucial role in lymphocyte proliferation and differentiation¹⁰. ADA has been reported to be a marker for insulin function^11,12. But its relation with the immune system in diabetic subjects is not established. Some studies reported elevated ADA activity in Type 2 DM (12) and some studies were inconclusive¹³. Adenosine formed by the activity of ADA increases the glucose uptake inside the cells. Thus, high ADA activity decreases adenosine levels and this decreases glucose uptake into cells¹⁴. Chronic hyperglycemia leads to increased oxidative stress by forming enediol radicals and superoxide ions by NADPH Oxidase system and increases ADA levels, Both these lead to insulin resistance.⁹ As there is relationship between cell mediated immunity, ADA and Type 2 DM, the present study was undertaken to determine S.ADA levels in Type 2 DM and highlight its importance in the immunopathogenesis of Type 2 DM. The study also aims to find correlation between S. ADA and Fasting Blood Sugar (FBS), Glycated hemoglobin (HbA1c).

 

MATERIALS AND METHODS

Hundred (100) individuals of established Type 2 DM attending the OPD of Medicine department of Gandhi Hospital were included in the study group.

Inclusion Criteria

1. Known Diabetics in the age group of 35 to 65 yrs.
2. Individuals on oral hypoglycemic drugs.

Exclusion Criteria

1. Diabetics on insulin treatment
2. patients with history of chronic inflammatory conditions, Liver diseases, Renal failure, Rheumatoid arthritis, Tuberculosis, Gout, Diabetic complications like neuropathy, nephropathy, retinopathy and cardiovascular diseases.

Study group is further divided into 2 groups with relation to HbA1c. Group Ia includes 50 Diabetics with HbA1c <7 % and Group Ib includes 50 individuals with HbA1C >7 %. A group of 50 Normal healthy individuals in the age group 35 to 65 yrs served as the control group. After due consent fasting venous blood 5 ml is collected from both study and control group individuals and following parameters are estimated. FBS is estimated by GOD POD Method. HbA1C by Mispa-I nephelometer (15). ADA levels were estimated using erba kit¹⁶. The 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 uric acid and H2O2 by xanthine oxidase. Hydrogen peroxide is further reacted with N-Ethyl N-2 -Hydroxy 3 sulf- opropyl -3 methyl aniline (EHSPT) and 4 aminoantipyrine in the presence of peroxidise to generate Quinine dye which is monitored at 546 nm. One unit of ADA is defined as the amount of ADA that generates one micromole of inosine from adenosine per min at 37⁰ c.

Statistical Analysis: Was performed by SSPS Software. Analysis was done by student t test. All variables were expressed as mean +SD. To find out the correlation between two variables Pearsons correlation coefficient was used. A p value of <0.05 was considered as statistically significant.

RESULTS

Diabetics attending OPD of Medicine department were screened by measuring HbA1c to get a study population of 50 Diabetics with <7% HbA1c and 50 individuals with >7%cHbA1c. The study group is divided into 2 groups, group I (n=50) with HbA1C < 7% and into Group II (n=50) HbA1c > 7 %. FBS and S.ADA levels are measured in both control and study groups. Mean age of control group was 52+3.25 yrs and mean age of study population Group I is 53+8.50 yrs and Group II is 56+12.15 yrs. Mean FBS among all groups is compared. Mean FBS in control group is 86 +4.21 which is less than mean FBS in Group I (N= 50) 128.1+11.2 (p<0.05) Table-1, fig-1.

 

Table 1: Comparison of FBS, HBA1c and S.ADA between control group and Study group I

Parameter	Controls(n=50)	Group I (n=50)	P value
FBS (mg/dl)	86.82+4.21	128.1+11.2	<0.05
HBA1c (%)	4.69+0.62	6.62+0.3	<0.01
S.ADA(U/L)	18.23+8.2	43.82+16.12	<0.001

Figure 1: Comparison of Mean FBS, HbA1C and S.ADA between control and study group I

The increase in FBS in Group II when compared to control group is more significant (p<0.001) than increase of FBS in Group I compared to control (p< 0.05) Table2.Fig-2

 

Table 2: Comparison of FBS, HbA1c and S.ADA levels between control group and study group II

 

Figure 2: Comparison of Mean FBS, HbA1c and S.ADA levels between Control and Study Group II

S.ADA levels are compared among the 3 Groups. S. ADA mean values in Group I and Group II are 43.82+16.12 and 100.24+13.2 respectively. Statistically Mean S.ADA in Group I is more than in control Group (p<0.001) Table1. Mean S.ADA in group II is more than in control group (p<0.0001). Table 2 Mean S.ADA is compared between Group I and Group II show a significantly high S.ADA levels in Group II (p<0.001) showing a significant difference between FBS and Mean ADA levels between subjects with poor glycemic control (HbA1c > 7%) than subjects with good glycemic control (HbA1c<7%) Table3, Fig 3

