Table of Content - Volume 9 Issue 3 - March 2019

 

Evaluation of serum gamma glutamyl transferase levels as a marker of oxidative stress in type 2 diabetes patients with and without retinopathy

 

 

Abstract               Background: Diabetic neuropathy is one of the common complications of diabetes mellitus. The pathophysiology of diabetic retinopathy is complex and recent studies have suggested the use of serum gamma glutamyl transferase (GGT) as an early marker of oxidative stress. In this study we investigated whether serum GGT may be useful in predicting diabetic retinopathy. Objective: This study was undertaken to evaluate whether serum GGT is a useful marker of oxidative stress in type 2 diabetic retinopathy. Methods: Our study included 60 patients with type 2 diabetes who were evaluated for the presence of retinopathy using clinical ophthalmologic examinations including fundus examination. We evaluated the association between serum GGT and the presence of diabetic retinopathy. Result: The serum GGT concentration were significantly elevated in type 2 diabetic patients with retinopathy compared to the patients without retinopathy (P < 0.01). Serum GGT was independently related with Malondialdehyde (MDA) according to linear regression analysis (P < 0.01).

Conclusion: This study shows that increased levels of serum GGT may have important role in the pathophysiology of retinopathy in patients with type 2 diabetes. Serum GGT can be used as a biomarker of oxidative stress in patients with diabetic retinopathy,

Key Word: Diabetes mellitus; Gamma glutamyl transferase; Oxidative stress; retinopathy

 

 

 

INTRODUCTION

Type 2 diabetes mellitus (DM) is a heterogeneous group of distinct genetic and metabolic disorder characterized by relative or absolute lack of insulin action and or secretion.¹ The overall prevalence is estimated to be approximately 285 million cases Worldwide and 50.8 million cases in India in 2010 due to population growth, aging, urbanization, lifestyle change, which may increase to 439 million globally and 87 million in India by 2030.² Long term vascular complications of uncontrolled hyperglycemia are broadly classified into micro vascular and macro vascular diseases in which Diabetic Retinopathy (DR) is one of the most feared micro vascular complications of DM. It is the leading cause for new cases of blindness in USA and found that people with DM have 25 times more risk of developing blindness and shorter life expectancy, than people without DM .³ Risk factors for DR are poor glycemic control, duration of diabetes, hypertension, elevated serum lipid levels and renal disease with an estimated Worldwide burden of DR around 93 million people in a recent study.⁴ The prevalence of diabetic retinopathy was 18% in urban Indian population.⁵ DR is broadly classified into non proliferative and proliferative, which may ultimately lead to vision loss. Altered retinal blood flow is the major pathophysiology of DR leading to loss of pericytes and basement membrane thickening. Chronic hyperglycemia affects the down regulation of glucose transport in endothelial cells compared to other cell types, leading to the intracellular accumulation of glucose. This induces overproduction of mitochondrial super oxides via four pathways such as polyol, hexosamine, diacylglycerol, advanced glycation end products (AGE) pathways that build up oxidative stress in endothelial cells, although there is no apparent link among these pathways.³ Since the retina is rich in high PUFA and oxygen content, it is more susceptible to oxidative stress and lipid peroxidation.⁶Malondialdehyde, a product of lipid peroxidation is a well-defined marker of oxidative stress.⁷ Gamma glutamyl transferase (GGT) has been recognized as a marker of liver disease and alcohol intake. But recent research has found an association between serum GGT and oxidative stress. In the CARDIA (Coronary Artery Risk Development in Young Adults) study diabetic and hypertensive adults showed a strong correlation between serum GGT levels and microalbuminuria.⁸Several studies have shown cellular GGT to be a marker of oxidative stress whose serum values are elevated along with serum MDA levels.^9-11 Serum gamma glutamyl transferase (GGT)is an ectoplasmic enzyme responsible for the extracellular catabolism of glutathione, which is synthesized in epithelial cells of the intrahepatic duct. It distributed indifferent cells with various secretory or absorptiveactivities.¹² GGT has an important role in glutathione homeostasis by initiating the breakdown of extracellular glutathione and turnover of vascular glutathione^13-15Consideringthe antioxidant activity of glutathione, increased level of GGT may be linked to greater oxidative stress. Increased oxidative stress has been implicated in insulin resistance by promoting ß-cell dysfunction and reducing insulinaction.^16’17 Therefore, serum GGT activity could reflect several different processes relevant to diabetes pathogenesis. Many epidemiological studies, have demonstrated high rates of elevated GGT levels among diabetic patients over past 40 years.¹²Recent prospective studies, have indicated that baseline serum GGT activity predicts occurrence of future diabetes, stroke and cardiovascular diseases^18-24and within reference interval, it strongly predicted incident type 2 diabetes^20-22 However, not all studies support this assumption.²⁵ A study by vijayalakshmi et al showed serum GGT can be used as a marker of oxidative stress in type 2 diabetic retinopathy. There are no studies relating serum GGT levels and the severity of diabetic retinopathy.²⁶ In this study serum Gamma Glutamyl Transferase levels will be compared in diabetics with retinopathy and in diabetics without retinopathy in rural population.

