Published by : PROFESSIONAL MEDICAL PUBLICATIONS
October - December 2009 (Part-I)
Evaluation of cardiac risk by oxidative stress and
inflammatory markers in diabetic patients
Dilshad Ahmed Khan1, Shazia Qayyum2
Objectives: To evaluate the diabetic patients for cardiac risk by measuring oxidative stress and inflammatory markers in relation with glycaemic control.
Methodology: A total of 140 subjects were included in this case-control study, comprising of 70 diabetic patients with coronary heart disease (CHD) and an equal number, age and sex matched controls. The patients were non-alcoholic and had age >40years, BMI < 30 kg/m2 and glycated hemoglobin (HbA1c) 7-10%. Serum total cholesterol (TC) and gamma glutamyltransferase (GGT) were analyzed on selectra-E auto analyzer. Serum nitrate was measured at 540nm on ELISA. HbA1c on was analyzed by using Human kit. Serum high sensitivity C-reactive protein (hS-CRP) was analyzed on immulite 1000.
Results: Patients mean age was 51 (range 40-73) years. Diabetic patients had significantly elevated median of HbA1c (7.9 vs 4.9), hS CRP (6.0 vs 2.12), TC (5.95 vs 4.45), nitrate (19.20vs 10.70) and GGT (29.50 vs 22.50) as compared to controls (p< 0.001). HbA1c showed a positive correlation (p <0.001) with hS-CRP (r=0.49), TC (r=0.69), nitrate (r=0.41) and GGT (r=0.30).
Conclusion: Oxidative stress and inflammatory markers should be used in addition to HbA1c for assessment of increased cardiac risk in un-controlled diabetic patients because of accelerated atherosclerosis due to free radical injury.
KEY WORD:Diabetes mellitus, Gamma glutamyltransferase, C-reactive protein, Nitrate, Total cholesterol, Oxidative stress.
Pak J Med Sci October - December 2009 (Part-I) Vol. 25 No. 5 776-781
How to cite this article:
Khan DA, Qayyum S. Evaluation of cardiac risk by oxidative stress and inflammatory markers in diabetic patients. Pak J Med Sci 2009;25(5):776-781.
1. Dr. Dilshad Ahmed Khan, FCPS,
Department of Pathology,
Army Medical College,
National University Sciences and Technology,
Rawalpindi - Pakistan.
2. Dr. Shazia Qayyum, M Phil (Chem Path)
Islamic International Medial College,
Rawalpindi - Pakistan.
Dr. Dilshad Ahmad Khan,
Dept. of Pathology, Army Medical College,
Abid Majeed Road, Rawalpindi-Pakistan,
* Received for Publication: April 11, 2009
* Accepted for Publication: September 3, 2009
Diabetes mellitus is commonly associated with both microvascular and macrovascular complications.1 Increasing evidence supports that atherosclerosis is a co-morbid condition in the diabetic patients.2 Impairment of vascular endothelial function is an initial step in the development of cardiovascular problems.3 Recently the important contribution of inflammation and oxidative stress to the pathogenesis of accelerated atherosclerosis in diabetic patients has been emphasized.4-5
Hypercholesterolemia causes focal activation of endothelium by infiltration and retention of LDL -cholesterol in arteries causing inflammatory response and activation of reactive oxygen species (ROS).6 Modification of LDL, through oxidation and enzymatic activity causes LDL oxidation. OxLDL when recognised by macrophages is converted into foam cells which is a key event in atherogenesis. The central role of dyslipidemia in causing progression of atherosclerosis in adults with diabetes has been elucidated. There are a few researcher who have reported higher levels of total cholesterol, LDL-cholesterol and triglyceride with higher HbA1c concentrations in diabetic patients.7-8
Elevated levels of inflammatory markers, including hS-CRP, interleukin-6, tumor necrosis factor-á and GGT have been reported in diabetic patients.5, 9 Recent research suggests that CRP is etiologically involved in the pathogenesis of diabetes.10 Hyperglycemia causes glycosylation of proteins and phospholipids. Advanced glycation end products (AGEs) generate reactive oxygen species (ROS) with consequent increased vessel oxidative damage and atherogenesis.11 Chronic over production of ROS in the diabetic patients leads to redox imbalance leading to increased GGT activity. Among the new emerging markers for evaluation of oxidative stress GGT is receiving increased attention and is an early marker of oxidative stress in humans.12-13
Nitric oxide (NO) is another important biomarker of inflammation and oxidative stress. It is a gaseous biological mediator first identified as the endothelium-derived relaxing factor (EDRF). Cytotoxicity attributed to NO is due to peroxynitrite and superoxide anion.14 Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms.14 Free radicals, such as superoxide anion, can rapidly react with NO, inactivating it and priming proatherogenic mechanisms.
