Predictive value of serum glycated albumin in coronary heart disease of type 2 diabetes mellitus
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https://www.eduzhai.net Public Health Research 2012, 2(3): 37-42 DOI: 10.5923/j.phr.20120203.01 Value of Serum Glycated Albumin in Prediction of Coronary Artery Disease in Type 2 Diabetes Mellitus Saba Irshad1,*, Rabia Riaz1, Farkhanda Ghafoor2 1Institute of Biochemistry and Biotechnology, University of the Punjab, New Campus, Lahore, 54590, Pakistan 2NHRC, Sheikh Zaid Medical Complex, Lahore, 54590, Pakistan email@example.com, firstname.lastname@example.org Abstract Coronary artery disease (CAD) is a major vascular complication of diabetes mellitus and reveals high mortality. Up to 30 % of diabetic patients with myocardial ischemia remain asymptomatic and are associated with worse prognosis compared to non-diabetic counterpart, which warrants routine screening for CAD in diabetic population. The purpose of this study was to evaluate the clinical value of serum glycated albumin level in predicting the presence of CAD in patients with type 2 diabetes. Ninety patients with type 2 diabetes were divided into four groups having the coronary artery disease with lumen diameter narrowing < 30 %, Group II with mild CAD contained those patients with lumen diameter narrowing 30-50 %, and Group III with Major CAD contained those subjects with lumen diameter narrowing 50-70 %. Finally group IV with severe CAD including the patients with lumen diameter narrowing > 70 %. Serum levels of glycated albumin was determined using ELISA as well as serum concentrations of glucose, lipids, were taken in questionnaire in all groups. Serum glycated albumin levels were significantly increased in diabetic patients with CAD. Keywords Diabetes Mellitus, Coronary Artery Disease, Glycated Albumin, ELISA, Glycated Hemoglobin 1. Introduction Diabetes mellitus is a group of metabolic disorder in which there is absolute or relative deficiency of insulin with resultant hyperglycemia, glycosuria, polyuria and polydipsia, which represent typical clinical manifestations. Diabetes can cause acute or chronic complications. The new classification system identifies three types of diabetes mellitus: type 1, type 2, "and gestational diabetes. 1.1. Diabetes Mellitus and Coronary Artery Disease Diabetes mellitus (DM) is common associated with both micro vascular and macro vascular complications. Macro vascular complications manifest themselves as accelerated arteriosclerosis, clinically resulting in premature coronary artery disease (CAD), increased risk of cerebrovascular disease, and severe peripheral vascular disease. Patient with type 2 diabetes mellitus (T2DM) have a two to four fold increase in the risk of CAD. Several independent factors, e.g. insulin resistance, hyperglycemia, hypertension, dyslipidaemia, abdominal obesity and low-grade inflammation, have all been associated with this condition in subjects with type 2 diabetes. Diabetic state also promotes the Amadori-modification of many circulating proteins, giving rise to concentrations of glycated proteins approximately one and a half to three times those found in non-diabetic persons that reflect integrated glycemia to which the protein has been exposed during its residence time in the circulation. 