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During pasteurization and storage of albumin products, Sodium octanoate (Oct) and N-acethyl-l-tryptophan (N-AcTrp) are used as the thermal stabilizer and the antioxidant for human serum albumin (HSA), respectively. We recently reported that N-acethyl-l-methionine (N-AcMet) is an antioxidant for HSA, which is superior to N-AcTrp when it is especially exposed to light during storage. The objective of the present study is to clarify the molecular mechanism responsible for the HSA protective effect of Oct and N-AcMet based on their ternary complex structure. Crystal structure of the HSA-Oct-N-AcMet complex showed that one N-AcMet molecule is bound to the entrance of drug site 1 of HSA, and its side chain, which is susceptible to the oxidation, is exposed to the solvent. At the same time, two Oct binding sites are observed in drug sites 1 and 2 of HSA, respectively, and each Oct molecule occupies the hydrophobic cavity in them. These results indicate the molecular mechanism responsible for the HSA stabilization by these small molecules as follows. N-AcMet seals the entrance of drug site 1 while it acts as an antioxidant for HSA. Oct is chiefly bound to drug site 2 of HSA and it increases the thermal stability of HSA because of the occupying the largest intra-cavity of sub-domain IIIA in HSA. These findings suggest that N-AcMet acts positively as useful stabilizer for albumin formulated products such as functionalized HSA and HSA fusion proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Silibinin is the main constituent of silymarin, an extract from the seeds of milk thistle (Silybum marianum). Because silibinin has many pharmacological activities, extending its clinical use in the treatment of a wider variety of diseases would be desirable. In this study, we report on the binding of silibinin to plasma proteins, an issue that has not previously been extensively studied. The findings indicated that silibinin mainly binds to human serum albumin (HSA). Mutual displacement experiments using ligands that primarily bind to sites I and II clearly revealed that silibinin binds tightly and selectively to site I (subsites Ia and/or Ic) of HSA, which is located in subdomain IIA. Thermodynamic analyses suggested that hydrogen bonding and van der Waals interactions are major contributors to silibinin-HSA interactions. Furthermore, the binding of silibinin to HSA was found to be decreased with increasing ionic strength and detergent concentration of the media, suggesting that electrostatic and hydrophobic interactions are involved in the binding. Trp214 and Arg218 were identified as being involved in the binding of silibinin to site I, based on binding experiments using chemically modified- and mutant-HSAs. In conclusion, the available evidence indicates that silibinin binds to the region close to Trp214 and Arg218 in site I of HSA with assistance by multiple forces and can displace site I drugs (e.g., warfarin or iodipamide), but not site II drugs (e.g., ibuprofen).

