Biochimica et Biophysica Acta 1794 (2009) 634–641

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Biochimica et Biophysica Acta
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a pAltered chain-length and glycosylation modify the pharmacokinetics of humanserum albumin
Yasunori Iwao a, Mikako Hiraike a, Ulrich Kragh-Hansen b, Keiichi Kawai c, Ayaka Suenaga a,
Toru Maruyama a, Masaki Otagiri a,⁎abcDepartment of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan
Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark
School of Health Sciences, Faculty of Medicine, Kanazawa University, Ishikawa 920-0942, Japana r t i c l e

i n f oArticle history:
Received 19 September 2008
Accepted 3 November 2008
Available online 8 December 2008
Keywords:
Human serum albumin
Genetic variant
Pharmacokinetics
Half-life
Hepatic uptake
Renal disposition
Spleen uptake

a b s t r a c t
Human serum albumin with modiﬁed plasma half-life will be useful for clinical purposes. Therefore, thepharmacokinetics of three of each of the following types of genetic variants, and of their corresponding
normal albumin, were examined in mice: N-terminally elongated, C-terminally truncated and glycosylated
albumins. Isoforms differing from the normal protein by three or more amino acids, especially two of thetruncated forms, had shorter half-lives. The effect of glycosylation depended on the position of attachment:in domain II it increased half-life, whereas in domain I and III it had no signiﬁcant effect. Liver, kidney andspleen uptake clearances were also modiﬁed. The pronounced changes in half-life of the two truncatedvariants and the glycosylated isoform could be explained, at least partly, by large changes in organ uptakes;
in the remaining six cases, different effects were registered. Such information should be useful when
designing therapeutical albumin products for, e.g., drug delivery systems. In addition to various types of cell
endocytosis, leading to intracellular destruction or recycling of the proteins, the metabolism of the
alloalbumins could be affected by plasma enzymes. No correlation was found between mutation-inducedchanges in the pharmacokinetic parameters and changes in α-helical content or changes in heat stability as
represented by ΔHv.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Human serum albumin (HSA) is produced in the parenchymal cellsof the liver, and it is the most abundant plasma protein. It is an
important circulating depot protein and transport protein for
endogenous and exogenous ligands in the blood, and contributes tothe maintenance of osmotic pressure, plasma pH and to the Donnaneffect in the capillaries [1,2]. The protein is formed by a single
polypeptide chain of 585 amino acids, and it has a molecular mass ofapproximately 66.5 kDa [2]. According to X-ray crystallographic
analyses of HSA and of its recombinant version (rHSA), albumin has
about 67% α-helix but no β-sheet. The analyses also showed that thepolypeptide chain forms a heart-shaped protein with three homologous domains (I–III), each comprised of two subdomains (A and B)
with distinct helical folding patterns that are connected by ﬂexible
loops [3,4]. A combined phosphorescence depolarization-hydrodynamic modeling study has proposed that the overall conformation ofAbbreviations: HSA, human serum albumin; rHSA, recombinant HSA; Alb, albumin;
Alb A, normal (wild-type) albumin; CD, circular dichroism; ΔHv, van't Hoff enthalpy;
RAGE, receptor for advanced glycation end products⁎ Corresponding author. Tel.: +81 96 371 4150; fax: +81 96 362 7690.
E-mail address: otagirim@gpo.kumamoto-u.ac.jp (M. Otagiri).
1570-9639/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2008.11.022

HSA in neutral solution is very similar to that observed in the crystal
form [5].
Clinically, HSA is used for urgent restoration of blood volume,
emergency treatment of chock, acute management of burns and othersituations associated with hypoproteinemia [2]. To date, albumin has
been produced by fractionation of whole blood. However, there is thepotential risk of HSA contamination with blood-derived pathogens. In
addition, human plasma can be in limited supply. Because of these
problems, rHSA, which is highly expressed by Pichia pastoris, mostprobably will be commercially available in the near future [6]. Anotherbeneﬁt of this approach is that protein engineering will enable thecreation of rHSAs with modiﬁed properties such as extended half-lifein the circulation. In this connection HSA dimers seem to be usefulcandidates. Matsushita et al. [7] found that rHSA dimers had a highretention rate in the circulatory blood and a lower vascularpermeability than native rHSA in normal rats and in mice with paw
edema. Similar observations have been made by Komatsu et al. [8],who examined the pharmacokinetics of chemically crosslinked rHSA
dimers in the rat. On the other hand, recombinant albumin domain(s)are cleared very fast. Shefﬁeld et al. [9] found that recombinantdomain I, I + II and III of rabbit serum albumin all had very short meanterminal catabolic half-lives in rabbits due to a fast elimination in theurine.

