Available online at www.sciencedirect.com

Biochimica et Biophysica Acta 1774 (2007) 1582 – 1590
www.elsevier.com/locate/bbapap

Changes of net charge and α-helical content affect the pharmacokinetic
properties of human serum albumin
Yasunori Iwao a , Mikako Hiraike a , Ulrich Kragh-Hansen b , Katsumi Mera a , Taishi Noguchi a ,Makoto Anraku a , Keiichi Kawai c , Toru Maruyama a , Masaki Otagiri a,⁎a

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japanb
Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmarkc
School of Health Sciences, Faculty of Medicine, Kanazawa University, Ishikawa 920-0942, Japan
Received 25 July 2007; received in revised form 1 September 2007; accepted 11 September 2007
Available online 19 September 2007

Abstract
The pharmacokinetics of 17 genetic variants of human serum albumin with single-residue mutations and their corresponding normal albumin werestudied in mice. In all cases, the plasma half-life was affected, but only variants with +2 changes in charge prolonged it, whereas changes inhydrophobicity decreased it. Good positive and negative correlations were found between changes in α-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
ΔHv. Liver and kidney uptake clearances were also modified: α-helical changes of domains I + III showed good negative correlations to both types ofclearances, whereas changes in domain II only had a good positive correlation to kidney uptake clearance. No correlation between the other molecularchanges and organ uptakes was observed. The relatively few correlations between changes in molecular characteristics and the organ uptakes of thevariants are most probably due to different handling by plasma enzyme(s) and the various types of cell endocytosis. Of the latter, most lead todestruction 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.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Human serum albumin; Genetic variant; Pharmacokinetics; Half-life; Hepatic uptake; Renal disposition

1. Introduction
Human serum albumin (HSA) is produced in the parenchymal cells of the liver and after removal of its pre–pro sequences,
it is secreted into the circulation, where it is the most abundant
plasma protein. It is an important circulating carrier of endogenous and exogenous ligands in the blood, and contributes to themaintenance 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 has a molecular massof approximately 67,000 [2]. According to X-ray crystallographic analyses of HSA and of its recombinant version,Abbreviations: HSA, human serum albumin; Alb, albumin; Alb A, normal(wild-type) albumin; ΔHv, van't Hoff enthalpy⁎ Corresponding author. Tel.: +81 96 370 4150; fax: +81 96 362 7690.
E-mail address: otagirim@gpo.kumamoto-u.ac.jp (M. Otagiri).
1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2007.09.001

albumin has about 67% α-helix but no β-sheet. The analyses
also showed that the polypeptide chain forms a heart-shaped
protein with three homologous domains (I–III), each comprisedof two subdomains (A and B) with distinct helical folding
patterns that are connected by flexible loops [3,4]. HSA has 35
cysteine residues, and all of these but one, Cys 34, are involvedin the formation of stabilizing disulfide bonds.
Clinically, HSA is used to treat severe hypoalbuminemia ortraumatic shock [2], and the usual dosages of HSA are in excess
of 10 g/dose. To date, albumin has been produced by fractionation of whole blood. However, there is the potential risk of HSA
contamination with blood-derived pathogens. In addition,
human plasma is in limited supply in countries like Japan.
Therefore, the development of an alternative method of industrial production of HSA is desired as this would greatly assist in
the general movement of, for example, Japan toward selfsufficiency in blood and blood products. Because of theseY. Iwao et al. / Biochimica et Biophysica Acta 1774 (2007) 1582–1590

problems, recombinant HSA, that is highly expressed by Pichia
pastoris, most probably will be commercially available in thenear future. Protein engineering will also enable the creation ofrecombinant HSAs with modified properties such as extended
half-life in the circulation. A modified half-life will also bebeneficial, when HSA is used as a fusion protein to extend thehalf-life of small proteins like soluble CD4 and hirudin [5,6].
Despite these possibilities, not much has been done to clarify theeffects of mutations on the pharmacokinetics of HSA.
Previous studies with chemically modified albumin havedemonstrated that changes in hydrophobicity and net charge on
the protein surface affect its in vivo clearance [7,8]. In a previous study in which we produced six recombinant HSAs, ourfindings suggested that even single-residue mutations of surface
or more internally placed amino acids can affect both the structural properties and the in vivo elimination of HSA [9]. Even
though these studies indicated that the elimination of HSA isdependent on its structural properties more detailed studies of
the relationships between structural and pharmacokinetic properties are needed to design useful recombinant HSAs withmodified half-lives in the blood.
In our search for such candidates, we paid our attention to
HSA genetic variants. Until now, more than 60 inherited variantsof HSA have been identified and structurally characterized [10].
Usually, these genetic variants are expressed in heterozygous
form and without any known association to disease [2]. There-

