The influence of fatty acids on determination of human serum albumin thiol group.

During investigation of the changes of the Cys34 thiol group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11-33%) than the total serum thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10(-3), 21.6×10(-3), and 11.2×10(-3) s(-1), respectively. The HSA-SH group of diabetics is more reactive compared with control individuals (rate constants 20.9×10(-3)±4.4×10(-3) vs 12.9×10(-3)±2.6×10(-3) s(-1), P<0.05). Recovery values of the SH group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum thiols, which make the determination of the thiol group unreliable.

Improving the reliability of human serum albumin-thiol group determination.

The thiol (Cys34) content of human serum albumin (HSA-SH) decreases during oxidative and carbonyl stress and, therefore, could represent a useful parameter in clinical practice. Nevertheless, the reliability of HSA-thiol determination with Ellman's method depends on the purity of isolated HSA. Determination of total serum thiols (mmol/L) and HSA-SH content (mmol -SH/mmol HSA) after HSA isolation from diabetic patient and control sera by a two-step precipitation with ammonium sulfate (AS), as well as HSA-SH contribution (%) to total serum thiols, was assessed. Purity and yield of isolated HSA were monitored spectrophotometrically and by native polyacrylamide gel electrophoresis. Precipitation of HSA from serum via a two-step method with AS produced HSA with 91.9±3.6% purity and 69.7±4.4% yield, allowing for precise (relative standard deviation of 3.2%) and reliable (comparing with total serum thiols) measurement of HSA-SH content with DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)]. The content of the HSA-SH group in patients with type 2 diabetes was significantly (P<0.05) lower compared with that of the healthy cohort (0.483±0.067 vs. 0.561±0.054 mmol -SH/mmol HSA). Because the proposed method of HSA isolation is simple, time-efficient, and technically less demanding, and it also enables reliable determination of HSA-SH content, it is suitable for clinical practice.

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

Carbonylation of the protein amino, guanidine, and thiol groups with α-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 ~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 < 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.