On account of its long circulating half-life, human serum albumin (HSA) is susceptible to posttranslational modifications that can alter its functions. Here, we comprehensively compared the degree of posttranslational modifications with the functional impairment of HSA derived from 5 different commercially available albumin preparations and clarified their relationships. We used electrospray ionization-time of flight mass spectrometry to evaluate the degree of posttranslational modification of the entire HSA molecule that was associated with disease development and found that the fraction of Cys34-cysteinylated HSA (Cys-Cys34-HSA), a major form of oxidative modification, varied substantially among the albumin preparations. Meanwhile, no remarkable difference was found in the degree of glycated or N-terminal truncated HSA among the preparations tested. The nonosmotic pressure maintenance functions of HSA, such as its antioxidative and ligand-binding activities significantly differed among the preparations. Interestingly, the alternations of these functions showed a significantly negative correlation only with the Cys-Cys34-HSA fraction. These findings suggest that the Cys-Cys34-HSA fraction, as estimated by electrospray ionization-time of flight mass spectrometry can be used as a predictive marker for the functional impairment of albumin preparations and that it would be preferable to use albumin preparations with higher contents of functionally effective albumin that correspond to a lower degree of cysteinylation of Cys34 in clinical practice., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Aims: To determine molecular information about the antioxidant properties of human serum albumin, which is an important extracellular antioxidant. To obtain this information, we studied this function of the protein by using H2O2 as the representative reactive oxygen species and two recombinant mutants and ten genetic variants with single-residue mutations., Main Methods: The antioxidant capabilities of the isoforms were registered as their ability to diminish the H2O2-induced conversion of dihydrorhodamine 123 to rhodamine 123, which can emit fluorescence at 536 nm. Structural properties were examined by circular dichroism and SDS-PAGE., Key Findings: Cysteine residues are important for the antioxidant function, but their effect depends on their position in the protein, with Cys410 > Cys34 ~ Cys169 (when not involved in forming a disulfide bond). Likewise, the substitution of a glutamic acid at position 122 or 541, but not at 240 or 560, improves the antioxidant effect, perhaps by making the methionine residues in their vicinity, Met123 and Met548, respectively, more accessible for the oxidant. A lysine at position 505, but not at 82 or 570, decreases the oxidative effect. Finally, the mutations D269G and K276N had no effect. In certain cases, albumin acts as a sacrificial antioxidant, as in the case of the mutants C34S and, in particular, R410C and E505K., Significance: The information gained is of protein chemical relevance, but it may also be helpful in understanding the function of proteins that act as antioxidants in biological systems subjected to oxidative stress in conditions such as inflammation and aging., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: The half-life of fatty acid-conjugated antidiabetic drugs are prolonged through binding to albumin, but this may not occur in diabetic patients with nephropathy complicated with hypoalbuminemia. We previously showed that human serum albumin (HSA) dimerized at the protein's Cys34 by 1,6-bis(maleimido)hexane has longer half-life than the monomer under high permeability conditions. The aim of this study was to investigate the superior ability of this HSA dimer as a plasma-retaining agent for fatty acid conjugated antidiabetic drugs., Methods: The diabetic nephropathy rat model was prepared by administering a single injection of streptozotocin (STZ) intravenously, and the pharmacokinetic properties of HSA monomer and dimer were evaluated. Site-specific fluorescent probe displacement experiments were performed using warfarin and dansylsarcosine as site I and site II specific fluorescent probes, respectively., Key Findings: The half-life of the HSA dimer in STZ-induced diabetic nephropathy model rats was 1.5 times longer than the HSA monomer. The fluorescent probe displacement experiment results for HSA monomer and dimer were similar, where fatty acid-conjugated antidiabetic drugs displaced dansylsarcosine but not warfarin in a concentration-dependent manner., Conclusions: The HSA dimer shows potential for use as a plasma-retaining agent for antidiabetic drugs due to its favourable pharmacokinetic properties., (© 2014 Royal Pharmaceutical Society.)

Background: Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products., Methods: Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration., Results: N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present., Conclusion: The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations., General Significance: N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA) clearly indicated that curcumin (Cur), demethylcurcumin (Dmc) and bisdemethoxycurcumin (Bdmc) bind to both Site I (sub-site Ia and Ib) and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds.

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.

Oxidative damage results in protein modification and is observed in numerous diseases. Human serum albumin (HSA), the most abundant protein in the plasma, exerts important antioxidant activities to prevent oxidative damage. This paper focuses on the characterization of chemical changes in HSA that are induced by oxidative damage, their relevance to human pathology, and the most recent advances in clinical applications. The antioxidant properties of HSA are largely dependent on Cys-34 and its contribution to the maintenance of intravascular homeostasis, including protecting the vascular endothelium under conditions related to oxidative stress. Recent studies have also evaluated the susceptibility of other important amino acid residues to exposure to free radicals. The findings suggest that a redox change in HSA is related to the oxidation of several amino acid residues by different oxidants. On the other hand, the ratio of the oxidized form to the normal form of albumin (HMA/HNA), which is a function of the redox states of Cys-34, could serve as a useful marker for evaluating systemic redox states, which would be useful for the evaluation of disease progression and therapeutic efficacy. This review provides new insights into our current understanding of the mechanism of HSA oxidation, based on in vitro and in vivo studies.

