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Indian Journal of Human Genetics, Vol. 9, No. 1, Jan-Jun, 2003, pp. 17-20 Serum Adenosine deaminase activity and C-reactive protein levels in unstable angina Surekha Rani H, Dayasagar Rao V,* Shiva Prakash M,** A. Jyothy Department of Cell Biology, Institute of Genetics, Hyderabad;
*Department of Cardiology, Durga Bai Deshmukh Hospital & Research Centre,
Hyderabad; and **Department of Endocrionology, National Institute of Nutrition,
Tarnaka, Hyderabad.
Code Number: hg03005 In unstable angina (USA) patients, immunological responses contributing to inflammation play a vital role in plaque rupture and thrombosis causing stroke. In the present study an attempt is made to estimate the levels of adenosine deaminase activity, an immunoenzyme marker and C-reactive protein, a marker of inflammation in USA patients. 45 patients presenting USA and 50 age and sex matched healthy controls were included in the study. Serum ADA activity was measured spectrophotometrically at 630nm and serum C-reactive protein was detected using Avitex CRP kit, which is a rapid latex agglutination test. The Mean ADA levels were 41.15 ± 11.04 in patients and 20.71±5.63 in controls and 66.6% of patients and none of the controls were positive to CRP. The present study observed the importance of ADA as a serum marker in addition to CRP for assessing the immune response in USA patients. Key Words: Adenosine deaminase, C-reactive protein, Unstable angina, Inflammation, Thrombosis, Atherosclerotic plaque. INTRODUCTION Coronary heart disease is a major cause of morbidity and mortality in the industrialized countries, and there is an international concern that heart diseases will become a major cause of death in developing countries by the year 2020. Based on the current trends India is on course of being the largest single contributor to global CHD mortality by the year 2015 and need for concern.1 Unstable angina (USA) is a clinical syndrome that lies in the spectrum of coronary heart disease which shares ill-defined borders with chronic stable angina, a presentation with lower risk, and with acute MI, a presentation with higher risk. It can be dangerous as it may quickly progress to heart attack. It occurs due to a sudden interruption of blood flow to the heart due to a partial or complete blockage of the artery. Coronary flow may be impaired by a thrombus in one of the coronary arteries or hemorrhage within or beneath an atherosclerotic plaque. Thus USA most often results from disruption of an atherosclerotic plaque and a subsequent cascade of pathologic processes that decrease coronary blood flow.2 The immunologic processes of the cellular and humoral type also contribute to the thrombotic component of the disease. The lesions of atherosclerosis contain a focal accumulation of macrophages and activated T cells, which secrete cytokines. Thus inflammatory response results in plaque rupture and thrombosis causing stroke.3 Adenosine deaminase, an enzyme catalyzes the conversion of adenosine and 2' deoxyadenosine to inosine and 2' deoxyinosine respectively.4 The major sources of serum ADA may be lymphocytes or the monocyte - macrophage cell system.5,6 It is required for lymphocyte proliferation and differentiation and it is thus considered as an important immunoenzyme marker of cell mediated immunity.7 Plasma C-Reactive Protein (CRP), a marker of low to moderate systemic inflammation, has received much attention in recent years. It is synthesized in the liver and is known as acute phase marker of tissue injury, infection and inflammation. Human CRP is a homogenous protein, free of lipid and carbohydrate. The CRP gene has been localized to chromosome 1 and codes for a mature, 206 amino acid polypeptide.8 Unstable angina results in congestive heart failure due to myocardial damage. The inflammation produced by myocarditis of viral or other origin may induce advanced myocardial damage, which results in heart failure with prognosis. Routine CRP is shown to identify patients with unstable coronary lesions who are previously unrecognized by traditional CHD markers. Therefore we measured the ADA activity as an index of cell-mediated immunity and CRP as a marker of inflammation in the patients with USA along with age and sex matched controls. MATERIAL AND METHODS Angiographically documented 45 patients of USA, admitted to the cardiology unit of Durga Bai Deshmukh Hospital & Research Centre, Hyderabad were included in the study. 50 age and sex matched healthy individuals with no known history of any disease were taken as controls. All the subjects were examined clinically and information pertaining to age, sex, habits and health status was recorded in special case proforma. Blood samples were collected from both controls and patients for the estimation of serum ADA and C-reactive protein. ADA Estimation The serum was assayed immediately for ADA activity at 370C by a spectrophotometric method using adenosine as the substrate. The principle of this method is based on the Bertholet reaction, that is, the formation of coloured indophenol complexes from ammonia liberated from adenosine and quantitated spectrophotometrically at 630 nm.9 One unit of ADA is defined as the amount of enzyme required to release one micromole ammonia per minute from adenosine at standard assay conditions. The activity of ADA is expressed in units/litre. Statistical analysis was carried out by Student t test. C-Reactive Protein Detection For the detection of CRP in serum, Avitex -CRP kit is used which is a rapid latex agglutination test. The test is based on the principle that Avitex-CRP latex paticles are coated with antibodies to human CRP, i.e when the latex suspension is mixed with serum containing elevated CRP levels on a slide, clear agglutination is seen within 2 minutes. Avitex -CRP has detection limit of 6 mg/litre of CRP in the patients serum. Positive results will be obtained at a CRP serum concentration above 6mg/litre and negative results will be obtained at 6 mg / litre and below. Semi-quantitative test A series of doubling dilutions of the patients serum in isotonic saline (1/2, 