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Volume 582, Issue 13 p. 1783-1787
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The antioxidant properties of serum albumin

Marjolaine Roche

Marjolaine Roche

Laboratoire de Biochimie et Génétique Moléculaire (LBGM), Université de La Réunion, Saint Denis de La Réunion, France

These two authors contributed equally to this work and should be considered co-first authors. Search for more papers by this author
Philippe Rondeau

Philippe Rondeau

Laboratoire de Biochimie et Génétique Moléculaire (LBGM), Université de La Réunion, Saint Denis de La Réunion, France

These two authors contributed equally to this work and should be considered co-first authors. Search for more papers by this author
Nihar Ranjan Singh

Nihar Ranjan Singh

Laboratoire de Biochimie et Génétique Moléculaire (LBGM), Université de La Réunion, Saint Denis de La Réunion, France

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Evelyne Tarnus

Evelyne Tarnus

Laboratoire de Biochimie et Génétique Moléculaire (LBGM), Université de La Réunion, Saint Denis de La Réunion, France

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Emmanuel Bourdon

Corresponding Author

Emmanuel Bourdon

Laboratoire de Biochimie et Génétique Moléculaire (LBGM), Université de La Réunion, Saint Denis de La Réunion, France

Corresponding author. Fax: +262 262 93 82 37.Search for more papers by this author
First published: 12 May 2008
Citations: 811

Abstract

Free radicals are a normal component of cellular oxygen metabolism in mammals. However, free radical-associated damage is an important factor in many pathological processes. Glycation and oxidative damage cause protein modifications, frequently observed in numerous diseases. Albumin represents a very abundant and important circulating antioxidant. This review brings together recent insights on albumin antioxidant properties. First, it focuses on the different activities of albumin concerning protein antioxidation. In particular, we describe the role of albumin in ligand binding and free radical-trapping activities. In addition, physiological and pathological situations that modify the antioxidant properties of albumin are reported.

1 Introduction

It is well established that free radicals and reactive oxygen species (ROS), nitrogen, and chlorine species contribute to the development of several age-related diseases by inducing oxidative stress and oxidative damage. Oxidative stress is commonly defined as a disturbance in the prooxidant and antioxidant balance leading to damage of lipids, proteins, and nucleic acids [1]. Oxidative stress can result either from low levels of antioxidants and/or from an increased production of reactive species [2]. What is an antioxidant? Halliwell and Whiteman defined an antioxidant as “any substance that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate”. The term “oxidizable substrate” corresponds to “every type of molecule found in vivo” [2].

Byproducts of oxygen metabolism, ROS, are reactive due to the presence of unpaired valence shell electrons. Production of ROS is not only intracellular but also concerns the surrounding area. Blood carries oxygen to tissues, and oxygen partial pressure is about 100 mm Hg in the arterial circulation and drops rapidly at the tissue levels with values between 4 and 20 mm Hg [3]. Then, blood is at least compble, if not more exposed to ROS than inside cells. Paradoxically, concentration of antioxidants is much lower in plasma than in cells [4]. Several models of oxidation indicate that albumin plays key role in antioxidant functions challenging the historical idea that only the intracellular compartment needs strenuous defense against oxidants [4-9].

2 Albumin, a multifunctional protein

Albumin contains 585 amino acids and has a molecular weight of 66 kDa. This highly soluble protein is present in human plasma at normal concentrations between 35 and 50 g/l (for a review see [10]). Albumin has several important physiological and pharmacological functions. It transports metals, fatty acids, cholesterol, bile pigments, and drugs. It is a key element in the regulation of osmotic pressure and distribution of fluid between different compartments. In normal conditions, its half-life is about 20 days, and its plasma concentration represents equilibrium not only between its synthesis in the liver and its catabolism, but also its transcapillary escape. In general, albumin represents the major and predominant antioxidant in plasma, a body compartment known to be exposed to continuous oxidative stress. A large proportion of total serum antioxidant properties can be attributed to albumin. Previous works have shown that more than 70% of the free radical-trapping activity of serum was due to human serum albumin (HSA) as assayed using the free radical-induced hemolysis test [11].

Usually, albumin concentrations remain very low in cerebrospinal, aqueous humor, and synovial and lung bronchoalveolar lining fluids when compared with plasma. Inflammation enhances vascular permeability mainly through chemicals released by activated neutrophils [12]. A beneficial effect arises from this apparent damage: albumin concentrations could be found enhanced in sites of inflammation, for the protein to exert its multiple antioxidant properties [13].