Background: Oxidative damage results in protein modification, and is observed in numerous diseases. Human serum albumin (HSA), the most abundant circulating protein in the plasma, exerts important antioxidant activities against oxidative damage., Scope of Review: The present review focuses on the characterization of chemical changes in HSA that are induced by oxidative damage, their relevance to human pathology and the most recent advances in clinical applications., Major Conclusions: The antioxidant properties of HSA are largely dependent on Cys34 and its contribution to the maintenance of intravascular homeostasis, including protecting the vascular endothelium under disease conditions related to oxidative stress. Recent studies also evaluated the susceptibility of other important amino acid residues to free radicals. The findings suggest that a redox change in HSA is related to the oxidation of several amino acid residues by different oxidants. Further, Cys34 adducts, such as S-nitrosylated and S-guanylated forms also play an important role in clinical applications. On the other hand, the ratio of the oxidized form to the normal form of albumin (HMA/HNA), which is a function of the redox states of Cys34, could serve as a useful marker for evaluating systemic redox states, which would be useful for the evaluation of disease progression and therapeutic efficacy., General Significance: This review provides new insights into our current understanding of the mechanism of HSA oxidation, based on in vitro and in vivo studies. This article is part of a Special Issue entitled Serum Albumin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The purpose of this study was to quantitatively evaluate the extent of contribution of cysteine-34 ((34) Cys) on the antioxidant effect of human serum albumin (HSA) and to elucidate the physiological implication in hemodialysis (HD) patients. The ratio of oxidized albumin correlated directly with the thiol content in plasma of the HD patients who received intravenous iron. Moreover, the degree of oxidation of (34) Cys in HSA purified from the HD patients' plasma correlated with the thiol content in plasma. The radical scavenging activity of purified HSA was dependent on the degree of (34) Cys oxidation. A recombinant mutant, C34S, was produced to confirm the role of (34) Cys. The activity of C34S was about half of that of wild-type HSA (WT-HSA). Consistent with this observation, the protective effects of C34S were significantly lower than those of WT-HSA in suppressing cytotoxicity and vascular endothelial growth factor production induced by fenton reaction in a human vascular endothelial cell model. These results indicate that the (34) Cys residue of HSA may account for more than 40% of the antioxidant effect of HSA in vivo, and thus may exert a protective effect on vascular endothelium function via an antioxidative mechanism in chronic renal disease., (Copyright © 2011 Wiley-Liss, Inc.)

Human serum albumin (HSA) is used clinically as a plasma expander in patients with hypoalbuminemia and can also function as a drug carrier. However, the administered HSA is readily eliminated from the blood circulation under pathological conditions, especially the nephrotic syndrome. In this study, we present data on the pharmacokinetics of a structurally defined HSA dimer [two HSA molecules that are cross-linked by reaction with 1,6-bis(maleimido)hexane via Cys34] in nephrotic rats and its superior circulation persistence, owing to the molecular size effect. The half-time (t(1/2)) of the HSA dimer persisted in the circulation 1.3 times longer than that of monomeric HSA in normal rats, primarily because of the suppression of the accumulation of the HSA dimer in the skin and muscle. In nephrotic rats, the t(1/2) of the HSA monomer decreased considerably, whereas the HSA dimer remained unaltered in the blood stream, similar to that for normal rats. As a result, the t(1/2) of the HSA dimer was 2-fold longer than that of the HSA monomer. This longer t(1/2) can be attributed to the fact that accumulation in the kidney and urinary excretion of the HSA dimer were significantly suppressed. The cross-linked HSA dimer shows a longer blood circulation than native HSA monomer in nephrotic rats, which can be attributed to the suppression of renal filtration and leakage into the extravascular space. This HSA dimer has the potential for use as a drug carrier, new plasma expander, and an artificial albumin-based oxygen carrier under a high glomerular permeability condition such as nephrosis.