This study aimed to determine the role of inflammation and oxidative stress markers for assessment of cardiac risk in patients with un-controlled type-II diabetese mellitus. We evaluated status of arteriosclerosis in diabetic patients by measuring oxidative stress, inflammatory markers and correlated with HbA1c in our clinical step-up.
Subject:This case-control study was conducted in the clinical pathology laboratory, Army Medical College, Rawalpindi, Pakistan. A total of one hundred thirty subjects, comprising of seventy diagnosed patients of coronary heart disease with type II diabetes mellitus and an equal number, age and sex matched healthy subjects were included after informed consent. The participants were included by purpose sampling technique. The patients had HbA1c between 7 to 10%, BMI< 30 kg/m2 and were non-alcoholic. Patient suffering from renal failure, hepatitis, acute illness, taking hormonal replacement therapy (HRT), anti-inflammatory drugs or statin were excluded. This study was carried out from April to Oct 2008, after approval of the protocol by institution review committee.
Methodology: Blood sample (5ml) was drawn at 7:00-9:00am following an overnight fast. All the assays were performed at Clinical Pathology Laboratory, Army Medical College, Rawalpindi Pakistan following the standard protocols and quality control. Serum hS-CRP was analyzed on Immulite 1000 (Seimen, USA) which is a solid-phase, two-site sequential chemiluminescent immunometric assay method.15 Two values of serum hS-CRP were obtained optimally two weeks apart, which were then averaged. Serum GGT activity was assessed by a kinetic colorimetric assay at 37oC and expressed as unit per liter. This method was standardized against recommended IFCC method.16 Serum nitrate was measured by using Griess reagent at 540nm on ELISA strip reader by colorimetric assay kit (Cayman, UK)17 Analysis for HbA1c was carried out at 415nm on microlab-200 by following the manufacture method.18 Serum total cholesterol was measured by cholesterol oxidase method (CHOD. POD) on selectra-E auto analyzer.19 Coefficient of variation was 4-5%.
Statistical Analysis: The data was analyzed by using standard SPSS software version-15 (SPSS Inc, Chicago) for statistical analysis. Descriptive statistics were applied and data were not following Gaussian distribution. Median and inter quartile range (25-75 percentile) were calculated. Comparison of data of both diabetic patients and control groups were done by applying Mann-Whitney t-test. The correlation between HbA1c, oxidative stress and inflammatory biomarkers was determined by Spearman’s correlation. A p-value <0.05 was considered significant.
Diabetic patients had mean (SD) age of 51 (6) years and controls 52 (5) years. Male to female ratio was 2:1. The patients had poor glycaemic control and had significantly raised HbA1c as compared to healthy subjects (Fig-1a).
The hS-CRP levels were also markedly elevated in the CHD patients with diabetes as compared to control indicating increased arthrosclerosis in these patients (Fig-1b).
Serum total cholesterol was also raised in type II diabetics. The patients had increased oxidative stress indicated by elevated GGT activity (Table–I) and high serum NO levels. Correlation of HbA1c levels with CRP in the diabetic patients is exhibit in Fig-2.