In the next section, various activities of albumin as a major antioxidant are examined. In the last section, some physiological or pathological functions modifying the antioxidant properties of albumin are reported.

3 Ligand-binding capacities for antioxidant properties

Albumin binds many types of molecules and was called a “sponge” or a “tramp steamer” of the circulation [10]. Many antioxidant activities of albumin result from its ligand-binding capacities. Albumin is well known for its ability to bind molecules, such as metals ions, fatty acids, drugs, and also hormones. The flexibility of the albumin structure adapts it readily to ligands, and its three domain design provides a variety of binding sites [10].

Among the cationic ligands, copper and iron deserve particular consideration because as transition metals, they are very potent to generate ROS after a reaction with oxygen. Free Cu(II) and Fe(II) ions can interact with hydrogen peroxide (H2O2) leading to the formation of the deleterious hydroxyl radical via the Fenton reaction. Bound to proteins, copper and iron are generally less susceptible to participate in the Fenton reaction. Concerning metals bound to albumin, hydroxyl radicals released from Fenton reaction are mostly directed to the protein sparing more important targets.

In plasma, most of the copper is bound to caeruloplasmin, but a high percentage of the metal ion may exist bounded to albumin [13]. HSA contains one high affinity site for copper, the N-terminal tripeptide Asp-Ala-His [14]. Protein sequestration of Cu(II) ions has been shown to prevent ROS-generating reactions. The first four amino acids of the N-terminus of human albumin, Asp-Ala-His-Lys (DAHK), form a tight-binding site for Cu(II) ions. Despite iron being present at higher physiological concentrations, copper can react with H2O2 to form hydroxyl radicals 60 times faster than iron [15]. Several studies have shown the antioxidant property of albumin by binding of copper using the copper-induced low-density lipoprotein (LDL) oxidation assay [6, 7, 16, 17]. In this assay, DAHK inhibited LDL lipid peroxidation and DAHK/Cu complex showed a superoxide dismutase-like activity by significantly preventing the formation of ROS [18]. Moreover, HSA and the tetrapeptide occupying its N-terminus (DAHK) were shown to prevent neuronal death in murine cortical cell cultures exposed to oxidative stress generated by H2O2 and by a mixture of copper and ascorbic acid [19].

Regarding iron, proteins involved in its transport are transferrin, caeruloplasmin, and lactoferrin. Nonetheless, high concentrations of circulating albumin indicate that the protein might be able to scavenge some hydroxyl radicals produced from iron reaction with H2O2 [13]. In addition, in iron-overload disease, a significant amount of iron-bound albumin was reported [20].

Among lipids, polyunsaturated fatty acids (PUFA) contribute largely to the pool of oxidizable biological compounds in plasma. Albumin binds with high affinity to long-chain fatty acids (LCFA) and PUFAs. The three main sites responsible for LCFA in albumin are shown in Fig. 1 [10]. There is no evidence that PUFAs bound to albumin are protected from lipid peroxidation. Nonetheless, the fact that effective lipid-phase antioxidants bind albumin (bilirubin and NO), indicates PUFAs bound to albumin, may be protected from oxidant-mediated damage [21, 22]. The binding of sterols by albumin is weak, and the esterified or non-esterified forms of cholesterol are carried in plasma by lipoproteins that are more specifically designed for the task [10]. Cholesterol undergoes oxidation in vitro and in vivo, forming biologically active derivatives known as oxysterols [23]. Interestingly, oxysterols, unlike cholesterol, distribute almost equally between lipoproteins and the lipoprotein-deficient serum (LPDS); in LPDS, the protein with which oxysterols are associated is albumin [24]. This higher affinity means that oxysterols carried by albumin are less rapidly released to cells than cholesterol. By this, albumin could limit detrimental effects of oxysterols on cells.

figure image
Main sites in albumin involved in its antioxidant activity. The structure of albumin molecule is shown as strands using a PDB file (PDB ID: 2BXF) of Ghuman et al. [61]. Lateral carbon chain of residues involved in albumin antioxidant properties are developed and colored. Amino terminus four amino acids of the protein are shown in blue. This sequence is involved in the metal binding of the protein. The sole free cysteine in the protein (Cys34) is shown in red. The free main sites of ligation of PUFA in albumin are shown in purple. Lys240 in albumin (yellow) is involved in bilirubin ligation. The six methionine residues are depicted in green. Molecular graphic image was produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).

