Drug Metab. Pharmacokinet. 21 (2): 140–146 (2006).

Regular Article
The Structural and Pharmacokinetic Properties of Oxidized Human Serum
Albumin, Advanced Oxidation Protein Products (AOPP)
Yasunori IWAO1, Makoto ANRAKU1, Mikako HIRAIKE1, Keiichi KAWAI2,
Keisuke NAKAJOU1, Toshiya KAI3, Ayaka SUENAGA1 and Masaki OTAGIRI1,*
1Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences,

Kumamoto University, Kumamoto, Japan
2School of Health Sciences, Faculty of Medicine, Kanazawa University, Ishikawa, Japan
3Pharmaceutical Research Center, Nipro Corporation, Kusatsu, Shiga, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: 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 mM;
mg protein) were similar to those of uremic patients. In
dityrosine content, 0.7±0.11 nmol of dityrosine W
structural analysis, the increases in AOPP and dityrosine content of HSA induced slight decreases in its
a-helical content. In pharmacokinetic analysis, oxi-HSA left the circulation rapidly, and organ distribution of oxi-HSA 30 min after intravenous injection was 51z for the liver, 23z for the spleen, and 9z 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 signiˆcantly greater than those of normal HSA(CLliver, 5058±341.6 vs 24±4.2 mL W
hr [pº0.01]; CLspleen, 2118±322.1 vs 32±2.7 mL W
hr [pº0.01]).
However, uptake by other organs was not signiˆcantly aŠected by oxidation. These results suggest thatthe liver and spleen play important roles in elimination of AOPP.

Key words: human serum albumin;
pharmacokinetics

structuralchange;advancedoxidation

proteinproducts;

products (AOPP) in CRF plasma. Witko-Sarsat et al.
showed that in vivo levels of AOPP strongly correlatewith creatinine clearance, indicating that AOPP areexcellent markers of progression of CRF.6) HOCl-treated HSA and in vivo-generated AOPP can trigger
oxidative bursts in neutrophils and monocytes in vitro,
indicating that they both can act as true in‰ammatory
mediators.7) Results of in vitro studies of mechanisms of
AOPP production indicate that HOCl-treated HSA cantrigger an oxidative burst.7) However, the mechanisms
by which AOPP are degraded and eliminated fromcirculating blood remain unclear.
Recently, there have been reports of several types ofreceptors that bind to modiˆed albumin, including
SR-A (human scavenger receptor class A, whichbinds LDL)8,9) and CD36 (scavenger receptor class B
family).10,11) Despite such important ˆndings, it remains
unclear whether physiologically oxidized HSA (oxi-Introduction
Recent evidence indicates that oxidative stress plays
an important role in the pathogenesis of chronic renal
failure (CRF).1) Oxidative stress is deˆned as increased
production of reactive oxygen species (ROS) due to an
imbalance of oxidant W
antioxidant systems. Activatedphagocytes are a major source of ROS, and play afundamental role in host defense.2) Neutrophils containthe heme enzyme myeloperoxidase (MPO), whichcatalyzes the reaction of chloride ion with hydrogen
peroxide (H2 O2 ), to generate the large amounts ofhypochlorous acid (HOCl) produced by neutrophils.3)
HOCl-modiˆed proteins have been detected in atherosclerotic lesions4) and plasma from glomerular nephritis
patients.5) To estimate the degree of oxidant-mediatedprotein damage in plasma of CRF patients, researchers
have assayed levels of advanced oxidation protein

Received; October 14, 2005, Accepted; November 25, 2005
*To whom correspondence should be addressed : Prof. Masaki OTAGIRI, Ph.D., Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan. Tel. ＋81-96-371-4150, Fax. ＋81-96-362-7690,
E-mail: otagirim ＠gpo.kumamoto-u.ac.jp

