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

Abstract

Introduction: Non-enzymatic glycation and enzymatic glycosylation of proteins have been proposed as a major factor in the pathogenesis of diabetic complications by altering structure-function of proteins. Though focus of previous research is primarily focussed on glycated adducts as glycated haemoglobin (HbA1c), Advanced Glycation End Products (AGEs) and glycated albumin. The effect of enzymatic glycosylation has been overlooked in monitoring of diabetes. Aim: The present study examines the relationship between glycemic status and serum levels of protein linked total carbohydrate using inexpensive and rapid methods. Materials and Methods: The present case-control study was conducted on 145 subjects that were divided into three groups i.e., normoglycemic controls (n=54), mild-moderate hyperglycemia (MHG) (n=44) and severe hyperglycemia (SHG) (n=47). Serum was subjected to precipitation and then precipitate was re-dissolved in urea/thiourea buffer. Protein content of re-dissolved precipitate was measured using Bradford assay. Serum total carbohydrate and carbohydrate content in resuspended pellet (mg carbohydrate per gram of protein designated as Carbohydate Protein Ratio or CPR) was measured using phenol-sulfuric acid method. Statistical analysis was done using SPSS statistics (V21.0) and Microsoft excel software, viz. Student-t-test for comparison of significant mean, binary logistic regression was used to measure Odds Ratio (OR). In all tests of significance, two sided p value have been reported and results were considered significant if p<0.05. Results: Serum total carbohydrate and CPR were significantly increased in MHG (p<0.001 and p=0.02, respectively) and SHG (p<0.001 and p<0.001, respectively) compared to controls. CPR levels increased with increase in glycemia i.e., lowest in controls, higher in MGH and highest in SHG. We also observed a statistically significant upward trend from 1st quartile through 4th quartile for CPR in SHG (p<0.001). Likewise OR for 4th quartile compared with inter-quartile range (IQR) as reference was statistically significant for SHG {OR (95%CI)=5.27 (2.09-13.25); p<0.001}. Conclusion: Glycated Haemoglobin (HbA1c) concentrations and self monitoring of blood glucose are widely used for monitoring of long term and short term glycemic control respectively, although overall glycation and glycosylation status of circulating or tissue proteins could also be affected by glycemic status of an individual. Therefore, it is desirable that an assay or biomarker would provide assessment of overall glycation/glycosylation process in between immediate and long term control. In this preliminary case-control study we found protein linked carbohydrates (PLC) levels and carbohydrate protein ratio (CPR) to be significantly elevated in SHG compared to controls (p<0.001 and p<0.01, respectively). The alterations in PLC levels and CPR may be attributed to difference in glycemic status between these two groups. The method used in current study is inexpensive, rapid and technically less demanding thus suitable for regions having limited resources.

Keywords

• advanced glycation end products
• glycoprotein
• glycosylation
• hyperglycemia