1.2. Glycated Albumin Early stage reaction product of albumin or serum protein is called Glycated albumin or Fructosamine. Glycated albumin exhibits potential atherogenic effects in various cell types, including mesangial, monocyte- macrophage, mesothelial, endothelial, and vascular smooth muscle cells. Glycated albumin is the predominant circulating Amadori -type glycated protein in vivo and plays a major role in the development of diabetic vascular complications. An increased serum level of glycated albumin is associated with the presence and severity of CAD, and may be useful in screening patients with T2DM. It is suggested that GA provides a significantly better measure to estimate glycemic control in HD patients with diabetes and that the assessment of glycemic control by HbA1c in these patients might lead to underestimation likely as a result of the increasing proportion of young erythrocyte by the use of erythropoietin. * Corresponding author: email@example.com (Saba Irshad) Published online at https://www.eduzhai.net Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved 1.3. ELISA To evaluate the clinical utility of a highly specific monoclonal antibody directed against the glycated epitopes re- 38 Saba Irshad et al.: Value of Serum Glycated Albumin in Prediction of Coronary Artery Disease in Type 2 Diabetes Mellitus siding in human albumin, Cohen and Hud developed an ELISA using this antibody to measure glycated albumin in plasma samples from non-diabetic and diabetic individuals. Relative percent concentration of glycated albumin in a sample is determined by dividing the microgram glycated albumin in the sample by the total microgram albumin in the sample. 2. Materials and Methods 2.1. Study Design and Study Population It was a Cross Sectional Analytical study. Among the study population, 90 cases of type II diabetes were registered in the institute of biochemistry and biotechnology at the University of Punjab Lahore. Inclusion Criteria was that individuals with type II diabetes and undergoing diagnostic coronary angiography to find out the presence and extent of CAD were included in this study group and all individuals with type 1 diabetes rheumatoid arthritis and any other inflammatory diseases were excluded from the study. 2.2. Data Collection 90 cases of type II diabetes undergoing coronary angiography for the diagnosis of CAD were taken from the Punjab institute of cardiology. All the patients were angiographically confirmed for coronary artery disease. The demographic information like (name, age sex, height, and weight), history of present illness including history of diabetes and cardiac history were taken. The severity of CAD was based on lumen diameter narrowing as < 30 % (Minor CAD), 30-50% (mild CAD) 50-70 % (moderate CAD) and > 70 % (severe CAD) on visual assessment by experienced observer. 5 ml of venous blood was drawn from each selected subject using standard venepuncture techniques. The sterilized needles were used for the collection of blood from all the patients. The blood was allowed to clot at the room temperature for 2-3 hours. The clot was then removed while the supernatant was centrifuged at 4000 rpm. 2.4. Quantitative Determination of Glycated Albumin and Total Albumin Glycated albumin and total albumin was determined in each samples by using commercially available Glycaben ELISA kit. Glycated albumin can be reported as an absolute concentration (mg / ml) or as a percent (%) of total albumin as calculated from the following equation using determined for each of sample: % Glycated Albumin sample = 100% x Glycated albumin sample / Total Albumin sample 3. Results 3.1. Composition of Study Groups Out of 90 total subjects 42 (46.7 %) were males and 48 (53.3 %) were females. In Group I (minor CAD), 11 (40.7 %) were males and 16 (59.3 %) were females. In Group II (mild CAD), 13 (52 %) were males and 12 (48 %) were females. In the Group III (Major CAD), 14 (54 %) were males and 12 (46 %) were females. In the Group IV (sever CAD), 4 (33.3 %) were males and 8 (66.7 %) were females (Table I). Mean age group and SD were calculated for each group. The mean age and SD of the group I (minor CAD) was 48 ±0.95. The mean age and SD of the group II (mild CAD) was 61 ±1.17. The mean age and SD of the group III (Major CAD) was 53 ±0.84 and of the group IV (severs CAD) was 55 ±0.87 (Table II) 2.3. Sample Collection