An indirect antioxidant activity of albumin comes from its ability to transport bilirubin, which binds with high affinity to the molecule at Lys240 [25]. Such albumin-bound bilirubin was shown to act as an inhibitor of lipid peroxidation [26]. Recently, bilirubin, bound to albumin in the primary site, was shown to protect α-tocopherol from damage mediated by peroxyl radicals [27] and to prolong the survival of human ventricular myocytes against in situ-generated oxidative stress [28].

Another aspect of antioxidant activity of albumin may come from its capacity to bind homocysteine, a sulfur-containing amino acid, which results from the catabolism of methionine residue. Elevated plasma homocysteine is a well-known risk factor for atherosclerosis and may act through oxidation of LDL [29].

4 Free radical-trapping properties in albumin

HSA contains one reduced cysteine residue (Cys34) which, due to the large amount of albumin in plasma, constitutes the largest pool of thiols in the circulation [30]. In healthy adults, about 70–80% of the Cys34 in albumin contains a free sulphydryl group, the rest forms a disulfide with several compounds like cysteine, homocysteine, or glutathione [30]. Through the reduced Cys34, albumin is able to scavenge hydroxyl radicals [31]. Oxidation of Cys34 leads to the formation of sulfenic acid (RSOH), which is further oxidized to sulfinic (RSO2H) or sulfonic acid form (RSO3H) [30, 32]. Sulfenic acid constitutes a central intermediate in both the reversible and irreversible redox modulation by reactive species. Sulfenic acid in HSA was recently involved in mixed disulfide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments [32]. Reactive nitrogen species (RNS) constitute nitrogen-centered species analogous to ROS. Some RNS, such as nitric oxide (NO), contribute to various biological processes. Other RNS, such as peroxynitrite (ONOO), constitute powerful oxidants and nitrating species [33]. The –SH group of albumin represents an important antioxidant against peroxynitrite as thiol group was shown to be oxidized to a sulfenic acid (HSA-SOH) [34]. Subsequently, HSA-SOH can be converted to a disulfide and then back to mercapto-albumin (HSA-SH) [22].

Methionine residues (six in HSA) also represent an oxidation-sensitive amino acid in albumin [7, 11]. Met is particularly susceptible to oxidation and a wide variety of oxidants lead to production of methionine sulfoxide [35]. Oxidation to sulfone, which is the next step, is only obtained under drastic conditions not occurring usually in biological systems. Methionine sulfoxide can be reversed back to Met with mild reductants or by methionine sulfoxide reductases, whereas sulfone formation is biologically irreversible. Levine et al. observed that preferential oxidation of exposed Met residues in enzymes, such as glutamine synthase, had little effect on their biological function [36]. They proposed the very attractive hypothesis that the oxidation and reduction cycle of Met residues in biological systems could serve as a ROS scavenging system to protect proteins from extensive modifications [36, 37].

Finally, hypochlorous acid (HOCl) constituted a strong oxidant compound. Activated phagocytes, such as neutrophils and monocytes, release myeloperoxidase enzyme, which catalyzes the formation of HOCl [38, 39]. Albumin is able to scavenge HOCl preventing alteration of its preferential biological target α1-antiprotease [13].

5 Impairments in albumin antioxidant capacities after oxidation

As previously mentioned, the albumin molecule exerts several antioxidant properties. These beneficial properties rely on the structure of the molecule. Damages in the albumin molecule and its antioxidant properties have been considered as “biologically insignificant” because of the large amount of this protein in plasma [13]. In addition, damaged albumin molecules was reported to be rapidly removed from circulation and degraded [40]. However, recent reports showed that antioxidant properties of impaired albumin may be related to pathological conditions.

During its long lifetime (more than 20 days), an albumin molecule makes about 15 000 passes through the circulation [10]. Albumin incurs some damage that affects its antioxidant properties. These modifications could occur in diabetes mellitus, which is one of the pathological condition associated with early occurrence of vascular complications, together with functional alterations of albumin [41]. In this complex pathology, albumin undergoes increased glycation [41]. This phenomenon corresponds to the non-enzymatic attachment of a glucose molecule to a free primary amine residue. Amadori rearrangement of the glycated protein gives rise to the deleterious advanced glycation end products (AGE) [42]. After showing that albumin antioxidant property was modified following in vitro glycation by methylglyoxal [43], Favier et al. showed impairment of the antioxidant properties of serum albumin in patients with diabetes [44]. The binding activity of glycated HSA to copper was shown to be lower than that of non-glycated HSAs [45]. In vivo, the contribution of albumin concentrations to the iron-binding antioxidant capacity was shown to be markedly reduced in diabetes [46]. Glycation of HSA induced a marked loss of antioxidant activity of this molecule to copper-mediated oxidation of LDL [6]. Furthermore, glycated albumin exacerbated copper-induced LDL oxidation, probably by the generation of superoxide [6, 45]. In vivo, albumin also transports tryptophan, the largest and the essential amino acid [10]. Binding of Trp is reduced in glycated albumin [47]. In addition, some impairments in the antioxidant properties of albumin after glycation and/or oxidation of the molecule was evidenced [6, 7]. Recently, we showed that in vitro glycated albumin enhanced oxidative damage in adipocytes from a primary culture [48, 49].

