Oxidation Of Human Serum Albumin Research Articles

Red Blood Cells Protect Albumin from Cigarette Smoke–Induced Oxidation

Different studies reported the presence of oxidized (carbonylated) albumin in the extravascular pool, but not in the intravascular one of cigarette smokers. In this study we attempted to explain this apparent discrepancy exposing human serum albumin (HSA) to aqueous cigarette smoke extract (CSE). CSE induces HSA carbonylation and oxidation of the HSA Cys34 sulfhydryl group. An antioxidant action of glutathione, cysteine, and its synthetic derivative N-acetylcysteine was observed only at supra-physiological concentrations, suggesting that physiological (plasma) concentrations of glutathione and cysteine in the low micromolar range are ineffective in preventing cigarette smoke–induced oxidation of HSA. Differently, human erythrocytes resulted to be protective towards CSE-induced oxidation (carbonylation and thiol oxidation) of both HSA and total human plasma proteins.

Hypochlorite scavenging activity of hydroxycinnamic acids evaluated by a rapid microplate method based on the measurement of chloramines.

Scavengers of hypochlorite (XOCl) could have beneficial effects in diseases in which this oxidant plays a pathogenic role. It has been reported that ferulic acid and chlorogenic acid, the quinic ester of caffeic acid, are good hypochlorite scavengers, but a systematic evaluation of the naturally occurring hydroxycinnamic acids (HCAs), which these substances belong to, has not been performed yet. Thus, in this work we studied, by two different in-vitro methods, the antioxidant activity of five HCAs: p-coumaric acid, ferulic acid, sinapinic acid, caffeic acid and chlorogenic acid. The methods applied in this study were based on the oxidation of human serum albumin (HSA) by XOCl, a new microplate method based on the measurement of chloramines and a previously described carbonyl assay. Firstly, lysine-derived chloramines, in the presence or absence of the HCAs, were detected using 5-thio-2-nitrobenzoic acid (TNB), measuring the absorbance at 415 nm by a microplate reader. To remove excess XOCl, Trolox, a known XOCl scavenger, was added before TNB. Secondly, lysine-derived carbonyls, in the presence or absence of the HCAs, were detected by using 2,4-dinitrophenylhydrazine. Hydroxycinnamic acids appeared active (caffeic >/= sinapinic > chlorogenic congruent with ferulic > p-coumaric acid) by both methods, suggesting possible pharmacological applications for these compounds, which are present at high concentrations in the plant kingdom.

Antioxidant properties of some different molecular weight chitosans

Chitosan, a cationic polysaccharide, is widely employed as dietary supplement and in pharmacological and biomedical applications. Although numerous studies have focused on its applications as pharmaceutical excipients or bioactive reagents, relationships between molecular weight (Mr) and biological properties remain unclear. The focus of this study was on the antioxidant properties of several Mr chitosans. We measured the ability of seven Mr chitosans (CT1; 2.8 kDa, CT2; 17.0 kDa, CT3; 33.5 kDa, CT4; 62.6 kDa, CT5; 87.7 kDa, CT6; 604 kDa, CT7; 931 kDa) to protect plasma protein from oxidation by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). A comparison of the antioxidant action of high Mr chitosans (CT6–CT7) with that of low Mr chitosans (CT1–CT5) showed that low Mr chitosans (CT1–CT5) were more effective in preventing the formation of carbonyl groups in plasma protein exposed to peroxyl radicals. AAPH substantially increases plasma protein carbonyl content via the oxidation of human serum albumin (HSA). We also measured the ability of these chitosans to protect HSA against oxidation by AAPH. Low Mr chitosans (CT1–CT5) were found to effectively prevent the formation of carbonyl groups in HSA, when exposed to peroxyl radicals. Low Mr chitosans were also good scavengers of N-centered radicals, but high Mr chitosans were much less effective. We also found a strong correlation between antioxidant activity and the Mr of chitosans in vitro. These activities were also determined by using the ‘TPAC’ test. These results suggest that low Mr chitosans (CT1–CT3) may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patients plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia.

Plasma Oxidation-Reduction Potential and Protein Oxidation in Traumatic Brain Injury

The amount of oxidative stress in patients with an isolated traumatic brain injury (ITBI) can be estimated by measuring several biochemical parameters, such as total antioxidants, lipid peroxidation, protein oxidation, and others. Unfortunately, measuring these parameters is time-consuming, impractical in a clinical setting, and may miss important factors contributing to the overall redox balance. Here we suggest that the overall oxidative status in ITBI patients can be assessed by measuring plasma oxidation-reduction potential (ORP). Daily whole blood samples were obtained from severe ITBI patients (abbreviated injury score [AIS] >or=3, n = 32), and demographically similar non-head injury traumatized patients (n = 26) until discharge. Whole blood was also collected from patients with minor to moderate ITBI (AIS <or= 2, n = 18) and healthy volunteers (n = 22). Admission plasma ORP was significantly elevated in all traumatized patients compared to controls. Maximum ORP was detected on day 6 for severe ITBI and non-head injury traumatized patients. However, maximum ORP values were significantly higher (p < 0.05) in the severe ITBI group (+8.5 mV +/- 3.4 SEM) compared to the non-head injury group (-5.2 mV +/- 2.9 SEM). Additionally, a significantly higher oxidation of human serum albumin (HSA) was measured in all trauma patients compared to controls. These results demonstrate the presence of an oxidative environment in the plasma of traumatized patients, specifically in severe ITBI patients. Therefore monitoring ORP is a potentially useful tool for assessing the degree of oxidative stress, inflammation, severity of injury, and potential efficacy of treatment in ITBI patients.