Table 3: Comparison of FBS, HbA1c and S.ADA levels between Group I and Group II

 

Figure 3: Comparison of Mean FBS, HbA1c and S.ADA LEVELS between Study Group I and Study Group II

 

It is observed that the mean HbA1c levels in control Group is 4.69+0.62 and that in Group I is 6.62+0.3 and in Group II is 8.1+1.2. There is significant difference in levels of HbA1c between control group and Group I (p<0.01) and Group II (p <0.001). Table1,2,3 Correlation coefficient showing the relationship between S.ADA, FBS and HbA1c showed positive correlation between S.ADA and FBS in Group I and Group II but there was no significant correlation between S.ADA, FBS and HBA1c in control group. Positive correlation between S.ADA and HBA1c is more significant in group II (p<0.001) than in Group I (p< 0.01). Table-4

 

Table 4: Pearsons Correlation and significance between S.ADA and FBS, HbA1C in control and study groups

Parameter		Control		Group I		Group II
ADA		FBS	HbA1c	FBS	HbA1c	FBS	HbA1c
	r value	0.048	0.062	0.28	0.36	0.523	0.38
	P value	0.641	0.52	0.01	0.009	0.002	<0.001

 

DISCUSSION

In our study the Mean S.ADA levels are significantly higher in Group I and Group II when compared to controls (p<0.01 and p< 0.001 respectively). S. ADA levels when compared between Group I and Group II also showed significant higher levels in Group II than in Group I (p<0.01). This is in accordance with previous studies, Kurtal et al¹¹ and Kaur et al¹⁷. Recent Studies showed that in type 2 DM, disturbed immune responses may result from the defects in insulin action which is required for T-Lymphocyte function¹⁸. ADA plays an important role in lymphocyte proliferation and differentiation¹⁰. The high S.ADA activity observed in our study in type 2 DM may be due to altered insulin related T-Lymphocyte function⁹. Adenosine plays a role in mediating insulin action of glucose uptake in skeletal muscle and so high ADA activity decreases glucose uptake into cells and contributes to insulin resistance¹⁹. Previous studies concluded that the increase in adenosine content has similar effect to insulin on glucose and lipid metabolism in adipose tissue^{20, 7}. The increase in S.ADA levels in our study may be due to insulin resistance or increased secretion of Adenosine²¹. Increase in S.ADA levels in Type 2 DM may be explained by extracellular cAMP Adenosine pathway. ADA excess activity inactivates adenosine and enhance lipolysis. This leads to increase in cAMP. Deficiency of insulin in case of Type 2 DM leads to increase in free fatty acids and glucose in circulation which are taken up by Liver and adipose tissue. This causes steatohepatitis, adipocyte hypertrophy and insulin resistance^22,23. Hyperglycemia leads to increased oxidative stress by formis with NADPH oxidase system and increases ADA levels both leading to insulin resistance. Adenosine absence causes down regulation of GLUT 4 receptors which may be another cause for insulin resistance.²⁴. In our study a positive correlation has been observed between HbA1c and ADA indicating S.ADA level as a marker for short and long term glycemic control and its role in glucose and lipid metabolic derangements seen in type 2 DM patients. This is in accordance with the previous studies Kurtul et al¹⁹, Ramani et al²⁵.

 

CONCLUSIONS

Increased S.ADA activity may be an important indicator of immunopathogenesis of Type 2 DM and hence can serve as a marker for diagnosis of DM. Positive Correlation between S.ADA and HbA1c suggests that SADA levels can be used for assessing the shortterm and long term Glycemic status in Type 2 Diabetes Mellitus.

 

