THE EFFECT OF METFORMIN ON NON-ENZYMATIC GLYCOSYLATION OF RECOMBINANT HUMAN SERUM ALBUMIN
Keywords:
Recombinant human serum albumin (rHSA), Metformin hydrochloride, glycosylation of albumin, Protein binding, LC-MSMSAbstract
Objective: The present research work was aimed to observe the effect of Metformin hydrochloride (MET) on non-enzymatic glucosylation of recombinant human serum albumin (rHSA).
Methods: Albumin was incubated at 37 ° in 10 mM phosphate buffer (pH 7.4) using 0.2% sodium azide for 7 d with different concentrations of D-(+)-glucose and MET. Incorporation of D-(+)-glucose (5-12 mM) into rHSA was calculated in the presence of metformin hydrochloride (1-5 µg/ml) following extensive dialysis of the incubation mixture. The binding experiments were exercised at respective plasma concentration of rHSA, D-(+)-glucose and MET in corresponding to that of diabetic and non-diabetic population. Glycosylation of rHSA was quantified using 2-thiobarbituric acid (2-TBA). Whereas, metformin hydrochloride was quantified using a validated LC-MS/MS method with negative ion electrospray ionization using multiple reactions monitoring (MRM) mode. MET was eluted isocratically on X-Bridge BEH Phenyl column with a mobile phase consisting of mixture of water (containing 50 mM ammonium acetate) and acetonitrile in a ratio of 70:30.
Results: The weighted (1/X2) calibration curve from 20 to 10, 000 ng/ml was employed for the calculation ofMET in sample with line equation of Y= 0.0020584x+0.0037475. The binding pattern ofMET to rHSA, was specific and pH dependent, possibly due to a change in ionization state of MET and change in conformation of rHSA during the binding process.
Conclusion: All the described parameters were in accordance to the FDA Guideline (inside 85-115% for the accuracy and less than 15% for the precision), thus it can be concluded that the bioanalytical method is were fully validated as per USFDA guideline. These experiments showed the affinity ofMETtoward the rHSA proved to be higher than its affinity toward the glucose.
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