The Effect of Storage on the Accumulation of Oxidative Biomarkers in Donated Packed Red Blood Cells

Transfusion-related acute lung injury (TRALI) is a life-threatening condition characterized by oxidative stress. Longer storage times of packed red blood cells (PRBC) and other blood products have been implicated with an increased risk in developing TRALI in transfused patients. A total of 10 units of blood containing PRBC stored in citrate-phosphate-dextrose buffer at 4 degrees C were included in the study. At Bonfils Blood Center (Denver, CO), samples were collected on storage day 1 and day 42. Samples were immediately centrifuged, and the supernatants were collected and stored at -80 degrees C until further analysis. Oxidation-reduction potential and protein oxidation were measured in both the day 1 and day 42 samples. Oxidation-reduction potential significantly increased (p < 0.05) in the day 42 sample (98.1 mV +/- 21.9 SD) versus the day 1 sample (62.6 mV +/- 21.5 SD). The oxidation of human serum albumin increased by 63.6% during the storage time. Other serum proteins such as apolipoprotein A1 and transthyretin demonstrated similar increases in oxidation. Also, proteins with a cleaved C-terminal amino acid were observed indicating the presence of carboxypeptidase activity, a marker of inflammation. The presence of an oxidative environment in transfused PRBC increases with storage time. This could partially explain the increased risk of developing TRALI related to the transfusion of older blood products.

Role of Cu2+ in Free Radical Oxidation of Human Serum Albumin and L-Tyrosine Dipeptide with Multicomponent Metal-Containing Xenobiotic

Cu(2+) entering the composition of multicomponent metal-containing xenobiotic in vitro initiated free radical oxidation of human serum albumin and L-tyrosine dipeptide. Oxidative modification was accompanied by the formation of derivatives of amino acids tyrosine and tryptophan and structural changes in human serum albumin.

Stabilizing mechanisms in commercial albumin preparations: octanoate and N-acetyl- l-tryptophanate protect human serum albumin against heat and oxidative stress

The capability of octanoate, N-acetyl- l-tryptophanate ( N-AcTrp) and other ions of fatty acids and amino acids to stabilize human serum albumin (HSA) against thermal and oxidative stress was studied. Native-PAGE showed that octanoate, and more hydrophobic fatty acids anions, stabilizes the monomeric form of HSA during heating at 60 °C for 30 min. Heating in the presence of octanoate did not change the far-UV CD-spectrum. The stabilizing role of octanoate also showed as an increase in denaturation temperature and calorimetric enthalpy, determined by differential scanning calorimetry (DSC). N-AcTrp, which was found to compete with octanoate for a common high-affinity site, has only a minor stabilizing effect. By contrast, no effect was found for l-tryptophanate or N-acetyl- l-cysteinate. Any ligand effect on oxidation was examined by using 2,2′-azobis(2-amidino-propane)dihydrochloride (AAPH) as oxidizing agent. One hour of incubation resulted in the formation of the same number of carbonyl groups, whether octanoate or one of the abovementioned amino acids was present or not. However, the number of groups formed after 24 h of incubation was significantly decreased in the presence of l-tryptophanate and, especially, N-AcTrp. The effect of 1-h incubation with AAPH on the oxidative status of 34-Cys was studied by the HPLC technique. It was found that N-AcTrp, but not octanoate, has a large protecting effect on the sulfhydryl group. Thus, octanoate has the greatest stabilizing effect against heat, whereas the presence of N-AcTrp diminishes oxidation of HSA.

Validation of the chloramine-T induced oxidation of human serum albumin as a model for oxidative damage in vivo.

The validity of using chloramine-T as a model compound for mimicing oxidative stress was examined using human serum albumin (HSA) as a model. Important sites of oxidation were studied by mild treatment with chloramine-T and by mutating 34Cys for a serine (C34S). High-performance liquid chromatography (HPLC) combined with fluorescence detection to confirm the validity of chloramine-T as an oxidizing agent was used. Oxidized amino acid residues were detected by reaction with 5,5'-dithiobis(2-nitro benzoic acid), digestion with cyanogen bromide, followed by capillary electrophoresis. Protein conformation was examined by spectroscopic techniques. From the HPLC analysis of human serum, the validity of using chloramine-T as an oxidizing agent was confirmed. At low chloramine-T concentrations (CT0.1-HSA, CT1-HSA), 34Cys and Met residues were oxidized, at medium concentrations (CT10-HSA), the tryptophan residue also appeared to be oxidized, and at the highest concentration (CT50-HSA), the net charge of Site II of HSA was found to be more negative. The two highest levels of oxidation of HSA (CT10-HSA, CT50-HSA) resulted in conformational changes with an increased exposure of hydrophobic regions, decreased high-affinity bindings of warfarin and ketoprofen and a reduced esterase-like activity. The latter protein also has a shorter plasma half-life and an increased liver clearance. We succeeded in imitating oxidative damage to HSA using chloramine-T and the findings show that Site II is more affected than Site I and 34Cys, when HSA is exposed to oxidative stress.