REFERENCES

1. Nathan DM, DCCT/EDIC Research Group (2014) The diabetes control and complications trial/epidemiology of diabetes interventions and complications study at 30 years: overview. Diabetes Care 37: 9-16.
2. S. Wild, G. Roglic, A. Green, R. Sicree, and H. King, “Global prevalenceofdiabetes: estimates for the year 2000 and projections for 2030,” Diabetes Care, vol.27,no.5,pp.1047–1053,2004.
3. H. King, R. E. Aubert, and W. H. Herman, “Global burden of diabetes, 1995–2025: prevalence, numerical estimates, and projections,”DiabetesCare, vol.21,no.9,pp.1414–1431,1998.
4. American Diabetes Association. ‘Diagnosis and classification of diabetes mellitus’, Diabetes Care 2014; 37:S81-S90.
5. Vanitha Gowda M. N. andVasudha K. C. and Reshma S. and Sujatha K. J. Serum Adenosine deaminase activity in type 2 Diabetes Mellitus patients. Int J Diabetes Dev Ctries 2012; 32(3): 176–181.
6. A.VaagandS.S.LundNon-obesepatientswithtype2diabetes and prediabetic subjects: distinctphenotyperequiringspecial diabetes treatment and (or) prevention?” Applied Physiology, NutritionandMetabolism,vol.32,no.5,pp.912–920,2007
7. M.S. Prakash, S. Chennaiah, Y.S.R. Murthy, E. Anjaiah, S.A. Rao, and C. Suresh, “Altered adenosine deaminase activity in type 2 diabetes mellitus,” Journal, Indian Academy of Clinical Medicine,vol.7,no.2,pp.114–117,2006.
8. M. B. van der Weyden and W. N. Kelley, “Human adenosine deaminase. Distributionandproperties,”TheJournalofBiologicalChemistry, vol.251, no.18,pp.5448–5456,1976.
9. Sullivan JL, Osborne WR, Wedgewood RJ. Adenosine deaminase activity in lymphocytes. Br J Haematol. 1977 Sep;37(1):157-8.
10. Hovi T, Smyth JF, Allison AC, Williams SC. Role of adenosine deaminase in lymphocyte proliferation. ClinExp Immunol 1976; 23: 395-403.
11. Kurtul N, Pence S, Akarsu E et al. Adenosine deaminase activity in the serum of type 2 diabetic patients. Acta Medica (Hradec Kralove) 2004; 47 (1): 33-5.
12. Hoshino T, Yamada K, Masuoka K et al. Elevated adenosine deaminase activity in the serum of patients with diabetes mellitus. Diabetes Res ClinPract 1994; 25: 97-102.
13. Angielski S, Jakubowski Z, Pawelczyk T, Piec G, Redlak M. Renal handling and metabolism of adenosine in diabetic rats. ContribNephrol. 1989; 73:52-7; discussion 57-8.
14. Vineet Kumar Khemka, DebajitBagchi, Arindam Ghosh, Oishimaya Sen, Aritri Bir, Sasanka Chakrabarti, and Anindita Banerjee. Raised Serum Adenosine Deaminase Level in Nonobese Type-2 Diabetes Mellitus. The Scientific World Journal Volume 2013: 1 - 5.
15. Jeppson JO. Approved IFCC Reference Method for the measurement of HbA1c in human blood. ClinChem Lab Method 2002; 40(1):78-89.
16. Delacour H, Sauvanet C, Ceppa F, Burnat P. Analytical performances of the Diazyme ADA assay on the Cobas ® 6000 system. ClinBiochem. 2010 Dec; 43(18):1468-71. doi: 10.1016/j.clinbiochem. 2010. 09. 005. Epub 2010 Sep 17.
17. Amandeep Kaur, SahibaKukreja, Naresh Malhotra, Neha. Serum Adenosine Deaminase Activity and Its Correlation with Glycated Haemoglobin Levels in Patients of Type 2 Diabetes Mellitus. Journal of Clinical and Diagnostic Research 2012 April; Vol-6(2): 252-256.
18. Frankie B, Abbas E. Activated T-lymphocytes in type2 diabetes: Implications from in vitro studies. Curr Drug Targets 2003; 4: 493-503.
19. L.Vergauwen,P.Hespel,andE.A.Richter,“Adenosinereceptors mediatesynergisticstimulation of glucose up take and transport by insulin and by contractions in rat skeletal muscle,” The JournalofClinicalInvestigation,vol.93,no.3,pp.974–981,1994
20. Ramani NS, Krishnamurthy N, Prasad BN, Ashakiran S, Sumathi ME, et al. (2012) Role of adenosine deaminase to predict glycemic status in type 2 diabetes mellitus. J Clin Biomed Sci 2: 123-133.
21. Ogbu IS, Nebo NC, Onyeanusi JC (2006) Adenosine deaminase activities and fasting blood glucose in obesity. J Coll Med 11: 115-119.
22. Ferranti S, Mozaffarian D. The perfect storm obesity, Adipocyte dysfunction, and metabolic consequences. Clinical Chemistry 2008; 54: 945-55.
23. Hafer GR, Haften TWV, Visseren FLJ. Adipose tissue dysfunction in obesity. Diabetes and vascular diseases. European Heart Journal 2008; 29: 295971.
24. Havilah P., Pandit Vinodh B, Durga Prasad K. Adenosine Deaminase Activity in Type-2 Diabetes Mellitus – An Independent Marker of Glycemic Status and Stimulator of Lipid Peroxidation. Int. J. Chem. and Life Sciences.2013; 2(6):1175-8. 7
25. Priti Singh, Salman Khan, Mittal Rabindra Kumar. Adenosine Deaminase Activity and its Relation with Glycated Hemoglobin and Uric Acid in Type 2 Diabetic Patients. Iranian Journal of diabetes and obesity 2013; 5(1):1-6. 3