Objectives: Intravenous iron administration (IVIR) is effective for correcting anemia in hemodialysis (HD) patients, but it also enhances the generation of hydroxyl radicals. Previously we demonstrated that IVIR increases oxidized serum albumin levels in HD patients. However, the effect of IVIR frequencies on the oxidative stress has never been studied before. Therefore, we compared the two IVIR schedules recommended by the Japanese Society for Dialysis Therapy guideline 2004 by measuring oxidized albumin in chronic HD patients., Design and Methods: Twenty-two HD patients were divided into two IVIR protocol groups (group I: 40 mg of iron 3 times a week for 4 weeks, group II: 40 mg of iron once a week for 3 months). These protocols differ in IVIR frequency, but receive the same amount of iron (total 520 mg). We compared these two regimens by determining the levels of hemoglobin, serum ferritin, advanced oxidation protein products (AOPP), and oxidized albumin at 0, 4, 8, 12, 16, and 20 weeks., Results: Both patient groups resulted in a similar and significant increase in hemoglobin levels, whereas group I markedly induced AOPP and oxidation of serum albumin than group II at 4 weeks (P<0.05). AOPP and oxidation of serum albumin was also gradually declined by 20 weeks, while the oxidized albumin and AOPP in group II was not significantly changed during the entire experimental period. Transferrin saturation and serum ferritin levels were also increased in group I compared with group II at 4 weeks (P<0.001). In addition, we found a strong positive correlation between oxidized albumin and serum ferritin levels (r=0.615, P<0.05), suggesting the possibility that the accumulation of iron stores has a causative role in the progression of oxidative stress in HD patients treated with IVIR., Conclusions: The results of this study indicate that lower frequency IVIR protocol is recommended to reduce IVIR-induced oxidative stress in HD patients.

Inhibition of protein oxidation by reactive oxygen species (ROS) would confer benefit to living organisms exposed to oxidative stress, because oxidized proteins are associated with many diseases and can propagate ROS-induced damage. We measured the ability of 2800Da chitosan, D-glucosamine and N-acetyl glucosamine to protect human serum albumin from oxidation by peroxyl radicals derived from 2,2'-azobis(2-amidinopropane)dihydrochloride and N-centered radicals from 1,1'-diphenyl-2-picrylhydrazyl and from 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Comparison with the antioxidant action of vitamin C showed that, on a molar basis, chitosan was equally effective in preventing formation of carbonyl and hydroperoxide groups in human serum albumin exposed to peroxyl radicals. It was also a potent inhibitor of conformational changes in the protein, assessed by absorption spectrum and intrinsic fluorescence. D-glucosamine was much less effective and N-acetyl glucosamine was not a useful antioxidant. Protection of the albumin from peroxyl radicals was achieved by scavenging of peroxyl radical. Chitosan was also a good scavenger of N-centered radicals, with glucosamine and N-acetyl glucosamine much less effective. The results suggest that administration of low molecular weight chitosans may inhibit neutrophil activation and oxidation of serum albumin commonly observed in patients undergoing hemodialysis, resulting in reduction of oxidative stress associated with uremia.

The pharmacokinetics of 17 genetic variants of human serum albumin with single-residue mutations and their corresponding normal albumin were studied in mice. In all cases, the plasma half-life was affected, but only variants with +2 changes in charge prolonged it, whereas changes in hydrophobicity decreased it. Good positive and negative correlations were found between changes in alpha-helical content taking place in domains I+III and domain II, respectively, and changes in half-lives. No correlation was found to type of mutation or to changes in heat stability as represented by DeltaH(v). Liver and kidney uptake clearances were also modified: alpha-helical changes of domains I+III showed good negative correlations to both types of clearances, whereas changes in domain II only had a good positive correlation to kidney uptake clearance. No correlation between the other molecular changes and organ uptakes was observed. The relatively few correlations between changes in molecular characteristics and the organ uptakes of the variants are most probably due to different handling by plasma enzyme(s) and the various types of cell endocytosis. Of the latter, most lead to destruction of albumin, but at least one results in recycling of the protein. The present information should be useful when designing recombinant, therapeutical albumins or albumin products with a modified plasma half-life.