Y. Iwao et al. / Biochimica et Biophysica Acta 1794 (2009) 634–641

Because of a relatively high in vivo half-life of ca. 19 days [2], HSA is
an attractive fusion partner to extend the half-life, and potentially thetherapeutic utility, of recombinant peptides and proteins. Among
recent examples are rHSA genetically fused to type 1 interferons [10],glucagon-like peptide-1 [11] and interleukin-2 [12]. However,
although an extension of the half-life of therapeutic peptides andproteins often is desirable, an extension to that of albumin could be
excessive.
Although HSA-preparations with a modiﬁed half-life thus could bevery useful, not much has been done to design or ﬁnd suchpreparations. In our search for useful candidates, we have paid ourattention to HSA genetic variants. Until now, 65 inherited variants of
HSA, including proalbumin variants, have been identiﬁed andstructurally characterized [13]. Usually, these genetic variants are
expressed in heterozygous form and without any known association
to disease [13]. Therefore, unlike lethal mutations, such may occur for
hemoglobin and coagulation factors, studying the pharmacokinetic
properties of HSA variants is a good way of gaining information which
can be used when designing recombinant HSAs, because we canconsider the effects of molecular variation without worrying aboutcomplications such as antigenic effects.
Recently, we have studied the pharmacokinetic properties inmice of 17 alloalbumins with single-residue mutations [14]. The
study showed that, for example, only a few of the variants had asigniﬁcantly modiﬁed half-life in the blood. In an attempt to ﬁnd
genetic variants with a more pronounced impact on pharmacokinetics, we now have expanded that study by determining theplasma half-lives and organ uptakes of three HSAs with a slightly
longer chain-length (proalbumin variants), three with a slightly
shorter chain-length (C-terminal variants) and three alloalbumins
N-glycosylated in domain I, II and III, respectively. For being able
to make a more detailed comparison between molecular characteristics and pharmacokinetic properties, we have estimated the effectof the molecular modiﬁcations on the α-helical content of the
alloalbumins by using circular dichroism (CD). Previously, the effectof genetic variation on the thermal stability of HSA has been
quantiﬁed in terms of, for example, changes in the van't Hoffenthalpy (ΔHv) [15]. In the present work, the pharmacokinetic
results have also been related to changes in ΔHv.
2. Materials and methods

635

Fig. 1. The crystal structure of HSA indicating the locations of the mutations of the three
C-terminal variants and the three proAlb variants used in this study. The locations of theglycosylated 63 Asn (Alb Malmö-95), 318 Asn (Alb Redhill) and 494 Asn (Alb Casebrook)
are also shown. The subdivision of HSA into domains is marked; N and C stand for the Nterminal and the C-terminal ends, respectively. The broken, black line added to the Nterminal end indicates the prosegment of HSA.

2.2. Chemicals and animals
111

InCl3 (74 Mbq/mL in 0.02 N HCl) was donated by Nihon MediPhysics (Takarazuka, Japan). All chemicals were of the highest grade
commercially available, and all solutions were prepared using
deionized, distilled water.
Male ddY mice (26–32 g) were purchased from the Shizuoka
Agricultural Cooperative Association for Laboratory Animals (Shizuoka, Japan), and were maintained under conventional housing
conditions. All animal experiments were conducted in accordancewith the principles and procedures outlined in the National Instituteof Health Guide for the Care and Use of Laboratory Animals.

2.1. Protein samples
2.3. In vivo experiments
The genetic variants of HSA and their normal (wild-type)
counterpart (endogenous Alb A) were isolated from serum fromheterozygous carriers by ion-exchange chromatography. The locations of the structural changes of the nine variants are indicated inFig. 1. After isolation, the albumins were checked for homogeneity
by native electrophoresis, and no denaturation or signiﬁcant (nomore than 5%) cross-contamination between variant and Alb A wasdetected. The proteins were donated to us by Drs. M. Galliano and L.
Minchiotti, University of Pavia, Pavia, Italy; Dr. S.O. Brennan,
Canterbury Health Laboratories, Christchurch, New Zealand; andDr. D. Donaldson, East Surrey Hospital, Redhill, UK. Before use, thealbumins were delipidated by treatment with hydroxyalkoxypropyldextran at pH 3.0, as described in a previous paper [16]. After
defatting, the albumins were dialysed extensively against deionizedwater, lyophilized and stored at −20 °C until used. Thus, thealbumins from a donor have been exposed to exactly the same
conditions from the time the blood samples were taken until thepresent experiments were performed.
Fraction V HSA (96% pure), assumed to be Alb A, was donated by
the Chemo-Sera-Therapeutic Research Institute (Kumamoto, Japan)
and defatted using the charcoal procedure described by Chen [17],deionized, freeze-dried and then stored at −20 °C until used.