Several receptors for AGEs, termed receptor for advanced glycation end products (RAGE), initiate intracellular signaling and enhance ROS formation in cells though its recognition and binding of AGEs [50]. The accumulation of N(epsilon)-(carboxymethyl)lysine (CML), a major antigenic AGE, was implicated in tissue disorders, in hyperglycemia and in inflammation [51]. Glycated albumin was shown to impair vascular endothelial NO synthase activity in vivo in aortas of rabbit [52]. Use of specific antibodies showed that these effects were mediated by CML and RAGE receptor [52]. The incubation of glycated HSA with HOCl led to the formation of CML [51]. The same group showed that ligand activity of AGE proteins to scavenger receptors was dependent on their rate of modification by AGEs [53]. Recently, hypochlorite-modified albumin was shown to colocalize with RAGE in the artery wall and to promote expression of monocyte chemotactic protein-1, promoting inflammatory complications in the arterial wall [54]. A toxic effect of glycated albumin was reported on microglial cells associated with impairments in cellular proteolytic systems [55]. Several studies has described the involvement of AGEs in neurodegeneration [41, 56, 57].

A total of 30–40% of patients with diabetes develop nephropathies that require hemodialysis treatment. Both dityrosine and carbonyl contents were found increased in HSA isolated from patients on hemodialysis [17]. Such HSA had impaired ligand-binding capacity [17]. Furthermore, impaired antioxidant properties of HSA from these patients was determined using two different systems, namely copper-mediated oxidation of human LDL and the free radicals-mediated hemolysis test [17]. Quinlan and Gutteridge [58] described an important occurrence in oxidative damages in patients with acute respiratory distress syndrome (ARDS). A beneficial effect of albumin administration was evidenced by the resulting enhancement in plasma thiol-dependent antioxidant status and in the reduction in protein oxidative damage [59].

Alterations in antioxidant properties of HSA was very recently identified in vivo in persons subject to obstructive sleep apnea syndrome [60]. This impaired antioxidant activity was associated with an enhanced glycation status of albumin in persons presenting this sleep disorder.

6 Conclusions

Albumin, the most abundant circulating protein in the plasma, exerts important antioxidant activities. The molecule acts through its multiple-binding sites and free radical-trapping properties. In physiological or pathological conditions, function associated with changes in the redox status, the albumin structure, and its beneficial antioxidant properties can be altered. In general, albumin constitutes the major plasma protein target of oxidant stress. Koch's postulate that a biological compound could constitute a causative agent in a disease appears to be fulfilled by oxidized albumin. If important beneficial actions are exerted by native albumin, very detrimental effects can be provoked by oxidized form of the protein and acting as a biomarker of oxidative stress. Critically, administration of albumin to patients with ARDS confers robust protection against oxidative stress and a favorable influence on redox-signaling processes regulating inflammation.

Additional studies, especially in vivo, are needed to document the modifications in albumin structure and antioxidant properties in pathologic conditions. Albumin has not yet showed all its secrets, and experiments are highly warranted to achieve a better understanding of its important antioxidant properties.

Acknowledgements

This work was supported by the Ministère de l'Enseignement Supérieur et de la Recherche et de l'Outre Mer, the Conseil Régional de la Réunion, and the Université de La Réunion. NRS and ET were supported by a fellowship from the Conseil Régional de La Réunion and from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche.

The corresponding author particularly acknowledges his former PhD director, Dr Denis Blache, for his valuable teachings concerning the very fascinating albumin. The authors would like to thank Dr Fabrice Gardebien for his advices concerning the use of Chimera freeware. The authors also acknowledge Valerie Systermans, Christian Lefebvre d'Hellencourt, Philippe Gasque, and Pierre Simon Petersson to read and correct the manuscript.