Our study also revealed a positive correlation of HbA1c with total cholesterol (r=0.69), nitrate (r=0.41) and GGT (r=0.30) in these diabetic patients (Table–II).
Our study has demonstrated that there is an increased inflammatory and oxidative damage of coronary vessels in type II diabetic patients. HbA1c is a marker of long-term glycaemic control and for every one-percent increase in HbA1c, increase the relative risk for cardiovascular events increase.20 Thus the measurement of HbA1c levels is important not only for monitoring of diabetes but also for assessment of the risk of CHD in diabetics.21 Cardiovascular morbidity is a major burden in patients with type II diabetes mellitus, with endothelial dysfunction as an early sign of diabetic vascular disease. Atherosclerosis in diabetes mellitus is more progressive than in non diabetic patients. Both type 1 and type 2 diabetes mellitus are associated with abnormalities of lipid metabolism. Several researchers reported similar abnormal lipid metabolism in un-controlled diabetic patients.7-8
We found that serum hs-CRP levels were much raised in patients with DM than in control group. Experimental evidence suggests that CRP is a direct participant in the progression of atherosclerosis and is an important cardiovascular risk marker in patients with diabetes mellitus.22 Inflammatory course of the atherosclerotic process is more severe in diabetic patients than in non-diabetic subjects and our study results strongly support it. 23 The identification of a genetic variant in the human CRP locus associated with a high serum CRP and an increased risk of diabetes support the hypothesis that CRP is also involved in the pathogenesis of diabetes.10 It is a liver – derived protein secreted under the influence of cytokines such as interleukin – 6 and TNF- a.24 Its levels increase with the stage of beta cell dysfunction and insulin resistance. It serves as a chemoattractant for monocytes. Increasing evidence suggests that CRP has a stronger predictive value for the risk of cardiovascular disease.25 Patients with CRP concentrations >5 mg/L at the time of hospital admission had a 50% to 330% increase in risk of death with diabetes.26
Nitric Oxide plays an important role in homeostatic vasodilatation and the regulation of blood flow. The increased plasma NO levels in patients with type II diabetes may be associated with the pathogenesis of vascular complications.27-28 Nitric oxide is an important protective molecule in the vasculature. Endothelial nitric oxide synthase (eNOS) is responsible for most of the vascular NO. produced. Uncoupling of the endothelial nitric oxide synthase (eNOS) in blood vessels of diabetic patients leads to excessive superoxide anion (O2·–) production and diminishes NO availability.29-30 It has also been suggested that CRP effects nitric oxide pathway and elevated serum CRP levels may cause endothelial dysfunction.31 Raised levels of both serum NO and hs-CRP in our diabetic patient indicate an acceleration in the atherosclerotic process.
We also studied levels of role of GGT for assessment of oxidative processes in relation to heart disease progression in diabetic patients. Baseline serum GGT activity most strongly predicts incident type 2 diabetes.9 Gamma glutamyltransferase is a surrogate marker of oxidative stress and a positive correlation is found between serum GGT and CRP.12, 32 It is the key enzyme that initiates the metabolism and turnover of glutathione (GSH) which is the main antioxidant in mammalian cells. Chronic over production of ROS lead to redox imbalance leading to increased GGT activity and represents early marker of oxidative stress in humans.13 The serum determination of GGT activity is a low-cost and sensitive laboratory test for assessment of oxidative in our patients. Population-based epidemiological studies showed a strong association of serum GGT activities within the reference interval in the metabolic syndrome.33 Vasular complications may have a defective cellular antioxidant response against the oxidative stress generated by hyperglycemia. Both oxidative stress and inflammation form a vicious cycle and measuring these markers can help in improvement of health status in diabetic patients.
The limitation of our study was that it mainly included type-2 diabetic patients so it can not be applied on type-I diabetic patients. This was one local study in Rawalpindi and need to be conducted as multicenter study in Pakistan with larger sample size. Future studies can be done by using other markers of inflammation and oxidative stress in the prediction of cardiac risk in type-I and type-II diabetic patients with CHD.