All proteins were radiolabeled with 111In using the bifunctionalchelating reagent DTPA anhydride according to the method of
Hnatowich et al. [18], as described elsewhere [19]. In previous
works, we found no signiﬁcant differences in pharmacokinetic
properties among these albumins, when 111In-labeled mouse, rat,bovine or human serum albumin was administered to mice (unpublished), suggesting that immunogenic behavior does not occur in mice.
Therefore, we chose the mouse as a reasonable model for the study ofthe pharmacokinetics of the HSAs. Mice received tail vein injections of
111
In-labeled proteins in saline, at a dose of 0.1 mg/kg and were housed
in metabolic cages to allow the collection of urine samples. Urine
samples were collected throughout the 120 min of the experimental
period. In the early period after injection, the efﬂux of 111In
radioactivity from organs is assumed to be negligible, because thedegradation products of 111In-labeled proteins using DTPA anhydridecannot easily pass through biological membranes [20]. This assumption was supported by the fact that no 111In was detectable in the urine
after 120 min. At 1, 3, 5, 10, 30, 60, 90 or 120 min after injection, blood
was collected from the vena cava under ether anesthesia and plasma
was obtained by centrifugation. After blood collection, the animals
were sacriﬁced, organs were excised, rinsed with saline and weighed.

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Y. Iwao et al. / Biochimica et Biophysica Acta 1794 (2009) 634–641

The radioactivity of each blood and tissue sample was measured in awell-type NaI scintillation counter (ARC-500, Aloka, Tokyo).
Pharmacokinetic analyses were performed as follows. The plasma
111
In radioactivity concentrations (Cp) were normalized with respect
to the percentage of injected dose and analyzed using the nonlinear
least-square program MULTI [21]. The two-compartment model was
ﬁtted according to the Akaike information criterion by Eq. (1).
−αtCp = Ae

−βt

þBe

ð1Þ

The half-lives of the HSAs were determined as β-phase elimination
within a 120-min period. The tissue distribution patterns were
evaluated using tissue uptake clearances (CLuptake) according to theintegration plot analysis. CLuptake was calculated using Eq. (2).
CLuptake =Xt =Ct
AUC0t =Ct

ð2Þwhere Xt is the tissue accumulation at time t, AUC0–t is the area under
the plasma concentration time-curve from time 0 to t, and Ct is theplasma concentration at time t. CLuptake was obtained from the slopeof the plot of Xt/Ct versus AUC0–t/Ct. We estimated the organ uptake
clearances within a 30 min period.
2.4. Far–UV CD spectra
The protein concentration was 1.5 μM, as determined by themethod of Bradford [22], and the buffer was 67 mM sodium phosphate,
pH 7.4, 25 °C. Far–UV intrinsic spectra were recorded from 200 to
250 nm using a Jasco J-720 spectropolarimeter (Tokyo, Japan). For
calculation of the mean residue ellipticity, [θ], the molecular masses
were assumed to be 65.8 kDa for Alb Venezia, 67.1 kDa for proAlb Lille
and Tradate and 66.5 kDa for the remaining variants and Alb A. The αhelical content of the proteins was estimated from the ellipticity values
at 222 nm as described by Chen et al. [23].
2.5. Analysis of experimental data
The effects of the molecular changes were evaluated by using thefollowing relationship:
Percent change =ðResult for variantÞ−ðResult for Alb AÞ
×100kðResult for Alb AÞð3ÞIn Eq. (3), the result can be a value determined for plasma half-life,
organ uptake clearance, α-helical content or for ΔHv.

