Microvascular complications of type 2 diabetes mellitus

BACKGROUND AND OBJECTIVES: It has been shown in studies that there are significant variations in glycation of various serum proteins in type 2 diabetes mellitus patients with and without microvascular complications. Present study aimed to evaluate whether correcting serum fructosamine for serum total protein level, serum glycated albumin for serum albumin level and deriving ratio of glycated β-lipoprotein to total βlipoprotein enhances their significance and correlation over and above serum fructosamine, glycated albumin and glycated β-lipoprotein respectively in type 2 diabetes mellitus patients with and without microvascular complications. METHODS: This was a cross sectional study involving 150 individuals at a tertiary care hospital in western India. 50 participants were healthy controls (group 1), 50 were type 2 diabetes patients without any evident microvascular complication (group 2) and 50 were type 2 diabetes patients with one or more microvascular complications (group 3). Serum fructosamine, FBS, PP2BS and other biochemical parameters were measured. Glycated albumin and glycated β-lipoprotein were measured by agarose gel electrophoresis followed by NBT staining. Corrected serum fructosamine, corrected glycated albumin and glycated β-lipoprotein/ β-lipoprotein ratio were calculated. Unpaired t-test was used to find out significance of difference between two groups and correlation coefficient to find out statistical correlation between two variables. RESULTS: Differences between the groups for corrected fructosamine, corrected glycated albumin and glycated β-lipoprotein/ β-lipoprotein ratio were significant (p<0.001). Corrected glycated albumin and corrected fructosamine correlated with FBS, PP2BS and with each other in all diabetic patients (groups 2 and 3). Glycated β-lipoprotein/ β-lipoprotein ratio was remarkably elevated in group 3 and it did not correlate either with corrected fructosamine or corrected glycated albumin. CONCLUSION: Glycated β-lipoprotein/ βlipoprotein ratio was markedly elevated in type 2 diabetes patients with microvascular compilations but neither of corrected fructosamine, corrected glycated albumin or glycated β-lipoprotein/ β-lipoprotein ratio provided any information in addition to that provided by the uncorrected parameters. Key-words: Type 2 diabetes mellitus, microvascular complications, corrected fructosamine, corrected glycated albumin, corrected glycated β-lipoprotein Microvascular complications of type 2 diabetes mellitus 140 Int J Res Med. 2016; 5(3); 139-145 e ISSN:2320-2742 p ISSN: 2320-2734 provide additional information as compared to glycated β-lipoprotein alone. The study was undertaken to assess levels of serum fructosamine corrected for serum total protein, serum glycated albumin corrected for serum albumin level and glycated β-lipoprotein to total βlipoprotein ratio in type 2 diabetes mellitus patients with and without microvascular complications and to find out their correlation with the complications over and above the uncorrected parameters. MATERIAL AND METHODS This was a hospital based cross-sectional study consisting of three groups and total 150 participants. Group 1 (control group, n=50) consisted of randomly selected age and sex matched non-diabetic subjects. They were free from any ailment which could affect the parameters under study (no clinical history or investigative result showing involvement of any organ system). They were selected from medical and paramedical staff, attendants of patients and persons coming to hospital for fitness purpose. Group 2 (type 2 diabetes mellitus patients without any microvascular complications, n=50) consisted of those with duration of diabetes 3 years or more, on life style modifications and oral anti-diabetic drugs and free from clinical or laboratory evidence of any microvascular complication of diabetes mellitus. Group 3 (type 2 diabetes mellitus patients with microvascular complications, n=50) consisted of those with duration of diabetes 3 years or more, on life style modifications, oral anti-diabetic drugs, insulin or combination of all three and diagnosed as having one or more microvascular complication of diabetes mellitus; either diabetic neuropathy (peripheral neuropathy diagnosed by 10 gm monofilament and reduced vibration perception using 128 Hz tuning fork), diabetic nephropathy (urine albumin ≥30 μg/ mg of creatinine with normal urine microscopy and not a known case of any other kidney disease) or diabetic retinopathy (diagnosed by direct ophthalmoscopy after mydriasis). Consecutive patients attending the medical outpatient department were enrolled in groups 2 and 3 unless they met any of the exclusion criterions. Patients with type 1 diabetes mellitus, pregnant females and patients with diseases unrelated to