AIM AND OBJECTIVES

1. To compare serum gammaglutamyl transferase activity in Type 2 diabetic patients with and without retinopathy.
2. To compare serum serum gammaglutamyl transferase level with serum malondialdehyde, (marker of oxidative stress)

 

MATERIALS AND METHODS

This study was conducted in the Department of Physiology at Sri Manakula Vinayagar Medical College and Hospital, Pondicherry, in collaboration with the Department of Opthalmology. This was a hospital-based case control study. This study was approved by the Institutional Ethics committee, Sri Manakula Vinayagar medical college hospital. The sample size was 60 subjects aged 35-55 years and they were divided into two groups

Inclusion criteria

1. Cases: Both males and females of age group between 30-55 years who were diagnosed to have Type 2 Diabetes with retinopathy.
2. Controls: Type 2 diabetic patients without retinopathy both males and females of age group 30-55 years.

Exclusion criteria

1. Cases: Diabetic Retinopathy patients with any other complications such as Ischemic Heart Disease, Nephropathy, Neuropathy, Infections.
2. Subjects with regular alcohol consumption, liver diseases, hypertension
3. Any subject who is not willing to give informed consent to this project.

 

Sample collection: Three ml of whole blood was collected from the controls and the study group after getting the informed consent. Then the serum was separated after subjecting the collected blood to centrifugation at 2500 rpm for 5min. The serum was divided into aliquots and stored at -20ºC for further analysis.

Estimation of serum gamma glutamyl transferase: Serum gammaglutamyl transferase was measured by a modified Szasz method using Cobas Miras plus Automated Chemistry Analyzer, made in USA.⁶

Estimation of fasting Plasma glucose: Fasting plasma glucose (FPG) was measured by glucose oxidase – peroxidase method. using Cobas Miras plus Automated Chemistry Analyzer, made in USA. ⁷

Estimation of HbA1c: HbA1c was estimated by turbidometric immunoassay method.⁸

Estimation of serum malondialdehyde: Serum malondialdehyde was estimated by thiobarbituric acid reactivity method.⁹

Clinical examination of Retina: were done for all participants. ¹⁰

Statistical analysis: The data collected were entered and analyzed using software Statistical Package for the Social Science 16.0 (SPSS 16.0). All parameters were presented as mean ± standard deviation (mean ± SD). Comparison of parameters was done with student ‘t’ test. Correlation analysis was done with Pearson’s correlation method. A linear regression analysis was performed to evaluate the independent predictor of oxidative stress. A p value of less than 0.05 was considered statistically significant.

RESULTS

The results were presented as mean ± standard deviation in table 1. The results showed that there is an increased serum GGT level in study group when compared to control group. Serum Malondialdehyde, fasting plasma glucose and HbA1c were significantly increased in the cases compared to the controls. There is significant association between raised GGT levels and fasting plasma glucose and HbA1c among the cases. Serum GGT was independently related with Malondialdehyde (MDA) according to linear regression analysis (P< 0.01).

 

Table 1: Comparison of various parameters between the cases and control group

Data are presented as Mean ±SD. * p<0.05 is considered statistically significant. MDA=Malondialdehyde, FPG=Fasting Plasma Glucose, GGT=Gammaglutamyl Transferase

 

Table 2- Correlation of GGT with various parameters among the cases

Pearson correlation analysis was performed to analyze the data. * p<0.05 is considered statistically significant. MDA=Malondialdehyde, FPG=Fasting Plasma Glucose, GGT=Gammaglutamyl Transferase.

