Association of Serum Free Haemoglobin in Normoglycemic and Hyperglycemic Individuals

Abstract

Introduction: Besides glycation induced injury, hyperglycemia in both type 1 and 2 diabetes can also increase oxidative stress leading to alterations in erythrocytic structural-functional organisation, shortening of life span and eryptosis. Free Haemoglobin (fHb) is released in circulation that could cause deleterious clinical effects. No study has associated fHb with hyperglycemia till now, thus it is pertinent to examine this aspect. Aim: The present study has associated serum fHB with hyperglycemia by simple, rapid, inexpensive spectrophotometric assay using Allen correction. Materials and Methods: The present case control study consisted of a total of 74 participants grouped into normoglycemic controls (n=37) and hyperglycemic subjects (n=37). Serum sample were analysed for haemoglobin (Hb) concentration by direct spectrophotometry using Allen correction. Concentration of Hb was calculated by Harboe’s Allen correction as Hb (mg/L)=1.68.A415-0.84.A380-0.84.A450 (absorbance in milliabsorbance). Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version V21.0 and Microsoft excel software employing independent unpaired student’s-t-test and pearson’s correlation coefficient and χ2 test for trend while Odds Ratio (OR) and corresponding 95% CI was calculated using binary logistic regression. Results: Mean serum fHb levels were significantly elevated in hyperglycemia (101.74±61.73 mg/L; p=0.001) as compared to controls (58.44±39.69 mg/L). A dose dependent relationship was evident between increased serum fHb with hyperglycemia (p<0.001) and OR was statistically significant in hyperglycemia [OR (95% CI)=2.95 (1.05–8.31); p=0.04]. Serum fHb concentration was positively and significantly correlated with fasting plasma glucose (FPG) (r=0.34, p=0.003). Conclusion: Hyperglycemia causes glycation of tissue and circulating proteins as well as oxidative stress to erythrocyte membrane leading to alterations in erythrocytic membrane fluidity and shortening of life span. Serum fHb is widely used as marker of intravascular haemolysis and fHb is estimated by immunoassay that is expensive and cyanide based spectrophotometry that is toxic. The current study has associated fHb with hyperglycemia with direct spectrophotometry using Allen correction that uses minimal reagents and is non-toxic besides being inexpensive. We found that levels of fHb were significantly increased in hyperglycemia than controls (p<0.05) and fHb was positively and significantly correlated with FPG (p=0.003). The findings of our study suggests that erythrocytes are susceptible to hyperglycemic injury and fHb could be estimated inexpensively as marker of haemolysis.

Keywords

• advanced glycation end products
• diabetes
• eryptosis
• haemolysis