and Serum Separation Sr. No. 1 2 3 4 5 Table I. Distribution of the study groups by sex Study groups Group I (minor CAD) Lumen diameter narrowing < 30 % Group II (mild CAD) Lumen diameter narrowing 30-50 % Group III (major CAD) Lumen diameter narrowing 50-70 % Group IV (sever CAD) Lumen diameter narrowing > 70 % Total Males n % 11 40.7 13 52 14 54 4 33.3 42 46.7 Females n % 16 59.3 12 48 12 46 8 66.7 48 53.3 Total n % 27 30.0 25 27.8 26 28.9 12 13.3 90 100 Table II. Distribution of the study groups by ages Sr. no. Study groups Mean age (yrs) ±SD 1 Group I (Minor CAD) 49 0.95 2 Group II (Mild CAD) 60 1.17 3 Group III (Major CAD) 52 0.84 4 Group IV (Sever CAD) 55 0.87 Public Health Research 2012, 2(3): 37-42 39 Out of 90 subjects 33 (36.7 %) have duration of diabetes less than 5 years and 57(63.3 %) have duration more than 5 years. And 37 (41.1 %) have the family history of diabetes and 53 (58.9 %) have no family history of diabetes (Table III and IV) and 15 (16.70 %) heaving family history of CAD, 75 (83.3 %) were without family history (Table V). Table III. Duration of diabetes Sr. no. Study groups Group I 1 (Minor CAD) Group II 2 (Mild CAD) Group III 3 (Major CAD) Group IV 4 (Sever CAD) 5 Total Duration of diabetes < 5 yrs > 5 yrs n % n % 5 18.5 22 81.5 12 48.0 13 52.0 14 53.84 12 46.16 2 16.7 10 83.3 33 36.7 57 63.3 Total 27 25 26 12 90 Sr. no. 1 2 3 4 5 Table IV. Family history of diabetes Study groups Group I (Minor CAD) Group II (Mild CAD) Group III (Major CAD) Group IV (Sever CAD) Total Family history of diabetes Yes No n % n % 4 14.8 23 85.2 8 32.0 17 68.0 20 77.0 6 23.0 5 41.7 7 58.3 37 41.1 53 58.9 Total 27 25 26 12 90 Table V. Assessment of smoking in each study group Sr. no. Study groups Assessment of smoking in each study group No Yes Total n % n % Group I 1 0 0 27 100 27 (Minor CAD) Group II 2 2 8 23 92 25 (Mild CAD) Group III 3 3 11.5 23 88.5 26 (Major CAD) Group IV 4 5 41.7 7 58.3 12 (Sever CAD) 5 Total 10 11.1 80 88.9 90 Among 90 subjects 10 (11.1 %) were smokers and 80 (88.9 %) were non-smokers and 37 (41.1 %) were hyper- tensive and 53 (58.9 %) were non-hypertensive (Table VI and VII). Table VI. Assessment of hypertension in each study group Sr. no. 1 2 3 4 5 Study groups Group I (Minor CAD) Group II (Mild CAD) Group III (Major CAD) Group IV (Sever CAD) Total Assessment of hypertension in each study group Yes No n % n % 6 22.2 21 77.8 10 40.0 15 60 14 53.8 12 46.2 7 58.3 5 41.7 37 41.1 53 58.9 Total 27 25 26 12 90 Sr. no. 1 2 3 4 5 Table VII. Family history of CAD Study groups Group I (Minor CAD) Group II (Mild CAD) Group III (Major CAD) Group IV (Sever CAD) Total Family history of CAD Yes No n % n % 92. 2 7.4 25 6 88. 3 12.0 22 0 80. 5 19.2 21 8 5 41.7 7 58. 3 83. 15 16.7 75 3 Total 27 25 26 12 90 3.2. Determination of Total Albumin and Glycated Albumi by ELISA There were three types of information sources for the total albumin and glycated albumin ELISA i.e. the calibration of the standards, quality control samples and the test specimens. These three were subjected to the analytical procedure and some response (color development) was obtained. After running the ELISA test concentration of total albumin and glycated albumin for standards were calculated (Table VIII and IX). Graphical representation is in Fig. 1 (A), (B). Table VIII. Standards for total albumin Standards No. Conc. of total albumin (mg/dl) Absorbance at 630nm A B C D 0.00 11.570 25.795 49.735 0.00 4.679 8.070 10.46 Table IX. Standards for glycated albumin Standards No. Conc. of glycated albumin ( mg/l) Absorbance at 450nm A B C D E 0.00 3.00 1.500 0.750 0.370 0.00 1.720 1.341 1.120 0.945 40 Saba Irshad et al.: Value of Serum Glycated Albumin in Prediction of Coronary Artery Disease in Type 2 Diabetes Mellitus Absorbance at 630nm 12 10 8 6 Absorbance at 4 630nm 2 0 0 20 40 60 Concentration of Total Albumin