140Pharmacokinetics of AOPP

HSA) containing AOPP behaves in the same way asother modiˆed albumins.
In the present study, using normal HSA and chloramine-T-treated HSA (oxi-HSA), we examined themechanisms by which AOPP are degraded and eliminated from circulating blood.
Material and Methods
Materials and animals: Chloramine-T (CT) was
purchased from Nacalai Tesque, Inc. (Kyoto, Japan).
The ‰uorescence probe 1, 1-bis-4-anilino-naphthalene5,5-sulfonic acid (bis-ANS) was purchased from Sigma(St Louis, MO, USA). 111InCl3 (74 MBq W
mL in 0.02 N
HCl) was donated by Nihon Medi-Physics (Takarazuka,
Japan). All chemicals used were of the highest grade
commercially available, and all solutions were preparedusing deionized, distilled water. Male ddY mice (24–26g) were purchased from the Shizuoka Agricultural
Cooperative Association for Laboratory Animals(Shizuoka, Japan). Animals were maintained underconventional housing conditions. All animal experiments were conducted in accordance with the principles
and procedures outlined in the National Institute of
Health Guide for the Care and Use of Laboratory
Animals.
Preparation of oxi-HSA: HSA (300 mM) was incubated for 1 h at 379C in an oxygen-saturated solution
containing 100 mM CT in phosphate buŠer (pH 8.0).
After incubation, the oxidation reaction was stopped byextensive dialysis of the solution against water. As anegative control, albumin was incubated in buŠer alone.
The oxi-HSA and negative control HSA were stored at－209
C until used.
Western blot analysis: Oxi-HSA was detected by
performing Western blot analysis. The antibodies usedwere an anti-HSA primary antibody raised in a rabbit
and an anti-rabbit secondary antibody conjugated tohorseradish peroxidase.
Determination of AOPP and dityrosine content:
AOPP content of oxi-HSA was determined using apreviously reported semi-automated method.6) Dityrosine content was assayed by measuring ‰uorescence
after the sample was diluted to 2 mM. Fluorescenceemission spectra of dityrosine were recorded at 410 nm
after excitation at 325 nm, using a spectro‰uorometer(FP-6200 Jasco) as described previously.12)
Amino acid analysis: The amino acid compositionof oxi-HSA was quantiˆed by performing amino acidanalysis after acid hydrolysis with 6 M HCl for 24 h
at 1109C, using an amino acid analyzer (L-8500A,
Hitachi, Tokyo, Japan) as described previously.13)
Structural properties of oxi-HSA: CD spectra wereobtained using a JASCO J-720 spectropolarimeter(JASCO, Tokyo, Japan) at 259C. The eŠectivehydrophobicity of oxi-HSA was estimated using the141‰uorescent characteristics of bis-ANS (10 mM) at 259
C.
Each compound was excited at 394 nm, and ‰uorescence spectra were recorded on a Jasco FP-770 ‰uorescence spectrometer (Tokyo, Japan).
In Vivo Experiments: All proteins were radiolabeledwith 111In using the bifunctional chelating reagent
DTPA anhydride, according to the method of
Hnatowich et al..14,15) Mice received tail vein injectionskg,of 111In-labeled proteins in saline, at a dose of 1 mg W
and were housed in metabolic cages to allow thecollection of urine samples. At appropriate intervals
after the injection, blood was collected from the vena
cava under ether anesthesia, and plasma was obtainedby centrifugation. The liver, kidney, spleen, lung, heartand muscle were excised, rinsed with saline andweighed. The radioactivity of each sample wasmeasured using a well-type NaI scintillation counter(ARC-500, Aloka, Tokyo). Pharmacokinetic analyses
were performed as follows. The plasma 111In radioactivity concentrations were normalized with respect to thepercentage of the dose per mL, and were analyzed
using the nonlinear least-square program MULTI.16)
Tissue distribution was evaluated using the organs
uptake clearance method (CLorgans), as described
previously.17)
Cellular assays: Endocytic uptake was determined
as described previously.18,27) RAW 264.7 cells were seeded in each well of a 24-well culture plate in 1.0 mL
RPMI 1640 medium, containing 10z FCS, 100 U W
mL
penicillin, and 100 mg W
mL streptomycin, and cultured
for 12 hr to subcon‰uence. The cells were washed with
1.0 mL PBS and replaced with Dulbecco's modiˆed
Eagle's medium containing 3z BSA, 100 U W
mL
mL streptomycin (medium A).
penicillin, and 100 mg W
The cells in each well were incubated at 379C for 6 hr
in 0.5 mL medium A with various concentrations of
125
I-control- or oxi-HSA in the presence (non-speciˆc) or
absence (total) of 50-fold unlabeled each ligands. At theindicated time, 0.375 mL of the culture medium wastaken from each well and mixed with 0.15 mL 40z
trichloroacetic acid (TCA) in a vortex mixer. We added
0.1 mL of 0.7 mol W
L AgNO3 to this solution, which was
followed by centrifugation. The resulting supernatant(0.25 mL) was used to determine TCA-soluble
radioactivity, which was taken as an index of cellulardegradation, since proteins are endocytosed by the cells
and delivered to lysosomes where they are degraded andexcreted into the culture medium in a TCA-soluble
form. Then, each well was washed three times with
1.0 mL ice-cold PBS. The cells were lysed with 1.0 mL
of 0.1 N NaOH for 1 hr at 379
C to determine thecell-associated radioactivity. Speciˆc cell-association ordegradation were determined by subtracting nonspeciˆc from total.
Statistics: Statistical analyses were performed using