Sodium octanoate (Oct) and N-acetyl-l-tryptophanate (N-AcTrp) are widely used as stabilizers during the pasteurization of albumin products. However, N-AcTrp has a possible side effect of intracerebral disease. To provide safe and risk-free albumin products, we validated N-acetyl-methioninate (N-AcMet) as a new stabilizer for albumin products. The effect of N-AcMet on oxidation was examined using 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) as an oxidizing agent. Carbonyl content in the presence of N-AcMet, as well as that in the presence of N-AcTrp after 24h (Anraku et al., 2004), was significantly decreased. The effect of AAPH on the oxidative status of 34-Cys on human serum albumin was also studied by HPLC. It was found that N-AcMet as well as N-AcTrp, has a large protective effect on the sulfhydryl group after 1h. Further, N-AcMet was found to be a superior radical scavenger to N-AcTrp using 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radicals. The thermal stabilizing role of N-AcMet manifested as an increase in denaturation temperature and calorimetric enthalpy, as determined by differential scanning calorimetry (DSC). In the present study, we suggest that use of N-AcMet in albumin preparation is safe and free of risk of side effects.

The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.

To determine the pharmacokinetic properties of advanced oxidation protein products (AOPP), we prepared oxidized human serum albumin (oxi-HSA) using chloramine-T (a hypochlorite analogue) in vitro. The AOPP and dityrosine content of oxi-HSA (AOPP content, 244.3+/-12.3 microM; dityrosine content, 0.7+/-0.11 nmol of dityrosine/mg protein) were similar to those of uremic patients. In structural analysis, the increases in AOPP and dityrosine content of HSA induced slight decreases in its alpha-helical content. In pharmacokinetic analysis, oxi-HSA left the circulation rapidly, and organ distribution of oxi-HSA 30 min after intravenous injection was 51% for the liver, 23% for the spleen, and 9% for the kidney, suggesting that the liver and spleen were the main routes of plasma clearance of oxi-HSA. The liver and spleen uptake clearance of oxi-HSA were significantly greater than those of normal HSA (CLliver, 5058+/-341.6 vs 24+/-4.2 microL/hr [p<0.01]; CLspleen, 2118+/-322.1 vs 32+/-2.7 microL/hr [p<0.01]). However, uptake by other organs was not significantly affected by oxidation. These results suggest that the liver and spleen play important roles in elimination of AOPP.

Hemodialysis (HD) patients are frequently in a state of increased oxidative stress, and hyperglycemia appears to be a major factor. We recently found that oxidized human serum albumin (HSA) is a reliable marker of oxidative stress in HD patients. However, the issue of whether oxidized HSA is associated with the progression of oxidative stress in HD patients with or without diabetes is not clear. In the present study, we examined the effect of a qualitative modification of HSA in HD patients with or without diabetes. Blood samples from 10 HD patients with diabetes, 7 HD patients without diabetes, and 10 healthy age-matched controls were examined. The increase in plasma protein carbonyl content and advanced glycation endproducts (AGEs) in HD patients was largely due to an increase in the levels of oxidized HSA. Furthermore, these increases were greatest in HD patients with diabetes. Purified HSA from HD patients (non-DM-HSA) was carbonylated and AGE-modified. The amount of modified HSA was the highest in HD patients with diabetes (DM-HSA). Carboxy methyl lysine (CML)-modified HSA triggered a neutrophil respiratory burst, and this activity was closely correlated with the increase in the CML/HSA ratio. These findings indicate that uremia plays an important role in the progression of oxidative stress in HD patients via an increase in CML-modified HSA. They also indicate that diabetic complications further exacerbate the progression of oxidative stress by further increasing the amount of these modified HSA molecules.