2.6. Statistical analysis
Statistical analyses were performed by using the Student t-test. A
probability value of P b 0.05 was considered to indicate statisticalsigniﬁcance.
3. Results
3.1. The genetic variants
The alloalbumins used in this study have been named after theplace from where the ﬁrst detected carrier originates, and theirmolecular changes are summarized in Table 1.
Proalbumin (proAlb) is an albumin molecule to which the propeptide, Arg-Gly-Val-Phe-Arg-Arg-, is still bound at the N-terminus (Fig. 1).
The positions of the propeptide are numbered from −6 to −1 (thejuxtaposition to albumin). Normally, proAlb does not occur in detectableamounts in the circulation, because the propeptide is cleaved off bypropeptidase within the liver cells. However, substitution of −1 Arg or −2
Arg (as in proAlb Lille [24] and proAlb Tradate [25]) inhibits theproteolytic cleavage of the propeptide but not the secretion of theprotein, and such proalbumin variants, in contrast to wild-type
proalbumin, can be isolated from the serum. In vivo, the prepro-formof proAlb Tradate (−2 Arg→ Cys) is often cleaved after the mutatedresidue giving rise to HSA retaining −1 Arg (Arg-Alb) [25].
Among the C-terminal variants most are truncated albumins (Table1). Thus, Alb Catania is three amino acids shorter than Alb A, and thethree last residues in the new C-terminal end are changed from GlnAla-Ala to Lys-Leu-Pro [26]. Alb Venezia has been shortened by sevenamino acids, and the new C-terminal end is changed from Gly-Lys-LysLeu-Val-Ala-Ala to Pro-Thr-Met-Arg-Ile-Arg-Glu [26]. Alb Bazzano has
been shortened by three amino acids, and 14 of the last 16 amino acidsin the new C-terminal end have been substituted: from Cys-Phe-AlaGlu-Glu-Gly-Lys-Lys-Leu-Val-Ala-Ala-Ser-Gln-Ala-Ala to Ala-Leu-ProArg-Arg-Val-Lys-Asn-Leu-Leu-Leu-Gln-Val-Lys-Leu-Pro [27]. Here the567 Cys → Ala substitution has caused the loss of the C-terminal
disulﬁde bridge.
It is uncommon for an amino acid substitution to result in theformation of an oligosaccharide attachment sequence. However, thathas happened to Alb Malmö-95 [28], Alb Redhill [29,30] and Alb
Casebrook [31,32], which are glycosylated in domain I, II and III,
respectively (Fig. 1). In all three cases, the glycan is a disialylated(mainly or totally) biantennary complex type oligosaccharide Nlinked to an asparagine residue [30]. Alb Redhill is unique, because it isTable 1
Half-lives and organ uptake clearances of 111In-labeled HSA variants and corresponding Alb A in mice
Variant name (mutation)

DomainproAlb Lille (−2Arg –N His)

I

proAlb Tradate (− 2Arg –N Cys)I

Arg-Alb (Alb A having −1 Arg)I

Alb Bazzano (567–582 substituted, 583–585 deleted)III

Alb Venezia (572–578 substituted, 579–585 deleted)III

Alb Catania (580–582 substituted, 583–585 deleted)III

Alb Malmö-95 (63 Asp –N Asn, glycosylated at 63 Asn)I

Alb Redhill (−1 Arg retained, 320 Ala –N Thr,
glycosylated at 318 Asn)
Alb Casebrook (494 Asp –N Asn, glycosylated at 494 Asn)

II

a

III

Variant
Alb A
Variant
Alb A
Variant
Alb A
Variant
Alb A
Variant
Alb A
Variant
Alb A
Variant
Alb A
Variant
Alb A
Variant
Alb A

Half-lifea(min)

Clearance (μL/hr)aLiver

KidneySpleen251.1 ± 4.31⁎
264.3 ± 4.58
249.4 ± 5.89
252.4 ± 7.62
262.1 ± 6.53
253.5 ± 4.98
231.1 ± 5.31⁎
245.2 ± 6.93
225.1 ± 5.49⁎⁎
247.2 ± 6.83
248.6 ± 5.46
251.3 ± 3.99
261.3 ± 7.62
264.2 ± 6.14
260.3 ± 7.43⁎
245.2 ± 6.34
251.2 ± 4.88
249.1 ± 4.54

56.13 ± 8.07
42.66 ± 7.52
111.99 ± 14.99
109.99 ± 14.01
21.16 ± 3.72⁎⁎
94.38 ± 5.17
189.77 ± 26.11⁎⁎
41.35 ± 4.68
134.32 ± 11.41⁎⁎
41.24 ± 4.81
12.29 ± 1.18⁎
48.99 ± 18.18
145.82 ± 13.84⁎⁎
79.72 ± 11.74
25.69 ± 3.51⁎⁎
93.49 ± 8.95
134.79 ± 13.97⁎⁎
81.45 ± 9.19

60.06 ± 6.76
79.69 ± 8.04
89.14 ± 11.11⁎⁎
46.01 ± 6.89
99.98 ± 23.18
119.38 ± 10.29
253.69 ± 36.22⁎⁎
119.95 ± 20.05
136.11 ± 13.55⁎⁎
62.87 ± 3.28
140.09 ± 15.01⁎⁎
81.53 ± 12.02
113.96 ± 15.72
111.64 ± 18.18
62.62 ± 7.05⁎⁎
125.84 ± 15.21
141.01 ± 8.14
148.71 ± 21.18