 

Table 3: Correlation of GGT with various parameters among the controls

Pearson correlation analysis was performed to analyze the data. * p<0.05 is considered statistically significant. MDA=Malondialdehyde, FPG=Fasting Plasma Glucose, GGT=Gammaglutamyl Transferase

Figure 1: Linear regression analysis between MDA and GGT levels among the cases

 

DISCUSSION

Type 2 diabetes mellitus (DM) is a heterogeneous group of disorders characterized by variable degrees of insulin resistance, impaired insulin secretion, and increased glucose production, which has most serious micro vascular complication namely Diabetic Retinopathy.¹ The worldwide burden of DM has raised dramatically from an estimated 30 million cases in 1985 to 285 million cases in 2010. The International Diabetes Federation (IDF) projects the above figure may drastically increase to 439 million by the year 2030.³² In 2011, IDF estimates that India alone has 61.3 million people living with diabetes and showed India holds her place second to China.³³Diabetic retinopathy (DR) can be defined as damage to micro vascular system in the retina due to prolonged hyperglycemia. It is broadly classified into nonproliferative DR (NPDR) and proliferative DR (PDR). Progression from mild, characterized by increased vascular permeability, to moderate, and then to severe NPDR characterized by vascular closure and an increased risk for the development of PDR distinguished by the growth of new blood vessels on the retina and posterior surface of thevitreous.³⁴ The capillaries of retina are composed of endothelial cells lining the vessel, which rest on the basement membrane and surrounded by pericytes. Pericytes are important for angiogenesis, the structural integrity of the microvasculature, and blood flow regulation. The ratio of endothelial cells to pericytes is 2:1. However, in diabetes, this ratio is altered to 4:1. The endothelial cells act as a selective permeability barrier for certain molecules. The retina has high content of polyunsaturated fatty acids (PUFA) and has the highest oxygen uptake and glucose oxidation relative to any other tissue. This phenomenon renders retina more susceptible to oxidative stress.³⁵ Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of DM. Pericyte loss, endothelial damage, basement membrane thickening, and altered blood flow are the major pathogenesis.³⁶Chronic hyperglycemia leads to intracellular accumulation of glucose in endothelial cells as it affects the down regulation of glucose transport in endothelial cells compared to other cell types. This ultimately leads to mitochondrial superoxide production via four pathways such as polyol, hexosamine, diacylglycerol, AGEs pathways, although they are not interlinked. All the four pathways reflect single process at the end, namely oxidative stress at the molecular level. Oxidative stress is a cytopathic consequence of excessive production of ROS and defect in its removal by antioxidant defense system. Several studies were conducted to know exact pathogenesis of oxidative stress in DR. GGT is found in the kidneys, biliary system, pancreas, and intestine. Briefly, GGT protein catalyzes an enzymatic action, which is the transfer of a glutamyl residue to an acceptor through the glutamate’s gamma carboxylic acid to an amine or other amino acid. The most abundant natural substrate is glutathione. Glutathione is extracellular and cannot pass through the cell membrane. Glutathione can be broken down into 3 amino acids (including cysteine, which may be deficient in low-protein diets) at the cell membrane by GGT. These amino acids can be taken up in the cells by the γ-glutamyl cycle. Glutathione is then reformed in the cells, where it protects cells against oxidants that are produced during normal metabolism. An increased need for reduced glutathione occurs with oxidative stress.^37,38Glutathione is a known, powerful antioxidant that may mediate the inflammatory effect of increased glucose, possibly by decreasing cytokine production in response to spikes of hyperglycemia.³⁹ Our study showed increased levels of serum GGT in patients with diabetic retinopathy. A study by Arkkila et al showed the association between GGT levels and complications of type 1 diabetes including neuropathy and retinopathy.⁴⁰ The serum levels of GGT also showed significant positive correlation with serum malondialdehyde, which is a biomarker of oxidative stress. This suggests that serum gamma glutamyl transferase (GGT) may be an early marker of oxidative stress that may contribute to the pathophysiology of the onset and progression of diabetic retinopathy through defects in metabolic and vascular pathways. The results are in accordance with a study by Whitfield JB who reported that increased GGT activity may be a response to oxidative stress, which can increase the transport of glutathione precursors into cells.⁴¹ Gamma glutamyl transferase is an easily available, universally standardized and routinely done test as part of liver function tests. Serum GGT is a useful biomarker for oxidative stress like serum MDA. Hence it can be used as a surrogate marker of microvascular complications in diabetes mellitus like diabetic retinopathy. Further studies using a larger sample size will be required to validate the results.