142

Yasunori IWAO et al.

amino acids (Table 2).
Structural properties of oxi-HSA: To obtain information about protein structure, CD measurements were
performed in the far-UV regions (Fig. 2A). The molar
ellipticity of oxi-HSA was signiˆcantly less than that ofcontrol-HSA in the far-UV CD spectrum. The eŠects ofoxidation on the structural properties were examinedusing the ‰uorescence probe bis-ANS. The spectra
indicate that oxidation of HSA causes a decrease in its
accessible hydrophobic areas (Fig. 2B). These results
suggest that the structure of HSA is signiˆcantly
changed by oxidation.
Pharmacokinetic properties of oxi-HSA: Figure 3A
shows the time courses for radioactivity of 111In-labeled
preparations of oxi-HSA. Oxi-HSA had a shorter
half-life than control-HSA. To determine the reasonsfor the decreased plasma half-life of oxi-HSA, we
examined organ uptake. Liver, spleen and kidney
uptake of oxi-HSA increased with time, and were allmuch greater than those of control-HSA (Fig. 3B-D,
Table 3). Uptake by other organs was not signiˆcantly
aŠected by oxidation (data not shown).

Student's t-test.
Results
Western blot analysis of oxi-HSA: After exposure
to CT, protein aggregation or cross-linking was clearly
indicated by the presence of a smear in the highmolecular weight range. The high-molecular-weightfragments were recognized by the anti-HSA antibody(Fig. 1). These results suggest that treatment of HSA
with CT causes aggregation and cross-linking due tooxidation (oxi-HSA).
The characteristics of oxi-HSA: The AOPP contentof oxi-HSA was signiˆcantly greater than that ofcontrol-HSA (244.3±12.3 vs 10.3±6.3 mM; pº0.01;
Table 1). Dityrosine content of oxi-HSA was alsosigniˆcantly greater than that of control-HSA (0.7±
0.11 vs 0.32±0.13 nmol dityrosine per mg protein;
pº0.01). These characteristics of oxi-HSA are similar
to those of proteins in uremic plasma (Table 1). The
results of amino acid analysis indicate that oxidation of
HSA induced signiˆcant changes in its content ofcertain amino acid residues including tyrosine and basic

Discussion
Although in vivo studies indicate that the constant
plasma half-life of radioisotope-labeled native albuminis 2 to 2.5 days in rats,19) modiˆed albumin has been
Table 2.

Fig. 1.

AOPP(mM)
Dityrosine
mg protein)(nmol W

Amino acids

Literature
value

Control-HSA(n＝3)

Oxi-HSA
(n＝3)

Thr
Ser
Gly
Ala
Val
Met
Ile
Leu
Tyr
Lys
His
Arg

28
24
12
62
41
6
8
61
18
59
16
24

22.5±0.3
24.5±0.2
12.0±0.3
62.0±0.438.6±0.2
3.2±0.1
7.7±0.561.3±0.4
16.0±0.3
53.0±0.1
15.1±0.4
23.9±0.4

22.4±0.4
24.6±0.3
12.8±0.3
62.0±0.1
39.4±0.4
2.14±0.2
7.8±0.1
61.8±0.1
6.7±0.4*26.9±0.4*
14.5±0.4
18.6±0.3**Signiˆcantly diŠerent (Pº0.01) from control-HSA.
Data are mean±SD.

Western blot analysis of control-HSA and oxi-HSA.

Table 1.

Changes in amino acids composition of oxi-HSA.

AOPP and dityrosine content of HSA treated with chloramine T.

Control-HSA

Oxi-HSA

Normal subjects

HD patients

10.3±6.3

244.3±12.3*

33.9±3.4a)

267.5±16.5a)

0.32±0.13

0.7±0.11*

0.36±0.05a)

1.03±0.12a)*Signiˆcantly diŠerent (Pº0.01) from control-HSA. Data are mean±SD.
a)Ref. 6.

Pharmacokinetics of AOPP

143

Fig. 2. Far-UV CD spectra (A) and relative ‰uorescence intensity of hydrophobicity using bis-ANS (B) of control-HSA (—) and oxi-HSA ().
Data are mean±SD.

Fig. 3.