82.47 ± 15.96
90.11 ± 7.97
74.83 ± 13.57
87.86 ± 6.36
105.77 ± 14.52
91.38 ± 6.51
84.66 ± 7.00⁎⁎
57.94 ± 5.11
88.48 ± 10.15
67.89 ± 15.03
45.97 ± 5.15
52.69 ± 7.64
53.21 ± 4.21⁎⁎
82.68 ± 4.09
45.68 ± 6.91⁎⁎
84.39 ± 7.26
56.94 ± 6.10
57.73 ± 4.25

The data are average values of 3–6 experiments (± SD). ⁎P b 0.05, ⁎⁎P b 0.01 as compared with endogenous Alb A.

Y. Iwao et al. / Biochimica et Biophysica Acta 1794 (2009) 634–641

the only example so far of an albumin with two mutations. One is the
320 Ala → Thr, which leads to glycosylation of 318 Asn; the other is −2
Arg → Cys, which results in abnormal hydrolysis of the prepro-form
within the liver cells and to the formation of albumin still possessing−1 Arg [29].
According to the literature cited [24–32], none of the mutations
seem to affect the oligomeric state of albumin.
3.2. Pharmacokinetic properties of HSA variants
Fig. 2 shows the time courses for radioactivity in mouse plasma
after intravenous administration of 111In-labeled preparations of the

637

variants and their corresponding Alb A. As seen, in all 9 cases themutation affected, to different degrees, the elimination of HSA. Table 1
gives the plasma half-lives, calculated by β-phase using the nonlinear
least-square program MULTI and Eq.(1), and liver, kidney and spleen
uptake clearances, determined by an integration plot analysis (Eq.(2)).
As a control we have compared the pharmacokinetic results obtainedfor endogenous Alb A (Table 1) with those obtained with commercial
HSA (not illustrated), because both types of preparations are assumed
to represent the normal protein. The average half-lives for Alb A andcommercial HSA are 252.5 min and 268.2 ± 7.2 min (n = 6), respectively.
The liver, kidney and spleen uptake clearances for Alb A are on anaverage 70.36 μL/h, 99.51 μL/h and 74.74 μL/h, respectively, whereas

Fig. 2. Relative plasma amounts of 111In-labeled proAlb variants (A), truncated HSA variants (B) and glycosylated HSA variants (C) and their corresponding Alb A after intravenous
administration in mice. 111In-albumin was injected as a bolus dose into the tail vein. Relative amounts are plotted against time after injection. The open and closed circles represent
variant and normal albumin, respectively. Each point represents an average value obtained for 3–6 mice (± SD).

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Y. Iwao et al. / Biochimica et Biophysica Acta 1794 (2009) 634–641