in mg/ml Figure 1(A). Standard curve for total albumin, whose absorbance is taken at 630 nm 3.3. Assessment of Mean BMI With % Age Glycated Albumin Mean BMI = height /weight In-group I (minor CAD) the mean BMI was 28.3, in-group II (mild CAD) the mean BMI was 27.4. In-group III (Major CAD) the mean BMI was 27.2 and in-group IV (sever CAD) the mean BMI was 18.2. The mean % age glycated albumin in the group I was 2.18, in the group II 3.3, in the group III 4.0 and in the group IV the mean % age glycated albumin was 5.3 (Table XI). There is negative association between mean BMI and % age glycated albumin (Fig. 3). 2 1.8 1.6 Absorbance at 450nm 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 0.5 1 1.5 2 2.5 3 3.5 Concentration of glycated albumin mg/ml Figure 1(B). Standard curve for total albumin, absorbance is at 450 nm The mean % age glycated albumin was calculated for each subject in each study group by using formula. The mean % age glycated in Group I was 2.18, in Group II was 3.3, in Group III 4.0, and in Group IV was 5.3 (Table X). Table X. Values of % age glycated albumin Sr. no. 1 2 3 4 Study Group Group I (Minor CAD) Group II (Mild CAD) Group III (Major CAD) Group IV (Sever CAD) Mean of %age glycated albumin 2.1800 ±SD 0.01000 3.3000 0.01000 4.0000 0.01000 5.3267 0.05508 The graphical analysis shows the relationship between severities of CAD with increase in % age glycated albumin (Fig. 2). 6 5 4 %age glycated albumin 3 2 1 0 1 2 3 4 Group no Group NO. %glycated albumin Figure 2. % age glycated albumin in each study group, groups are taken on x-axis and % age glycated albumin is on y-axis Sr no 1 2 3 Table XI. Mean BMI for each study group Study Groups Group I (Minor CAD) Group II (Mild CAD) Group III (Major CAD) Mean BMI 28.3 27.4 27.2 Mean %age Glycated albumin 2.18 3.3 4.0 4 Group IV (Sever CAD) 18.2 5.3 30 Mean BMI in Each Study Group 25 20 15 BMI 10 5 0 0 1 2 3 4 5 6 %age Glycated Albumin in Each Study Group Figure 3. Assessment of mean BMI with % age glycated albumin, % age glycated albumin decreases with increase in BMI values 3.4. Association between Mean Triglycerides and Mean % Age Glycated Albumin The mean % age glycated albumin and triglycerides level were calculated for each study group (Table XII). It was observed that % age glycated albumin level increases as the triglyceride level increases, providing a direct relationship between % age glycated albumin and TG (Fig. 4). Table XII. Mean Triglycerides and mean %age glycated albumin Sr No 1 2 3 4 Study groups Group I (Minor CAD) Group II (Mild CAD) Group III (Major CAD) Group IV (Sever CAD) Mean % age Glycated Albumin 2.18 3.3 4.0 5.3 Mean Triglycerides mg/dl 102 136 223 340 Public Health Research 2012, 2(3): 37-42 41 350 300 250 Mean triglycerid 200 e mg/dl 150 100 50 0 1 23 4 Mean % age glycated albumin Mean triglycerdies mg/dl Mean % age glycated albumin Figure 4. Association between mean triglycerides and mean % age glycated albumin 3.5. Association between Mean Glucose Level and Mean % Age Glycated Albumin The mean % age glycated albumin and glucose level were calculated for each study group (Table XIII). It was observed that % age glycated albumin level increases as the glucose level increases, providing a direct relationship between %age glycated albumin and glucose (Fig. 5). Table XIII. Mean glucose level and mean %age glycated albumin Sr. no 1 2 3 4 Study groups Group I (Minor CAD) Group II (Mild CAD) Group III (Major CAD) Group IV (Sever CAD) Mean %age Glycated Albumin 2.18 Mean Glucose mg/dl 192 3.3 201.3 4.0 223 5.3 240 Figure 5. Association between glucose level and mean % age glycated albumin 4. Discussion Diabetes has been recognized as an important risk factor for CAD, and diabetic patients are at 2-fold increased risk of cardiovascular mortality compared to their non diabetic counterparts. Previous