diabetes which might significantly alter serum protein profile e.g. hepatic diseases, hematological malignancy, chronic infections and inflammations like tuberculosis, sarcoidosis, rheumatological diseases, infectious mononucleosis, AIDS were excluded from the study. Objectives of the study were explained to all subjects eligible for this study. Informed written consent of all the subjects was obtained for voluntary participation in study group, sample collection and for data utilisation for the purpose of publication. Data were recorded in a questionnaire designed for the study and it included sociodemographic data, presenting complains, detailed diabetes history including treatment and complications, history of other ailments, past, personal and family histories as well as findings of a thorough physical examination. For each subject, overnight fasting blood sample was collected in a fluoride vacutainer for FBS and in a plain vacutainer for other biochemical parameters. Urine sample was collected in universal container for urine creatinine and urine protein estimation. Post prandial (2 hour) sample was collected in a fluoride vacutainer for PP2BS estimation. Serum and plasma were separated within an hour of collection and stored at 2 to 8°C temperature till analyses were performed. Electrophoresis was performed within 5 days whereas all the other parameters were estimated on the same day. FBS and PP2BS were estimated by Glucose Oxidase-Peroxidase (GOD-POD) enzymatic end point method. Serum fructosamine was estimated by NBT colorimetric kinetic assay. Serum Creatinine concentration was measured by modified Jaffe’s kinetic method. Serum total cholesterol was estimated by Cholesterol Oxidase-Peroxidase (CHODPAP) enzymatic end point method. Serum LDL cholesterol was measured by direct enzymatic method. Urinary creatinine was Microvascular complications of type 2 diabetes mellitus 141 Int J Res Med. 2016; 5(3); 139-145 e ISSN:2320-2742 p ISSN: 2320-2734 estimated by modified Jaffe’s kinetic method, urinary protein was measured by Pyrogallol red end point method and urinary protein/creatinine ratio was calculated. All biochemical investigations were performed on a fully automated analyzer. Serum glycated β-lipoprotein was measured by agarose gel film electrophoresis at pH 8.6 followed by colour development with nitro-blue tetrazolium (NBT) by a modification of staining method adapted from Kunio Kobayashi et al [4]. Dried and compressed electrophoresis plate was immersed for 20 hours in glycated protein colour development reagent (5 mmol/l NBT in 50 mmol/l sodium carbonate buffer pH 10.3) at room temperature followed by two washes of stop solution (0.3% citric acid). NBT stained blue bands on the glycated protein film were scanned at 540 nm (Olympus glass filter plate) using a densitometric scanner (Nikon Super COOLSCAN 9000 ED). Image was analysed by free electrophoresis image analysis software GelAnalyzer 2010a. Proportions (%) of glycated albumin and glycated lipoprotein were represented as the percentage of total area under the scanned profile attributable to each peak. Concentration (in μmol/l) was calculated by multiplying percentage with serum fructosamine value. Staining in β region was considered to represent glycation of βlipoproteins. Serum protein (stained with 0.5% acid blue 29 in 5% acetic acid) and lipoprotein (stained with 0.05% fat red 7B and 0.05% oil red O in methanol: water, 4:1) electrophoresis was performed simultaneously for all the samples to aid in quantitation by comparison of mobility of respective proteins (figure 1). Figure 1: Correlation of serum corrected fructosamine with corrected glycated albumin (n=150) Corrected serum fructosamine (μmol/gm of total protein) was calculated by dividing serum fructosamine value (μmol/l) by serum total protein (gm/l). Corrected glycated albumin (μmol/gm of serum albumin) was calculated by dividing serum glycated albumin value (μmol/l) by serum albumin (gm/l). Similarly, glycated βlipoprotein/ β-lipoprotein ratio was calculated by dividing glycated βlipoprotein value (μmol/l) by serum LDL (after converting to gm/l). Data collected were recorded and analysed statistically to determine the significance of different parameters by MedCalc version 12.4. Statistical analysis was done by using unpaired t-test to find out significance of difference between two groups and correlation coefficient to find out statistical correlation between two variables and its significance. p value less than 0.05 was considered significant. Results and discussion Table 1: Comparison of study groups Group 1 Group 2 Group 3 Number of participants 50 50 50 Sex (M/F) 58/42 54/46 48/52 Average age (years) 47±10 53±9 52±9 Average duration of diabetes mellitus (in year) 6.3±2.8 8.2±3.8 FBS (mg/dl) 94±11 129±39 188±86 PP2BS (mg/dl) 119±11 184±57 271±109