Date Published: March 19, 2018
Publisher: Public Library of Science
Author(s): Anna Pieniazek, Lukasz Gwozdzinski, Zbigniew Zbrog, Krzysztof Gwozdzinski, Rodrigo Franco.
In chronic hemodialyzed (CH) patients the balance between production of reactive oxygen species and antioxidant defense system is disturbed and shifted towards oxidative conditions. The properties of albumin in CH patients were studied before hemodialysis (HD) and post-HD.
Two oxidants were applied, organic t-butyl hydroperoxide (t-BOOH) and inorganic hydroperoxide (H2O2), for oxidation of albumin molecules. By comparison, albumin from healthy donors was also modified by both oxidants. The thiol content in albumin was determined by the Ellman method. Albumin properties were evaluated with the spin labelling technique using two covalently bound spin labels, maleimide (MSL) and iodoacetamide (ISL), and fatty acid spin probe, 16-doxylstearic acid (16-DS).
A decrease in thiols level in HD albumin was greater than in control albumin. The t-BOOH modified the microenvironment at the binding site of MSL and ISL in control albumin molecules to a greater extent than hydrogen peroxide. Control albumin treated with t-BOOH and H2O2 showed an increase in the mobility of 16-DS. However, no changes were observed in albumin from CH patients treated with either of the oxidizing agents.
Both oxidants induced strong conformational changes in albumin from healthy volunteers, but were less effective or ineffective in modification of albumin derived from CH patients. These results show that albumin from CH patients is highly modified in vivo and is not vulnerable to oxidation in the same way as normal albumin.
Reactive oxygen species (ROS), including free radicals, have been suggested to contribute to many pathological conditions such as atherosclerosis, stroke, ischemia-reperfusion injury, cancer and others. Chronic hemodialysis (CH) is also associated with oxidative stress [1,2]. Oxidative stress in patients on chronic hemodialysis is manifested by an increase in lipid and protein oxidation in blood plasma, and lipid peroxidation products and protein carbonyl formation have been found in the plasma of these patients [3,4]. Hemodialyzed patients have decreased levels of antioxidant defense [5,6]. Antioxidant enzyme activities are generally lower and levels of low molecular weight antioxidants such as GSH, α-tocopherol and ascorbic acid were also lower . Patients hemodialyzed using unmodified cellulose membranes develop activation of neutrophils and monocytes, which leads to respiratory burst and the release of highly reactive oxygen free radicals [8,9]. Higher levels of toxic oxygen species and impaired antioxidant defense lead to damage of blood components [2,8]. Additionally, the higher level of urea in the blood of chronic hemodialyzed patients leads to carbamylation of proteins, glycoproteins, and other components in the plasma .
In our study, applied concentrations of hydrogen peroxide or t-BOOH did not oxidize all thiol groups in albumin. The thiol content in albumin samples was determined by spectrophotometric measurements using Ellman’s method. The obtained data showed the effect of hemodialysis on the level of –SH groups in CH patients. After HD the level of thiols was significantly decreased (approx. 15%). As expected, both oxidants led to a significant decrease in the thiol groups in albumin. However, a significantly greater decrease in thiol content was observed following t-BOOH treatment than H2O2 treatment, at 23% and 10% respectively in control (normal) albumin (Fig 1). In albumin from CH patients an even higher decrease was found, 33% upon t-BOOH treatment and 18% upon H2O2 treatment before hemodialysis, and 31% and 24%, respectively after hemodialysis.
Albumin plays a key role in the regulation of osmotic pressure, and the binding and transport of various substances including bilirubin, steroids, fatty acids and drugs. The Cys-34 in HPA is the most abundant plasma thiol which has central contribution of albumin redox processes . This protein also plays an important role as a scavenger of reactive oxygen species and as a transition metal binding protein, mainly copper . Albumin may be oxidatively modified in vivo in chronic kidney disease (hemodialyzed patients), diabetes mellitus liver disease and other diseases.
In this work we showed strong impact of both oxidants on conformational state of albumin only from healthy volunteers. These results showed that albumin from hemodialyzed patients is highly modified in vivo and is not vulnerable to oxidation as albumin from healthy volunteers in vitro. The different mechanism of action of both oxidants may be related to their different hydrophobicity.