 

CONCLUSION

The present study has shown that oxidative stress is associated with diabetic retinopathy. Poor glycemic control as reflected by increased HbA1c causes worsening of retinopathy. This study shows that increased levels of serum GGT may have important clinical implications in the presence of retinopathy in patients with type 2 diabetes. Serum GGT is a useful marker of oxidative stress in patients with diabetic retinopathy.

 

REFERENCES

1. Alvin C. Powers. Diabetes mellitus. Harrison’s endocrinology. 2^nded. New York: McGraw-Hill companies;267-313.
2. Ramachandran A, Das AK, Joshi SR, Yajnik CS, Shah S, Prasanna Kumar KM. Current status of diabetes in India and need for novel therapeutic agents. Journalof association of physicians of India 2010; 58:7-9.
3. Michael Brownlee, Llyod P. Aiello, Mark E. Cooper, Aaron I. Vinik, Richard W. Nesto, Andrew J. M. Boulton. Complications of diabetes mellitus. Williams textbook of endocrinology 11^th ed. Philadelphia: Saunders-Elsevier inc; 1417-1501.
4. Yau JW, Rogers SL, Kawasaki R, Lamoureux EL, Kowalski JW, Bek T et al. Meta-analysis for eye disease (META-EYE) Study Group. Global prevalence and major risk factors of diabetic retinopathy. Diabetes care 2012; 35:556-64.
5. Raman R, Rani PK, Reddi Rachepalle S, Gnanamoorthy P, Uthra S, Kumaramanickavel G, Sharma T. Prevalence of diabetic retinopathy in India: Sankaranethralaya diabetic retinopathy epidemiology and molecular genetics study report 2. Ophthalmology 2000; 116:311-8.
6. Kumari S, Panda S, Mangaraj M, Mandal MK, Mahapatra PC. Plasma MDA and Antioxidant vitamins in Diabetic Retinopathy. Indian journal of clinical biochemistry 2008; 235:158-62.
7. El-Mesallamy HO, Rizk KA, Hashad IM. Role of oxidative stress, inflammation and endothelial dysfunction in the pathogenesis of diabetic retinopathy. IIOABJ2011; 2:91-97.
8. Lee DH, Steffen LM, Jacobs DR. Association between serum gamma-glutamyl transferase and dietary factors: the Coronary Artery Risk Development in Young Adults (CARDIA) Study. Am J ClinNutr2004; 79:600-605.
9. Pieri L, Dominici S, Del Bello B, Maellaro E, Comporti M, Paolicchi A et al. Redox modulation of protein kinase/phosphatase balance in melanoma cells: the role of endogenous and gamma-glutamyl transferase dependent H2O2 production. Biochim Biophys Acta2003; 1621:72-83.
10. Lim J, Yang J, Chun BY, Kam S, Jacobs D, Lee D. Is serum gamma glutamyl transferase inversely associated with serum antioxidants as a marker of oxidative stress? Free RadicBiol Med2004; 37:1018-1023.
11. Drozdz R, Parmentier C, Hachad H, Leroy P, Siest G, Wellman M. Gamma-glutamyl transferase dependent generation of reactive oxygen species from a glutathione/transferrin system. Free Radic Biol Med1998; 25:786-772.
12. Whitfield JB. Gamma glutamyl transferase. Crit Rev Clin Lab Sci 2001; 38: 263-355.
13. Lieberman MW, Barrios R, Carter BZ, Habib GM, Lebovitz RM, Rajagopalan S, et al. gamma-Glutamyl transpeptidase. What does the organization and expression of a multi promoter gene tell us about its functions? Am J Pathol1995; 147:1175-85.
14. Lee DH, Blomhoff R, Jacobs DR Jr. Is serum gamma glutamyl transferase a marker of oxidative stress? Free Radic Res 2004; 38:535-9.
15. Lee DH, Ha MH, Kam S, Chun B, Lee J, Song K, et al. A strong secular trend in serum gamma-glutamyl transferase from 1996 to 2003 among South Korean men. Am J Epidemiol 2006; 163:57-65.
16. Blaha M, Elasy T. Clinical use of the metabolic syndrome: why the confusion? Clin Diabetes 2006; 24:125-31.
17. Evans J, Goldfine I, Maddux BA, Grodsky G. Are oxidative stress-activated signaling pathways mediators of insulin resistance and beta-cell dysfunction? Diabetes 2003; 52:1-8.
