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Comparative Analysis of Serum Levels of Total Carbohydrates and Protein Linked Carbohydrates in Normal and Diabetic Individuals

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Comparative Analysis of Serum Levels of Total Carbohydrates and Protein Linked Carbohydrates in Normal and Diabetic Individuals Original Article DOI: 10.7860/JCDR/2019/41432.12881 Comparative Analysis of Serum Levels Section Biochemistry of Total Carbohydrates and Protein Linked Carbohydrates in Normal and Diabetic Individuals PRAVEEN SABLANIA1, MONTOSH CHAKRABORTY2, MANISH MANDAL3, PRERNA SINGH4, JIUT RAM KESHARI5 ABSTRACT Results: Serum total carbohydrate and CPR were significantly Introduction: Non-enzymatic glycation and enzymatic increased in MHG (p<0.001 and p=0.02, respectively) and glycosylation of proteins have been proposed as a major SHG (p<0.001 and p<0.001, respectively) compared to factor in the pathogenesis of diabetic complications by controls. CPR levels increased with increase in glycemia i.e., altering structure-function of proteins. Though focus of lowest in controls, higher in MGH and highest in SHG. We previous research is primarily focussed on glycated adducts also observed a statistically significant upward trend from as glycated haemoglobin (HbA1c), Advanced Glycation End 1st quartile through 4th quartile for CPR in SHG (p<0.001). Products (AGEs) and glycated albumin. The effect of enzymatic Likewise OR for 4th quartile compared with inter-quartile range glycosylation has been overlooked in monitoring of diabetes. (IQR) as reference was statistically significant for SHG {OR (95%CI)=5.27 (2.09-13.25); p<0.001}. Aim: The present study examines the relationship between glycemic status and serum levels of protein linked total Conclusion: Glycated Haemoglobin (HbA1c) concentrations carbohydrate using inexpensive and rapid methods. and self monitoring of blood glucose are widely used for monitoring of long term and short term glycemic control Materials and Methods: The present case-control study was respectively, although overall glycation and glycosylation conducted on 145 subjects that were divided into three groups status of circulating or tissue proteins could also be affected i.e., normoglycemic controls (n=54), mild-moderate hyperglycemia by glycemic status of an individual. Therefore, it is desirable (MHG) (n=44) and severe hyperglycemia (SHG) (n=47). Serum was that an assay or biomarker would provide assessment of overall subjected to precipitation and then precipitate was re-dissolved glycation/glycosylation process in between immediate and long in urea/thiourea buffer. Protein content of re-dissolved precipitate term control. In this preliminary case-control study we found was measured using Bradford assay. Serum total carbohydrate protein linked carbohydrates (PLC) levels and carbohydrate and carbohydrate content in resuspended pellet (mg carbohydrate protein ratio (CPR) to be significantly elevated in SHG compared per gram of protein designated as Carbohydate Protein Ratio or to controls (p<0.001 and p<0.01, respectively). The alterations in CPR) was measured using phenol-sulfuric acid method. Statistical PLC levels and CPR may be attributed to difference in glycemic analysis was done using SPSS statistics (V21.0) and Microsoft status between these two groups. The method used in current excel software, viz. Student-t-test for comparison of significant study is inexpensive, rapid and technically less demanding thus mean, binary logistic regression was used to measure Odds Ratio suitable for regions having limited resources. (OR). In all tests of significance, two sided p value have been reported and results were considered significant if p<0.05. Keywords: Advanced glycation end products, Glycoprotein, Glycosylation, Hyperglycemia INTRODUCTION life span, presence of deglycating enzymes leading to variable Prolonged exposure to hyperglycemia is recognised as the principal glycation, uremia and haemoglobin variants [3] while AGEs assays aetiological factor in pathogenesis of diabetic complications. are either yet to be standardised or are expensive [4]. Glycosylation (enzymatic) and glycation (non-enzymatic) of tissue, All glycated serum proteins are fructosamines, measured by circulating and erythrocytic proteins can alter protein function as Nitroblue Tetrazolium (NBT) colorimetric assay, less expensive structural alterations, chemical modifications, cell behaviour and than HbA1c assay, and is considered to reflect concentration of interaction [1]. Though alterations in both glycosylation and glycation glycated albumin [5]. Nevertheless, glycation of albumin is affected are associated with hyperglycemia, focus of diabetes management by hypoalbuminemic states as nephrotic syndrome, cirrhosis, has traditionally been assessment of glycemic control in diabetes hyperuricemia, thyroid disease and hypertriglyceridemia [3]. by measurement of glycated adducts [2]. Glycated