In the present study, we investigated the esterase-like activity of human serum albumin (HSA) and the mechanism by which it hydrolyzes, and thereby activates, olmesartan medoxomil (CS-866), a novel angiotensin II receptor antagonist. CS-866 has previously been shown to be rapidly hydrolyzed in serum in which HSA appeared to play the most important role in catalyzing the hydrolysis. We found that the hydrolysis of CS-866 by HSA followed Michaelis-Menten kinetics. Compared with the release of p-nitrophenol from p-nitrophenyl acetate (PNPA), CS-866 showed lower affinity to HSA and a lower catalytic rate of hydrolysis. Thermodynamic data indicated that PNPA has a smaller value of activation entropy (deltaS) than CS-866; consequently, PNPA is more reactive than CS-866. Ibuprofen and warfarin acted as competitive inhibitors of hydrolysis of CS-866, whereas dansyl-L-asparagine, n-butyl p-aminobenzoate, and diazepam did not. These findings suggest that the hydrolytic activity is associated to parts of site I and site II for ligand binding. All chemically modified HSA derivatives (Tyr-, Lys-, His-, and Trp-modifications) had significantly lower reactivity than native HSA; Lys-HSA and Trp-HSA had especially low reactivity. All the mutant HSAs tested (K199A, W214A, and Y411A) exhibited a significant decrease in reactivity, suggesting that Lys-199, Trp-214, and Tyr-411 play important roles in the hydrolysis. Results obtained using a computer docking model are in agreement with the experimental results, and strongly support the hypotheses that we derived from the experiments.

The effect of telmisartan on ambulatory blood pressure, plasma neurohormonal parameters, and oxidation of serum albumin has not been investigated in hemodialysis (HD) patients. Thirteen hypertensive HD patients were treated with 40 mg telmisartan once daily, and 24-h ambulatory blood pressure monitoring was performed after 0, 4, and 8 weeks of treatment. Plasma renin activity, plasma aldosterone concentration (PAC), brain natriuretic peptide (BNP) level, and serum oxidized albumin level were determined at the same time points. Serum telmisartan concentration was also measured at 4 and 8 weeks. Telmisartan significantly reduced systolic blood pressure and diastolic blood pressure (both awake and sleeping) after 4 weeks, and these pressures showed a further significant decrease after 8 weeks. Plasma levels of aldosterone, BNP, and serum oxidized albumin were markedly decreased after 4 weeks and these lower levels were maintained at 8 weeks. The trough serum telmisartan concentration was not significantly different at 8 weeks compared with 4 weeks. Throughout the treatment period, there were no significant adverse effects. Telmisartan effectively lowers blood pressure and reduces PAC, BNP, and oxidative stress and is safe and well-tolerated by HD patients. A long-term study in a large population is required to determine the influence of telmisartan therapy on cardiovascular mortality and morbidity in HD patients.

Oxidized albumin is a reliable marker of oxidative stress in hemodialysis (HD) patients. However, oxidized albumin in vivo and its possible clinical significance has been rarely investigated. In the present study, the qualitative modification of albumin in HD patients (n = 20) was examined and their results were compared with healthy age-matched controls (n = 10). The increase in plasma protein carbonyl levels in HD patients was largely due to an increase in oxidized albumin. Human serum albumin (HSA) of HD patients, HSA of HD patients (HD-HSA) and normal subjects (Normal-HSA) were purified on a blue Sepharose CL-6B column. Spectroscopic analysis confirmed that the HD-HSA samples contained higher levels of carbonyls than Normal-HSA. An HPLC analysis also suggested that the state of the purified HSA used throughout the experiments accurately reflects the redox state of albumin in blood. HD-HSA was found to have a decreased the antioxidant activity, and was able to trigger the oxidative burst of human neutrophils, compared to Normal-HSA. HD-HSA was conformationally altered, with its hydrophobic regions more exposed and to have a negative charge. In binding experiments, HD-HSA showed impaired Site II-ligand binding capabilities. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in HD patients.