those for commercial HSA are 54.31 ± 8.23 μL/h, 83.12 ± 5.43 μL/h and63.65 ± 3.56 μL/h, respectively. The slightly lower half-life and theslightly higher organ uptake clearances found for Alb A are mostprobably due to differences in isolation and/or defatting procedures.
The effects of the mutations on plasma half-lives and organ uptake
clearances, calculated according to Eq.(3), are given in Table 2. As seen,
all the proAlb variants and the truncated variants have diminishedplasma half-lives, i.e., negative percent changes; this is most evident
for Alb Venezia. By contrast, the effect of glycosylation varied: Almost
no effect in the case of Alb Malmö-95 and Casebrook but a signiﬁcantprolongation of the half-life for Alb Redhill. The increase of the halflife for Alb Redhill can partly be explained by the presence of −1 Arg(Table 2). In all 9 cases, the molecular changes resulted in a modiﬁed
liver, kidney and spleen uptake clearance (Table 2). The mostpronounced effects were found for the truncated variants Alb Bazzano
and Venezia; the uptakes by the three organs were increased verymuch. At the other extreme, the uptakes of Alb Redhill were in allcases signiﬁcantly decreased. For all three variants, the changes inorgan uptakes can explain, at least partly, the changes in plasma halflives. For the remaining six alloalbumins, the effects on organ uptakes
are more complex, i.e., both increases and decreases in uptake were
found for the same variant. The most pronounced changes, i.e., morethan 100%, were observed for liver and kidney uptakes of Alb Bazzano
and Venezia.
3.3. Relationships between structure, stability and pharmacokinetic
properties of HSA variants
The effects of genetic variation on the albumin structure werestudied by far–UV intrinsic CD. From Fig. 3 it is apparent that thevariants have spectra which differ to varying extents from that of theircorresponding Alb A. In most cases, the structural modiﬁcation results
in a less negative spectrum. However, in two cases (Arg-Alb and Alb
Venezia) the variant has a more negative spectrum. The mostpronounced spectral changes were found for two variants having a
modiﬁed chain length (proAlb Tradate and Alb Bazzano). We used theellipticities at 222 nm to calculate the α-helical content of all thealbumins. The impact of genetic variation on that parameter is given ina quantitative way in Table 2. Because the alloalbumins had both
modiﬁed pharmacokinetic properties (section 3.2.) and modiﬁed αhelical contents we investigated, whether there is a direct correlation
between these parameters. That was found not to be the case. Whenplotting the changes in half-lives as a function of changes in α-helicalcontent a poor correlation was found; a straight line had a P-value of0.48 (not shown). Likewise, no correlations were found between thechanges in organ uptake clearances and changes in α-helical content;
the P-values were in the range 0.16–0.52 (not shown).
Previously, we have studied the effects of genetic variation on thethermal stability of HSA [15]. The stability was quantiﬁed bydetermining van't Hoff enthalpies (ΔHv-values), and the results ofthat study are included in Table 2. We also examined whether acorrelation exists between changes in the ΔHv-values and the changes
in pharmacokinetic properties. However, no such correlation was
found. When plotting the changes in half-lives versus the changes in
ΔHv-values, the P-value was only 0.68 (not shown). In addition, no
correlations were found between the changes in organ uptakes andchanges in ΔHv-values; the P-values were in the range 0.38–0.83 (notshown).
4. Discussion
Clinically, it would be useful, if protein engineering could result in
the production of rHSA preparations with a prolonged half-life in thecirculation. In addition, because of its half-life of 19 days in humans, itsease of synthesis and its known structure albumin is an attractive
candidate for use in recombinant fusion proteins and as a carrier indrug delivery systems. However, also in the two latter types ofexamples it would be advantageous to be able to modify the plasma
half-life of the protein product. For ﬁnding such isoforms, in thepresent study, we investigated the half-life and organ uptakes of aseries of genetic HSA variants with relatively large molecular changes,
i.e., modiﬁed chain-length or glycosylated. The most clear results wereobtained for two truncated alloalbumins (Alb Venezia and Bazzano)and a glycosylated variant (Alb Redhill). For the other six variants lessclear and individual results were obtained.
All the alloalbumins with chain-lengths deviating by three or more
residues from Alb A have diminished half-lives in the circulation(Table 2). This is most evident for Alb Bazzano and Venezia the halflives of which were reduced by 6–9%. These results must be due tomutation induced changes in protein charge and/or conformation. In
this connection it is relevant to note that Alb Bazzano has lost its Cterminal disulﬁde bridge because of the 567 Cys → Ala substitution(see Section 3.1.). Our previous study [14], making use of singleresidue mutations, revealed that the half-life of Alb Hawkes Bay isshortened by ca. 30%. This ﬁnding is due to the mutation 177 Cys → Phe[33], which results in the loss of the disulﬁde bond between 168 Cys
and 177 Cys. Thus, the lifespan of HSA is dependent on the existence ofits 17 stabilizing disulﬁde bridges [1–4].
The effect of glycosylation depends on the position of attachment.
Glycosylation of 63 Asn in domain I (Alb Malmö-95) or 494 Asn indomain III (Alb Casebrook) has no signiﬁcant effect on variant half-life.
By contrast, glycosylation of 318 Asn in domain II (Alb Redhill) results
in a signiﬁcant increase in half-life; however, part of the increment iscaused by the presence of −1 Arg. Shefﬁeld et al. [9] also observed
position-dependent effects of N-glycosylation when studying the halflife of mutated rabbit serum albumin in rabbits. These authors
reported no effect of glycosylation of 12 Asn in the 14 Val → Thrvariant. By contrast, a similar modiﬁcation of 494 Asn in the 494
Asp → Asn isoform resulted in a reduction of the mean terminal
catabolic half-life from 4.32 days to 2.87 days. The different results
obtained in our and their study for the latter variant is probably due tospecies differences between protein and/or test animal.