Our study strengthens the role of oxidative stress and inflammation as a common mediator in the pathogenesis of accelerated atherosclerosis in the diabetic patients. Oxidative stress and inflammatory bio-markers should be used in addition to HbA1c for assessment of cardiovascular status in the un-controlled type-2 diabetic patients.
This study was supported by Amcolians Alumni Association (AAA) C/O Army Medical College, Rawalpindi-Pakistan.
1. Shen R, Wiegers SE, Glaser R. The evaluation of cardiac and peripheral arterial disease in patients with diabetes; mellitus. Endocr Res 2007;32(3):109-42.
2. Wu JT, Wu LL. Linking inflammation and atherogenesis: Soluble markers identified for the detection of risk factors and for early risk assessment. Clin Chim Acta 2006;366(1-2):74-80.
3. Vitale C , Fini M, Leonardo F, Rossini P, Cerquetani E, Onorati D, et al . "Effect of estradiol valerate alone or in association with cyproterone acetate upon vascular function of postmenopausal women at increased risk for cardiovascular disease, Maturitas. 2001;40(3):239–45.
4. Farah R, Shurtz-Swirski R, Lapin O. Intensification of oxidative stress and inflammation in type II diabetes despite antihyperglycemic treatment. Cardiovasc Diabetol 2008;7(20):1-8.
5. de Rekeneire N, Peila R, Ding J, Colbert LH, Visser M, Shorr RI, et al. Diabetes, hyperglycemia, and inflammation in older individuals: the health, aging and body composition study. Diabetes Care 2006;29(8):1902-08.
6. Leitinger N. Oxidized phospholipids as modulators of inflammation in atherosclerosis. Curr Opin Lipidol 2003;14(5):421-30.
7. Petitti DB, Imperatore G, Palla SL, Daniels SR, Dolan LM, Kershnar AK. et al. Serum lipids and glucose control: the search for diabetes in youth study. Arch Pediatr Adolesc Med 2007;161 (2): 159 – 65.
8. Abdel-Gayoum AG. The effect of glycemic control in type II diabetic patients with diabetes-related dyslipidemia. Saudi Med J 2004;25(2):207-11.
9. Meisinger C, Lowel H, Heier M, Schneider A, Thorand B. KORA Study Group. Serum gamma-glutamyltransferase and risk of type 2 diabetes mellitus in men and women from the general population. J Intern Med. 2005;258(6):527-35
10. Dehghan A, van Hoek M, Sijbrands EJ, Stijnen T, Hofman A, Witteman JC. Risk of type 2 diabetes attributable to C-reactive protein and other risk factors. Diabetes Care 2007;30(10):2695-99
11. Esper RJ, Vilariño JO, Machado RA, Paragano A. Endothelial dysfunction in normal and abnormal glucose metabolism. Adv Cardiol 2008;45:17-43.
12. Simão AN, Dichi JB, Barbosa DS, Cecchini R, Dichi I . Influence of uric acid and gamma-glutamyltransferase on total antioxidant capacity and oxidative stress in patients with metabolic syndrome. Nutrition 2008;24(7-8):675-81.
13. Sedda V, Chaira BD, Parolini M, Caruso R, Campolo J, Cighetti G, et al. Plasma glutathione level are independently associated with Gamma Glutamyl transferase activity in subjects with cardiovascular risk factors. Free Rad Res 2008;42(2):135-41.
14. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007;87(1):315-424.
15. Roberts WL, Sedrick R, Moulton L, Spencer A, Rifai N. Evaluation of four automated high-sensitivity C-reactive protein methods: implications for clinical and epidemiological applications. Clin Chem 2000;46(4):461-8.
16. Schumann G, Bonora R, Ceriotti F, Clerc-Renaud, P, Ferard G, Ferrero CA. et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37o C. Part 6, Reference procedure for the measurement of catalytic concentration of gamma glutamyltransferase. Clin Chem Lab Med 2002;40(7):734-8.