Effect of oxidative stress on the structure and function of human serum albumin.

Human serum albumin (HSA) was mildly oxidized by a metal-catalyzed oxidation system (MCO-HSA), chloramine-T (CT-HSA) or H2O2 (H2O2-HSA), and the effects of these treatments on the structural, drug-binding and esterase-like properties were studied. Protein conformation was examined by calorimetric, chromatographic, electrophoretic and spectroscopic techniques. Drug binding was studied by ultrafiltration method, and esterase-like activity was determined using p-nitrophenyl acetate as a substrate. Far-UV and near-UV CD spectra indicated that significant structural changes had occured as the result of treatment with MCO-HSA and CT-HSA but not with H2O2-HSA. However, SDS-PAGE analysis does not provide precise information on gross conformational changes such as fragmentation, cross-linking and SDS-resistant polymerisation. The results of differential scanning calorimetry, the fluorescence of the hydrophobic probe 1,1-bis-4-anilino-naphthalene-5,5-sulfonic acid and the elution time from a hydrophobic HPLC column indicated that MCO-HSA and CT-HSA in particular, have a more open structure and a higher degree of exposure of hydrophobic areas than unoxidized HSA. In all cases, high-affinity binding of warfarin remained unchanged for all the oxidized HSAs. However, high-affinity binding of ketoprofen to CT-HSA and, especially, MCO-HSA was diminished. In addition, the esterase-like activity of these proteins were all decreased to the same low level. Mild oxidation of HSA has no detectable effect on the binding of drugs to site I in subdomain IIA. In contrast, both the ligand binding property of site II and the esterase-like activity of oxidized HSAs are decreased, most probably due to conformational changes in subdomain IIIA.

Kinetics of Peroxynitrite Reaction with Amino Acids and Human Serum Albumin

An initial rate approach was used to study the reaction of peroxynitrite with human serum albumin (HSA) through stopped-flow spectrophotometry. At pH 7.4 and 37 degreesC, the second order rate constant for peroxynitrite reaction with HSA was 9.7 +/- 1.1 x 10(3) M-1 s-1. The rate constants for sulfhydryl-blocked HSA and for the single sulfhydryl were 5.9 +/- 0.3 and 3.8 +/- 0.8 x 10(3) M-1 s-1, respectively. The corresponding values for bovine serum albumin were also determined. The reactivity of sulfhydryl-blocked HSA increased at acidic pH, whereas plots of the rate constant with the sulfhydryl versus pH were bell-shaped. The kinetics of peroxynitrite reaction with all free L-amino acids were determined under pseudo-first order conditions. The most reactive amino acids were cysteine, methionine, and tryptophan. Histidine, leucine, and phenylalanine (and by extension tyrosine) did not affect peroxynitrite decay rate, whereas for the remaining amino acids plots of kobs versus concentration were hyperbolic. The sum of the contributions of the constituent amino acids of the protein to HSA reactivity was comparable to the experimentally determined rate constant, where cysteine and methionine (seven residues in 585) accounted for an estimated 65% of the reactivity. Nitration of aromatic amino acids occurred in HSA following peroxynitrite reaction, with nitration of sulfhydryl-blocked HSA 2-fold higher than native HSA. Carbon dioxide accelerated peroxynitrite decomposition, enhanced aromatic amino acid nitration, and partially inhibited sulfhydryl oxidation of HSA. Nitration in the presence of carbon dioxide increased when the sulfhydryl was blocked. Thus, cysteine 34 was a preferential target of peroxynitrite both in the presence and in the absence of carbon dioxide.

Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging.

Mild oxidative stress, as elicited by ascorbate, oxygen, and trace metals, affects the binding properties of human serum albumin via purely conformational changes. In fact, no gross alteration can be observed in the electrophoretic and chromatographic patterns of albumin, whereas localized modifications are indicated by the changes in absorption and fluorescence spectra and in polarization degree. The oxidized protein presents a small increase of bityrosine production and a time-dependent increase in the content of carbonyl groups, whereas proteolytic susceptibility is unchanged. A higher affinity for cis-parinaric acid and a slight loss of solubility in high salt indicate a greater surface hydrophobicity. Pinpoint denaturation of the albumin molecule is also suggested by a decreased "esterase" activity in the presence of p-nitrophenyl acetate. Conformational stability evaluated through thermal shock and addition of moderate amounts of guanidine indicate that the oxidized protein is more heat-resistant, less flexible, and more rigid than the native one. Although limited, structural damages afforded by the oxidative stress cause alterations of albumin binding properties as documented by experiments with probes and physiological ligands. The loss of biological activity of human serum albumin induced by ascorbate system appears of medical relevance, because it can affect drug metabolism and particularly drug tolerance in the elderly.