haemoglobin The other spectrum of carbohydrate linkage to protein ie, enzyme can reflect glucose concentration over the preceeding 8-12 weeks mediated protein glycosylation differ between persons but is whereas that of glycated albumin over preceeding 2-3 weeks. remarkably constant per individual. Glycosylation status changes Glycation of proteins can progress to form AGEs and hyperglycemia remarkably with lifestyle changes, in pathological & inflammatory accelerates protein linked AGEs [3]. HbA1c is measured by states and some congenital disorders of glycosylation [6]. immunoassay/High Performance Liquid Chromatography (HPLC) Glycation and glycosylation status in an individual could be affected and AGEs by immunoassays/mass spectrometry but various factors by hyperglycemia and homeostatic changes. Though most abundant influence either measurement or interpretation of HbA1c results serum protein i.e., albumin, is not glycosylated, most serum protein such as chronic renal failure, iron deficiency anemia, erythrocyte are glycoproteins. It is pertinent to determine alteration in total 32 Journal of Clinical and Diagnostic Research. 2019 May, Vol-13(5): BC32-BC35 www.jcdr.net Praveen Sablania et al., Comparative Analysis of Serum Levels of Total Carbohydrates and Protein Linked Carbohydrates in Normal and Diabetics carbohydrate (of glycation or glycosylation origin) linked to protein Statistical analysis was done using Statistical Package for Social in hyperglycaemic state inexpensively and rapidly in technically less Sciences (SPSS) version V21.0) and Microsoft excel software. demanding manner in resource poor regions of world. An attempt Variables in controls were compared with those of cases for was made to address these issues in current study. The aim of significant mean with independent unpaired student t-test and the present study is to examine the relationship between glycemic pearson correlation coefficient. Mild-moderate and severe status and serum levels of protein linked total carbohydrate using hyperglycaemic cases were categorised into quartiles according methods that are inexpensive and rapid. to distribution in controls. Risk was estimated using binary logistic regression while odds ratio (OR) was calculated for 4th quartile of MATERIALS AND METHODS mild-moderate and severe hyperglycemia using interquartile range The present case control study was conducted in Department of (IQR) as reference. In all tests of significance, two sided p value Biochemistry, Indira Gandhi Institute of Medical Sciences, Patna and have been reported with p-value significant at <0.05. Andaman and Nicobar Islands Institute of Medical Sciences, Port Blair on serum specimen collected for various diagnostic purposes RESULTS including fasting and post-prandial plasma glucose from July, 2018 Mean Fasting Plasma Glucose (FPG) levels were 86.72±9.29 mg/ to January, 2019. The study was approved by Institutional Ethics dL in normoglycemia, 164.81±15.59 mg/dL in Mild-moderate Committee (registration no. ECR/640/Inst/BR/2014/RR-17) and Hyperglycemia (MHG) and 256.71±52.67 mg/dL in Severe informed consent was taken for both cases and control. Power of Hyperglycemia (SHG). FPG levels were significantly increased in the study was 85% associated with 95% confidence interval. Fasting MHG and SHG compared to controls (p<0.001 and p<0.001, plasma glucose (FPG) was categorised as normoglycemic (80 to respectively). Mean Serum Total Carbohydrate (STC) levels 120 mg/dL), mildly to moderately hyperglycemic (121 to 200 mg/dL) and severe hyperglycemic (>200 mg/dL) [7]. A total of 145 subjects were significantly elevated in MHG and severe hyperglycemia were included in the study that were subdivided into three groups; SHG compared to controls (243±29.90 mg/dL in MHG Vs normoglycemic controls (n=54), mild-moderate hyperglycemia (n=44) 179.42±21.79 mg/dL in controls; p=0.01 and 323.26±60.75 and severe hyperglycemia (n=47). Hyperglycemic samples were mg/dL in SHG Vs 179.42±21.79 mg/dL in controls; p<0.001, obtained from freshly diagnosed type 2 diabetes mellitus subjects. respectively). Protein Linked Carbohydrate (PLC) levels were Individual that came to attend OPD for routine check-up were recruited not significantly increased in MHG compared to controls as controls and were matched with respect to age and gender with (146.38±24.67 mg/dL in MHG and 140.09±20.09 mg/dL in hyperglycemic cases. For both controls and hyperglycemic cases, controls; p=0.17) while it was significantly higher in SHG than only those subjects were included that aged between 18-65 years in controls (172.46±54.79 mg/dL and 140.09±20.09 mg/dL in with normal liver and renal function tests. Subjects with long term controls; p=0.002). Alterations in mg of Carbohydrate Per Gram medication, active smokers, alcoholic individuals, any concurrent of Protein (CPR) were statistically significant both in MHG and illness, pregnant females
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