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have Tm values (midpoint of denaturation) higher than, and nine have Tm values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have DeltaHv values (van't Hoff enthalpy) higher than, and 14 have DeltaHv values lower than, normal albumin. All types of combinations of positive and negative DeltaTm values and Delta(DeltaHv) values were found. Good linear correlations between mutation-induced changes of alpha-helical content and Delta(DeltaHv) values, but not DeltaTm values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on Tm and DeltaHv was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive DeltaTm values and negative Delta(DeltaHv) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.

The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6-bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pI: 4.8) and denaturing temperature (65 degrees C). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL(-1) dimer solution has the same COP with the physiological 5 g dL(-1) rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the 125I-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis[(1-methylcyclohexanamido)phenyl]porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin-heme hybrid in dimeric form. It can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O2-recombination reaction. The O2-binding affinity and the O2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated.

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 degrees 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 above mentioned 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.

The binding properties of the disulfide covalent bond between N-acetyl-L-cysteine (NAC) and human serum albumin (HSA) were investigated. HSA, purified from either healthy subjects or renal failure patients, was incubated with NAC in buffer and analyzed by 4VP-EG-Me column chromatography, which can distinguish between the redox states of the only free thiol of HSA. Although intact HSA was found to consist of mainly three sub-types, marcaptoalbumin (HMA), cysteine-bound nonmercaptoalbumin (HNA(Cys)) and a further oxidized form (HNA(oxy)), the formation of a new type of nonmercaptoalbumin (HNA(NAC)) was confirmed after incubation with NAC. Interestingly, NAC rapidly dissociated Cys from HNA(Cys) and NAC itself bound very slowly to HSA. These findings suggest that the interaction between NAC and HSA proceeds in a 2-step processes. The first-order binding and dissociation rate constants of NAC to healthy HSA (k(on,NAC)) and Cys from healthy HNA(Cys) (k(off,Cys)) were approximately 0.0032 and 1.3 (h(-1)), respectively. On the other hand, HSA from renal failure patients showed decreased HMA and increased HNA(Cys). The k(on,NAC) and k(off,Cys) were 0.0094 and 0.45 (h(-1)), respectively, suggesting that the pathological state may affect the binding properties of HSA and NAC.

Background: Intravenous iron administration (IVIR) is effective for correcting anemia in hemodialysis (HD) patients. However, it may also enhance the generation of hydroxyl radicals. Recently, plasma proteins have been demonstrated to be extremely susceptible to oxidative stress. Therefore, we investigated the effect of IVIR on the oxidative status of albumin, a major plasma protein, in HD patients., Methods: Eleven hemodialysis (HD) patients were treated with 40 mg of saccharated ferric oxide intravenously after every dialysis session for four weeks, and 11 age-/gender-matched HD patients were treated with vehicle. We performed high performance liquid chromatography (HPLC) analysis of serum albumin and determined the levels of reduced and oxidized albumin. Carbonyl formation of plasma proteins were also measured using an anti-2,4 dinitrophenylhydrazine antibody in patients with or without IVIR., Results: IVIR resulted in an increase in both disulfide form (f(HNA-1)) and oxidized form (f(HNA-2)) of albumin in HD patients (36.0 +/- 6.03 vs. 41.7 +/- 6.27; 5.46 +/- 1.50 vs. 8.7 +/- 2.22, respectively, P < 0.05). The findings here also show that IVIR substantially increased plasma protein carbonyl content by oxidizing albumin. In addition, we found a strong correlation between plasma carbonyl content and the levels of oxidized albumin (f(HNA-1) and f(HNA-2)) in HD patients (R= 0.674 and R= 0.724, respectively, P < 0.01)., Conclusion: The results of this study indicate that the HPLC analysis of serum albumin represents a potentially useful method for the quantitative and qualitative evaluation of oxidative stress in HD patients, and strongly suggest the possibility that oxidative stress, generated by IVIR, enhances the oxidation of albumin in those patients.

Purpose: 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)., Methods: 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., Results: 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., Conclusions: 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.

Purpose: 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., Methods: 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., Results: 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., Conclusions: 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.