17. Giovannoni G, Land JM, Keir G, Thompsion EJ, Heales SJR. Adaption of nitric reductase and Griess reaction methods for measurement of serum nitrate plus nitrite levels. Ann Clin Biochem 1997; 34:193-8
18. Goldstein DE, Little RR, Wiedmeyer HM, England JD, Rohlfing CG. Glycated haemoglobin estimation in the 1990’s: A review of assay methods and clinical interpretation. In: Marshall SM, Home PD, editor. The Diabetes Annual. New York (NY): Elsevier Science B.V; 1994;p.193–212.
19. Ellerbe P, Myers GL, Cooper GR, Hert HS, Sniegoski LT, Welch MJ, et al. A comparison of results for cholesterol in human serum by the reference method and by the definitive method of the National Reference System for cholesterol. Clin Chem 1990; 36(2): 370-5.
20. Selvin E, Marinopoulos S, Berkenblit G, Rami T, Brancati FL, Powe NR, et al. Meta-analysis: glycosylated hemoglobin and cardiovascular disease in diabetes mellitus. Ann Intern Med 2004;141(6):421-31.
21. Middelbeek RJ, Horton ES. The role of glucose as an independent cardiovascular risk factor. Curr Diab Rep 2007;7(1):43-9.
22. Schulze MB. Rimm EB, Li T, Rifai N, Stampfer MJ, Hu FB. C-reactive protein and incident cardiovascular events among men with diabetes. Diabetes Care. 2004;27(4):889–94.
23. Pereira FO, Frode TS, Medeiros YS. Evaluation of tumour necrosis factor alpha, interleukin-2 soluble receptor, nitric oxide metabolites, and lipids as inflammatory markers in type II diabetes mellitus. Mediators Inflamm 2006;1:1-7
24. Pfutzner A, Forst T. High-sensitivity C - reactive protein as Cardiovascular risk marker in patients with diabetes mellitus. Diabetes Technol Ther 2006;8(1):28-36.
25. Ridker PM. C-reactive protein: eighty years from discovery to emergence as a major risk marker for cardiovascular disease. Clin Chem 2009; 55(2): 209–215.
26. Marsik C, Kazemi-Shirazi L, Schickbauer T, Winkler S, Wagner OF, Endler G. C-reactive protein and all-cause mortality in a large hospital-based cohort. Clin Chem 2008;54(2):234-7.
27. Izumi N, Nagaoka T, Mori F, Sato E, Takahashi A, Yoshida A. Relation between plasma nitric oxide levels and diabetic retinopathy. Jpn J Ophthalmol 2006;50(5):465-8
28. Ramakrishna V, Jailkhani R. Oxidative stress in non-insulin-dependent diabetes mellitus (NIDDM) patients. Acta Diabetol 2008;45(1):41-6.
29. Bauersachs J, Schaefer A. Tetrahydrobiopterin and eNOS dimer/monomer ratio: a clue to eNOS uncoupling in diabetes? Cardiovasc Res 2005;65(4):823-31.
30. Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, et al. Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation 2002;105:1656–62.
31. Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badiwala MV, et al. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation 2002; 106(8):913-9.
32. Lee MY, Koh SB, Koh JH, Nam SM, Shin JY, Shin YG. et al. Relationship between gamma-glutamyltransferase and metabolic syndrome in a Korean population. Diabet Med 2008;25(4):469-75.
33. Lee DS, Evans JC, Robins SJ, Wilson PW, Albano I, Fox CS. et al. Gamma glutamyl transferase and metabolic syndrome, cardiovascular disease, and mortality risk: The Framingham Heart Study. Arterioscler Thromb Vasc Biol 2007;27(1):127–33.
HOME | SEARCH | CURRENT ISSUE | PAST ISSUES
Room No. 522, 5th Floor, Panorama Centre
Building No. 2, P.O. Box 8766, Saddar, Karachi - Pakistan.
Phones : 5688791, 5689285 Fax : 5689860