18. Trell E, Kristenson H, Peterson B, Fex G, Henningsen N, Berntorp K, et al. Two-hour glucose and insulin responses after a standardized oral glucose load in relation to serum gamma-glutamyl transferase and alcohol consumption. Acta Diabetol Lat 1981; 18:311-7.
19. Wannamethee G, Ebrahim S, Shaper A. Gamma-glutamyl transferase: determinants and association with mortality from ischemic heart disease and all causes. Am J Epidemiol 1995; 142:699-708.
20. Perry J, Wannamethee G, Shaper A. Prospective study of serum gamma-glutamyl transferase and risk of NIDDM. Diabetes Care 1998; 21:732-7.
21. Jousilahti P, Rastenyte D, Tuomilehto J. Serum gamma-glutamyl transferase, self-reported alcohol drinking, and the risk of stroke. Stroke 2000; 31:1851-5.
22. Lee D, Ha M, Kim Jet al. Gamma-glutamyltransferase and diabetes--a 4 year follow-up study. Diabetologia 2003; 46:359-64.
23. Lee DH, Jacobs DR Jr, Gross M, Kiefe CI, Roseman J, Lewis CE, et al. Gamma glutamyl transferase is a predictor of incident diabetes and hypertension: the Coronary Artery Risk Development in Young Adults (CARDIA) Study. Clin Chem 2003; 49:1358-66.
24. Meisinger C, Löwel H, Heier M, Schneider A, Thorand B; KORA Study Group. Serum gamma-glutamyl transferase and risk of type 2 diabetes mellitus in men and women from the general population. J Intern Med 2005; 258:527-35.
25. Vozarova B, Stefan N, Lindsay R, Saremi A, Pratley RE, Bogardus C, et al. High alanine aminotransferase is associated with decreased hepatic insulin sensitivity and predicts the development of type 2 diabetes. Diabetes 2002; 51:1889-95.
26. Vijayalakshmi B, Ravikiran P. Serum gamma-glutamyl transferase as a marker of oxidative stress in diabetic retinopathy. IJPBS 2013; 33:496-503.
27.  Szasz G. A kinetic photometric method for serum gamma-glutamyl transpeptidase. Clin Chem. 1969; 15(2):124-136.
28.  Trindler P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem. 1969; 6:24–27.
29.  Jeppsson JO, Kobold U, Barr J, Finke A, Hoelzel W, Hoshino T et al. Approved IFCC reference method for the measurement of HbA1c in human blood. Clin Chem Lab Med. 2002; 40(1):78-89.
30. Satok K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978; 90(1):37-43.
31. American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus. Diabetes care 2011; 34(1):62-69.
32. Richard S, Jonathan S, Paul Z. The global burden, diabetes and impaired glucose tolerance. IDF Diabetes atlas 4^th ed. p. 1-105.
33.  International Diabetes Federation. The IDF Diabetes Atlas. Fifth Edition. The global burden 2011.
34. John W. Baynes, Suzanne R. Thorpe. Role of oxidative stress in diabetic complications. Diabetes 1999; 48:1-9.
35. Jennifer K. Sun, Hillary A. Keenan, JerryD, Bela F. Asztalos, Ernst J. Schaefer, David R. Sell, et al. Protection from retinopathy and other complications in patients with type 1 diabetes of extreme duration. Diabetes care 2011; 34:968–74.
36. Lawrence J. Mandarino. Current hypotheses for the biochemical basis of diabetes retinopathy. Diabetes care 1992; 15:1892-01.
37. Lee D, Blomhoff R, JacobsD. Is serum gamma glutamyl transferase a marker of oxidative stress. Free Radical Research2004; 38:535-539.
38. Whitfield J. Gamma glutamyl transferase. Critical Reviews in Clinical Laboratory Sciences, 2001; 38:263-355.
39. Wright E, Scism B. Oxidative stress in type 2 diabetes: The role of fasting and postprandial glycaemia. International Journal of Clinical Practice2006; 60:308-314.
40. Arkkila PE et al. Diabetic complications are associated with liver enzyme activities in people with type 1 diabetes. Diabetes Res Clin Pract. 2001; 52(2):113-118.
41. Whitfield J. Gammaglutamyl transferase. Crit Rev Cli Lab Sci. 2001; 38:263-355.