1/4,1/8 and 1/16) were prepared and the test procedure for each dilution was repeated. The serum CRP concentrations were calculated approximately by multiplying the dilution factor (i.e. 2, 4, 8 or 16) by the detection limit i.e. 6 to give the mg/litre concentration e.g. if the agglutination titre is at 1/8 the approximate serum CRP levels will be 8x6=48 mg /litre. RESULTS Age & Sex: Among 45 patients presenting unstable angina, 30 patients were males with mean age 51.46±11.12 and 15 were females with mean age 55.31±11.25 , while among 50 healthy controls 30 were males with mean age 50.46±8.45 and 20 were females with mean age 52.36±9.76 respectively as shown in Table 1. Habits: Out of 30 male patients 19 (63.3%) of them were smokers and 14 (46.6% ) were alcoholic; all the female patients and healthy controls were non smokers and non- alcoholic as shown in Table 2. Health status: Out of 45 patients, 25 (55.5%) were hypertensive and 13 (28.8%) were diabetic as shown in Table 2. Serum ADA levels: Mean ADA levels estimated in patients with USA and controls are presented in Table 3. The mean ± SD of ADA levels in serum of USA patients was 41.15 ± 11.04 and that of controls was found to be 20.71 ± 5.63. The difference in the mean values was statistically significant at p< 0.05. Serum CRP levels: CRP levels estimated in the USA, and controls are presented in Tables 4 & 5. In the present study 30/45 cases of USA were found to be positive to CRP while all the controls were negative for test. Among positive cases 13/30, 9/30,6/30 and 2/30 of USA cases showed a concentration of 12mg/litre, 24mg/litre, 48 mg/litre and 96mg/litre respectively. DISCUSSION In unstable angina patients, transient decrease in blood supply is associated with intracoronary thrombus formation or yellow plaque which is present in most unstable culprit arteries and has been characterized as grayish white and platelet rich while in myocardial infarction it is red. A positive correlation exists between the severity of the unstable angina presentation and the presence of an intracoronary thrombus or complex lesion.10 The thrombotic component of the disease is also associated with immunologic processes of the cellular and humoral type. Macrophages and activated T cells accumulate in the lesions of atherosclerotic plaques causing inflammation that leads to plaque rupture. Macrophages and mast cells in unstable coronary plaques secrete metalloproteinases that induce degradation of the extracellular matrix and may cause plaque rupture and thrombosis causing stroke.11 The major mechanisms by which T cells contribute to the pathogenesis of inflammatory disease are via the release of specific patterns of cytokines. Evidence is indicated for an LDL- induced pathway of type 1 cytokine activation in atherosclerosis, which is regulated by the local production of IL-12 and IL-10. Thus the degree of T-cell and macrophage infiltration in atherosclerotic plaques has been shown to correlate with the occurrence of plaque rupture.12 It is reported that the ADA levels are elevated whenever cell mediated immunity is stimulated and thus reflects the activity of stimulated T-lymphocytes. Cytokines produced by Th1 and Th2 cells also regulate membrane adenosine deaminase on human lymphocytes. IFN-g activates the monocyte -macrophage cell system, which may also contribute to the regulation of plasma ADA activity.13 Hence, ADA levels have been estimated in the USA patients to assess the cell mediated immune response. The results of the present study showed highly significant mean levels of ADA in USA patients when compared to controls. The elevated levels of ADA reflect the changes in the immune response in the pathogenesis of unstable angina. Elevated levels of ADA have also been reported in other diseased conditions like tuberculosis,14 acute nephrotic syndrome,15 leukemia, Behcet's Disease,7 typhoid,5 and in patients with renal transplants. Large number of studies showed the evidence that local and systemic inflammation plays a role in the initiation and progression of atherosclerosis. C-reactive protein, a sensitive and nonspecific marker of inflammation has been widely studied. Peptides produced by the proteolysis of CRP, with both anti- inflammatory and pro inflammatory functions modulate the local immune disregulation that characterizes atheroma progression and plaque rupture. In addition CRP may induce human monocytes to synthesize tissue factor, a potent procoagulant. It may also contribute to endothelial dysfunction. Serum levels of CRP were found to be elevated in febrile illness and various diseases with inflammatory status like hepatic disease, autoimmune disorder, malignancy etc.8 In patients with USA, CRP is an important predictor of MI and CHD death and it identifies the subjects at high risk for recurrent vascular events who were previously thought to be at relatively low risk. Haverkate et al17 has also observed raised circulating concentrations of CRP as predictors of coronary events in patients with stable or unstable angina. Keshavamurthy18 also showed that 65% of patients with USA had elevated levels of CRP as compared to only 13 % with chronic stable angina and observed that higher levels of CRP correlate with a twofold increase in coronary events. Similar observations were made in the present study which showed 66.6% patients to be positive to CRP and a majority of them had higher values. Although the results of the present study on CRP are in accordance with the earlier reports, the study suggests the inclusion of another easily measurable and cost effective marker ADA along with CRP as vital in assessing the immune response in unstable angina patients for better management and for developing new treatment strategies. ACKNOWLEDGEMENTS Authors are grateful to the Department of Biotechnology (DBT), New Delhi for providing financial assistance. REFERENCES
Copyright 2003 - the Indian Society of Human Genetics The following images related to this document are available:Photo images[hg03005t2.jpg] [hg03005t3.jpg] [hg03005t5.jpg] [hg03005t1.jpg] [hg03005t4.jpg] |
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