studies have demonstrated that silent myocardial ischemia, which is mainly caused by autonomic neural dysfunction, occurred in about 20 % to 25 % of diabetic patients, and the prevalence may be as high as 60 in those at high-risk. All proteins in the body can be modified by non- enzy- matic glycation. In diabetes mellitus the extent of the nonenzymatic glycation of proteins increases, compared with non-diabetic subjects, which may comprise at least a part of diabetic complications. Among these modified proteins, measurement of HbA1c has been applied for clinical use in order to monitor chronic glycemic control in diabetic patients. But now we try to provide the better indicator of glycemic control in CAD patients which is Glycated albumin, is a kind of early-stage amadori-modified reaction products formed from Schiff's base adducts and has been implicated in the pathogenesis of diabetic complications and better indicator[11, 3]. Stable fraction of glycated hemoglobin (HbA1c) is routinely measured in the majority of patients with diabetes around the world; Since HbA1c reflects glycemic control over the preceding 2-3 months. However HbA1c may not be suitable for evaluation of short term variation in glycemic control because of long life span of erythrocytes (120 days). Because the turnover of human serum albumin is much more rapid (half life of 15-20 days) than that of hemglobin, the measurment of glycated albumin (GA) provides an index of glycemic control over a short period of time than the measurment of HbA1c[13, 14]. There are several risk factors associated with coronary artery disease, such as family history of diabetes and coronary artery disease, smoking, hypertension, hyperglycemia, hypertriglyceridemia, BMI. The prevalence of CAD increased with age and duration of diabetes and in this study 57 (63.3 %) subjects have duration of diabetes more than 5 years and mean age is 55±0.87. Cigarette smoking is a leading risk factor for CVD. Patients with diabetes who are smokers are doubly at risk. Unfortunately, many patients continue to smoke despite having diabetes; for these patients, the benefits that can be derived from modifying other risk factors are mitigated. In this study, 80 (88.9 %) subjects were smokers. High blood pressure accounts for 20 to 25 % of all CAD deaths. Subjects with hypertension have a two-fold higher risk of CAD. In this study, Out of 90 subjects, 53 (58.9 %) patients were hypertensive. Serum glycated albumin levels are low in obese. The result of this study shows the negative association of % age glycated albumin with the BMI. And we found that serum GA levels are influenced by BMI in diabetic patients. Hyperglycemia, leading to the formation of advanced glycation end products (AGE), on vascular function. Glycated albumin is a kind of Amadori-modified derivatives, accounting for about 80 % of the circulating glycated proteins in vivo. Hyperglycemia increases serum glycated albumin level, and the extent of increase reflects glycemia status of diabetic patients over a retrospective period. Although relationship between diabetic atherogenesis and several common risk factors plus non traditional risk markers have been studied extensively and the data is having some controversies. 42 Saba Irshad et al.: Value of Serum Glycated Albumin in Prediction of Coronary Artery Disease in Type 2 Diabetes Mellitus 5. Conclusions Previous studies showed that glycated albumin was more sensitive than glycosylated hemoglobin (HbA1c) in the evaluation of the severity of diabetes. The present study showed that glycated albumin was an independent risk factor for CAD in patients with type 2 diabetes, with and predicts the coronary artery disease in type 2 diabetes mellitus. ACKNOWLEDGMENTS The authors acknowledge the enabling role of Miss. 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