Table 2
Percent changes of half-life and organ uptake clearances of HSA variants in mice
Variant name (mutation)

Half-life(%)

Liver clearance(%)

Kidney clearance(%)

Spleen clearance(%)

α-helical
content (%)ΔHva (%)

proAlb Lille (−2Arg –N His)
proAlb Tradate (− 2Arg –N Cys)
Arg–Alb (Alb A having −1 Arg)
Alb Bazzano (567–582 substituted, 583–585 deleted)
Alb Venezia (572–578 substituted, 579–585 deleted)
Alb Catania (580–582 substituted, 583–585 deleted)
Alb Malmö-95 (63 Asp –N Asn, glycosylated at 63 Asn)
Alb Redhill (−1 Arg retained, 320 Ala –N Thr, glycosylated at 318 Asn)
Alb Casebrook (494 Asp −N Asn, glycosylated at 494 Asn)

−4.99
−1.19
3.39
−5.73
−8.94
−1.07
−1.10
6.16
0.84

31.58
1.82
−77.58358.94225.74−74.92
82.91−72.5265.49−24.6393.74−16.25111.49116.5071.83
2.07−50.24− 5.17−8.48−14.84
15.75
46.10
30.32
−12.75− 35.65−45.63−1.36−9.60−24.155.64−20.09
5.41
−1.09
−0.31
−3.26
−7.06

−6.08
N.D.b
3.80
−4.49
34.50
− 19.71− 30.75
−4.48
17.54

a
b

The values for ΔHv are taken from Kragh-Hansen et al. [15].
ND, Not determined.

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639

Fig. 3. Far–UV CD spectra of proAlb variants (A), truncated HSA variants (B) and glycosylated HSA variants (C) and their corresponding Alb A. The dotted and full curves represent
variant and normal albumin, respectively. The curves are averages for three experiments.

In addition to change in chain-length and glycosylation we
investigated whether the modiﬁed plasma half-lives could becorrelated to other molecular parameters. Therefore, we estimatedthe effect of genetic variation on the α-helical content of albumin andrelated any changes to the changes in plasma half-life and organ
uptakes. However, no clear correlation could be found between thechanges in α-helical content and the different pharmacokinetic
parameters (not shown). The changes in half-life and organ uptakes
were also compared to mutation-induced changes in heat stability;
quantiﬁed by using ΔHv [15]. However, also in this case no correlations
were found (not shown).
For being able to explain, at least in part, the modiﬁed plasma halflives of the genetic variants we investigated their liver, kidney andspleen uptake clearances (Table 1). The diminished plasma half-livesof Alb Bazzano and Venezia are in full accordance with pronounced
increases in liver, kidney and spleen uptake clearances. Likewise, theincreased half-life of Alb Redhill can fully, or partly, be caused bydiminished uptake by the three organs. Although most of theremaining variants have an increased liver clearance and a reduced
spleen uptake individual results were obtained for these six
alloalbumins. However, for all variants also other metabolic factors
could be modiﬁed, see below.
Uptake of HSA by liver, kidney and spleen is mainly due to thepresence of cell membrane receptors which recognize the protein andthen internalize it by endocytosis. Some of these receptors interact
with native protein, whereas other receptors interact with modiﬁedprotein, and both types of interactions could be affected by genetic
variation of albumin. Thus, binding of HSA to the membrane-boundreceptor gp60 initiating a transcellular pathway for albumin across theendothelial cell wall to the underlying interstitium [34] could be
affected. Sometimes, a glycosylated albumin variant looses one orboth of the sialic acid residues on the antennae. Since the remaining of640

