Monitoring of the human serum albumin carbonylation level through determination of guanidino group content

Abstract

Carbonylation of the protein amino, guanidine, and thiol groups with alpha-oxoaldehydes (which are produced in higher quantities in diabetes, uremia, oxidative stress, aging, and inflammation) is one of the important causes of vascular complications. For monitoring of the human serum albumin (HSA) carbonylation level, a spectrophotometric method based on the formation of colored adduct between guanidine group and thymol-sodium hypobromite reagent in the alkaline medium was investigated. Beer's law is obeyed in the concentration range of Arg and protein guanidine groups from 1 to 40 mM. Precision of the method (relative standard deviation) was in the range of 0.9 to 2%. Accuracy was examined by the standard addition method (recovery similar to 100%). The method was applied for monitoring of the carbonylation level of HSA with methylglyoxal in vitro and of HSA isolated (using affinity chromatography) from sera of 21 patients with type 2 diabetes and 12 healthy persons. The content of guanidine groups in HSA isolated from diabetics (19.64 +/- 1.07 mM/mM albumin) was significantly lower (P lt 0.001) in comparison with a control group (21.87 +/- 1.02 mM/mM albumin). The method is simple and fast, has good accuracy and precision, and is suitable for clinical practice as well for in vitro protein carbonylation experiments.