Plasma and organ levels of 111In-labeled control-HSA () and oxi-HSA () after i.v. administration to mice. Data are mean±SD.

shown to have an extremely short half-life in circulating
blood.20) As one of reasons for the formation ofmodiˆed albumin in vivo, free radical species have beenfound to induce conformational changes, degradation

and aggregation of proteins in several diseases.21) In
uremic patients, AOPP are produced by HOClmodiˆed albumin cross-linking in vivo. Further, AOPP
can act as mediators of oxidative stress, and they have

144

Yasunori IWAO et al.

been implicated in the immune dysregulation associatedwith chronic uremia.6,7) However, the mechanisms bywhich AOPP is degraded or eliminated from circulating
blood remain unclear. Therefore, in the present study,
we used CT-treated HSA (oxi-HSA) to examine themechanisms involved in elimination of AOPP.
The AOPP and dityrosine content of oxi-HSA weresimilar to those of proteins from uremic plasma(Table 1). In the Western blot analysis, oxi-HSA wasmostly present in the form of multimolecular aggregates, probably resulting from dityrosine cross-linking and W
or disulˆde bridges (Fig. 1). Further, the aminoacid analysis indicates that oxidation of HSA induced
signiˆcant modiˆcation of tyrosine and basic aminoacid residues such as lysine and arginine (Table 2).
These results suggest that the modiˆcations of thoseamino acid residues are responsible for the conformational changes observed in HSA (Fig. 2).
The pharmacokinetic analysis consistently showedthat oxi-HSA left the circulation rapidly. The organ
distribution 30 min after the intravenous injection was
51z for the liver, 23z for the spleen, and 9z for thekidney, suggesting that the liver was the main route forplasma clearance (Fig. 3). Since many of the scavenger
receptors are expressed in sinusoidal endothelial cells
and KupŠer cells in the liver,22,23) plural scavenger
receptors may be responsible for the hepatic uptake of
oxi-HSA. In general, most scavenger receptors can binda variety of polyanionic ligands, including negatively
charged albumins.24,25) In some diseases including
diabetes, AGE-albumins have well known as major
Table 3. Uptake clearance of control- and oxi-HSA labeled with
111
In after i.v. administration to mice.
hr)( mL W

Liver

KidneySpleenControl-HSA
Oxi-HSA

24±4.25058±341.6*

48±5.31188±208.2*

32±2.72118±322.1**Signiˆcantly diŠerent (Pº0.01) from control-HSA.
Data are mean±SD.

Fig. 4.

negatively charged proteins in plasma. AGE-albumins
are nonenzymatically glycosylated proteins that accumulate in vascular tissue with aging and accumulate
at accelerated rates in diabetic patients. Schmidt et al.22)
reported that AGE-albumins plasma clearance in mice
was rapid, with 70z of the injected dose removed fromthe circulation within 5 min. In their study, liver was
also the main route for plasma clearance of AGEalbumin. Their data are very similar to the present
ˆndings for oxi-HSA. Interestingly, oxi-HSA has anegatively charged molecule due to the modiˆcationsof basic amino acid residues in structural analysis(Table 2). Further, oxi-HSA has known to contain astructural motif of AGE-proteins as a result ofoxidation.6,7) Therefore, oxi-HSA are possible to be
distributed for liver nonparenchymal cells (endothelial
and KupŠer cells) as same as those of AGE-albumins.
However, the evidence for scavenger receptor involvedin the elimination of modiˆed albumin, including
AGE-albumin and oxi-HSA, on liver nonparenchymal
cells has been rarely found.26,27) Unfortunately, we werealso unable to clarify the receptor responsible forhepatic uptake in this study, but we should examine theelimination mechanism of oxi-HSA by using cell line
highly expressed scavenger receptors in the future.
On the other hands, scavenger receptors are expressed
not only on liver nonparenchymal cells but also various
macrophages. Given the fact that the organ distributionof oxi-HSA was 23z for the spleen, oxi-HSA may bealso taken up by macrophage of spleen via scavenger
receptor-mediated endocytosis. Macrophage has known
to express highly SR-A, one of the scavenger receptor.8)
To examine the relationship between oxi-HSA and
SR-A in macrophage, we used the macrophage-derived
cell line RAW 264.7. Signiˆcant amounts of 125I-oxiHSA were associated with these cells and underwent
endocytic degradation within the cells, whereas no suchˆndings were observed for control-HSA (Fig. 4). Thus,
SR-A may be involved in the spleen clearance of oxiHSA.

Endocytic uptake of 125I-labeled control-HSA () or oxi-HSA () by RAW 264.7 cells. Data are mean±SD.

Pharmacokinetics of AOPP

In summary, the kinetics of chloramine-T-treated
HSA (oxi-HSA) are similar to those of AOPP in uremic
patients. Oxi-HSA left the circulation rapidly andaccumulated in the liver, spleen and kidney. The liverand spleen appear to play particularly important roles inelimination of AOPP.

13)

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