Y. Iwao et al. / Biochimica et Biophysica Acta 1794 (2009) 634–641

the glycans has galactose and mannose units, these forms could
interact with increased afﬁnity with the galactosyl receptor-mediated(asialoglycoprotein receptor-mediated) endocytotic pathway of thehepatocytes and the mannose receptor-mediated endocytotic pathway of non-parenchymal cells in the liver. The liver also possesses
receptors for rapid uptake of oxidized albumin and albumin withadvanced glycation end products. Whether the present molecularchanges can initiate endocytosis by scavenger receptors such as gp18,
gp30, stabilin-1 or stabilin-2 is at present only speculative. However,
liver uptake by adsorptive endocytosis could be inﬂuenced by themolecular changes, because this type of uptake is dependent on the
net charge of the protein.
Normally, glomerular ﬁltration of HSA in the kidneys is followedby its return into the venous circulation without degradation (thealbumin retrieval pathway). However, part of the protein reabsorbedby the proximal tubule cells, most probably via the endocytic
receptors megalin and cubulin, is degradated in the lysosomes.
Whether genetic modiﬁcation of HSA affects glomerular ﬁltration
and/or reabsorbtion of the protein remains to be clariﬁed. However,since the size and charge of albumin inﬂuence glomerular ﬁltration,
especially the glycosylated variants could have an altered handlingby the glomeruli. It should be noted that no radioactivity was
detected in the urine during the present experimental period. An
alternative, or supplementary, explanation for the modiﬁed uptakeof the albumin isoforms by the kidney could be the presence of
tubular RAGE which is known to cause the internalization ofproteins with advanced glycation end products. Irrespective of thedetailed mechanism, the net effect of deleting three or more Cterminal amino acids is an increased kidney uptake clearance(Tables 1 and 2). The effect of glycosylation depends on the positionof attachment, because glycosylation of domain II (Alb Redhill)decreases uptake, whereas glycosylation of domain I (Alb Malmö95) and domain III (Alb Casebrook) has no signiﬁcant effect on
kidney uptake.
Glycosylation diminishes uptake by the spleen (Tables 1 and 2).
This is most evident for Alb Redhill, especially when taking into
account that the presence of −1 Arg increases uptake. The presence ofa modiﬁed propeptide also decreases clearance uptake. By contrast,
the truncated variants with the most pronounced molecular changes(Alb Bazzano and Venezia) have signiﬁcantly increased uptakes.
Apparently, less is known about endocytosis-associated membrane
receptors in the spleen than in the liver and kidneys. However, theorgan most probably also has endothelial cells with the receptor gp60
[34]. In addition, it harbors scavenger receptors such as stabilin-1 andstabilin-2. To what extent albumin-receptor interactions are affectedby the mutations remains to be elucidated.
More recently, another type of endocytosis of HSA has been
identiﬁed in virtually all nucleated cells which results in reuse of theprotein [35–37]. After pinocytosis, albumin binds intracellularly and ina pH-dependent manner to the receptor FcRn. Thereby the protein isdiverted from the lysosomal degradation pathway and exocytosed
back to the circulation in an intact form extending its plasma half-life.
Anderson et al. [35] have proposed that the intracellular binding of
HSA to FcRn is due to a hydrophobic interaction, whereas Chaudhury
et al. [36] suggested interaction(s) between histidine residue(s) in thereceptor and histidine residues in domain III of albumin. By contrast,
Andersen et al. [37] suggested that FcRn interacts with negatively
charged and surface exposed residues on domain III of HSA. However,
the authors cited agree that domain III of albumin seems to contain allof the FcRn binding activity, why especially genetic variants withdomain III changes could have modiﬁed plasma half-lives due to a
modiﬁed HSA–FcRn recycling process.
The metabolism of HSA can be affected by enzymes such asaminopeptidase(s) and carboxypeptidase(s) in the circulation. That
has been observed in, for example, a patient with a severe traumatic
injury, who had an increased activity of carboxypeptidase A resulting

in hydrolysis of the C-terminal leucine and a fast elimination ofalbumin from the blood; the half-life was changed from ca. 19 days toless than 80 h [38]. In addition, modiﬁed albumin can be a substrate
for endopeptidase(s) in the blood. Thus, oxidized bovine serum
albumin, but not the native protein, can be cleaved by oxidized proteinhydrolase [39]. Because the hydrolase is found in the blood, also thisenzyme could hydrolyse some of the genetic variants in the mouse
circulation and thereby render them more exposed to organ uptake.
In conclusion, the pharmacokinetics of HSA can be modiﬁed bychanges in chain-length and by glycosylation. Three of the alloalbumins with a modiﬁed chain-length had a signiﬁcantly shorter half-lifein the circulation, whereas a glycosylated protein had an increased
half-life (Table 1). These ﬁndings are useful when trying to construct
an isoform with a modiﬁed stability in plasma. If an isoform with ashorter half-life is wanted, it is probably preferable to choose one witha C-terminally shortened chain-length rather than one with a Nterminally elongated chain-length, because the presence of a propeptide is known to block the high-afﬁnity binding site for metal ionssuch as Cu++ and Ni++ [40]. It is a disadvantage to block this binding
site, because albumin binding of Cu++ is an element in our antioxidative defense. Liver, kidney and spleen clearances were determined, and eight of the nine genetic variants had one or more
modiﬁed organ uptakes. The uptakes measured are net effects oforgan uptake leading to destruction in the lysosomes or to recycling
via the FcRn receptor. The uptakes themselves and the two
intracellular pathways could be affected differently by the protein
modiﬁcations. The results revealed that if an increased uptake in thethree organs is wanted for, for example, drug delivery systems, thentruncated variants like Alb Bazzano and Venezia are good candidates.
If the opposite is wanted, namely decreased uptakes in the organs,then the glycosylated Alb Redhill could be useful. Finally, weinvestigated whether blood half-lives or organ uptakes are directly
related to mutation-induced changes in the proteins α-helical content
or to changes in ΔHv, representing thermal stability. However, thatwas found not to be the case.
Acknowledgements
This work was supported, in part, by Grants-in-Aid for ScientiﬁcResearch from the Ministry of Education, Science, Sports and Cultureof Japan (14370759) and by Fonden af 1870.
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