1583

fore, unlike lethal mutations, such as 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
about complications such as antigenic effects.
Previously, we have shown that inherited single-residue
substitutions affected the structure (α-helical content) andthermal stability (van't Hoff enthalpy (ΔHv)) of HSA [11]. In
addition, good linear correlations between mutation-inducedchanges of α-helical content and ΔHv were found. In the present
study, we examined the pharmacokinetics of 17 structurally
different genetic variants with single-residue mutations isolated
from heterozygote carriers, namely Alb Malmö-95 (D63N), Alb
Tregasio (V122E), Alb Hawkes Bay (C177F), Alb Herborn(K240E), Alb Niigata (D269G), Alb Caserta (K276N), Alb
Canterbury (K313N), Alb Brest (D314V), Alb Roma (E321K),
Alb Sondrio (E333K), Alb Parklands (D365H), Alb Milano
Slow (D375H), Alb Kashmir (E501K), Alb Maku (K541E), Alb
Church Bay (K560E), Alb Verona (E570K) and Alb Milano Fast
(K573E). We labeled the variants and their corresponding,endogenous normal albumin with 111In, and examined thepharmacokinetic properties of these albumins in mice. Then, we
determined the effects of the natural mutations on plasma halflife and organ uptakes. Finally, we related these results to thepreviously reported changes in α-helical content and ΔHv.

Fig. 1. The crystal structure of HSA showing the locations of the single-residue mutations of the 17 genetic variants used in this study. N and C stand for the N-terminal
and the C-terminal ends, respectively.

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2. Materials and methods
2.1. Protein samples
The 17 HSA genetic variants used in this study have been named after theplace from where the first detected carrier originates. The variants have singleresidue substitutions, and the location of the mutations is shown in Fig. 1. Allvariants, except one, have net charges which differ from that of normal (wildtype) HSA (endogenous Alb A) at physiological pH: Alb Roma (E321K) [12],Sondrio (E333K) [13], Kashmir (E501K) [14] and Verona (E570K) [15] are all+2 variants (i.e., they have two positive charges more than Alb A); Alb Malmö95 (D63N) [16], Niigata (D269G) [17], Brest (D314V) [18], Parklands (D365H)[19] as well as Milano Slow (D375H) [20] are +1 variants; Alb Tregasio(V122E) [21], Caserta (K276N) [22] and Canterbury (K313N) [23] are − 1variants; and, finally, Alb Herborn (K240E) [24], Maku (K541E) [25], Church
Bay (K560E) [26] and Milano Fast (K573E) [27] are −2 variants. The only
variant with no change in net charge is Alb Hawkes Bay (C177F) [28]. Most of
the substitutions are placed in domain II (residues 200–391), namely 9. Domains
I (residues 1–199) and III (residues 392–585) are represented by three and fiveexamples, respectively.
After isolation from heterozygous sera by ion-exchange chromatography,
the individual albumins were checked for homogeneity by native electropho-

resis, and no denaturation or significant (no more than 5%) cross-contamination
between variant and Alb A was detected. The proteins were donated to us byDrs. M. Galliano and L. Minchiotti, University of Pavia, Pavia, Italy; Dr. S.O.
Brennan, Canterbury Health Laboratories, Christchurch, New Zealand; Dr. A.L.
Tárnoky, University of Reading, Reading, UK; and Dr. O. Sugita, Niigata
University School of Medicine, Niigata, Japan. Before use, the albumins weredelipidated by treatment with hydroxyalkoxypropyldextran at pH 3.0, as
described elsewhere [29]. After defatting, the albumins were dialysed
extensively against deionized water, lyophilized and stored at − 20 °C until
used. Thus, the albumins from a donor have been exposed to exactly the same
conditions from the time the blood samples were taken until the present 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 [30], deionized, freeze-dried
and then stored at − 20 °C until used.

2.2. Chemicals and animals
111
InCl3 (74 Mbq/mL in 0.02 N HCl) was donated by Nihon Medi-Physics(Takarazuka, Japan). All chemicals were of the highest grade commerciallyavailable, and all solutions were prepared using deionized, distilled water.

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

Y. Iwao et al. / Biochimica et Biophysica Acta 1774 (2007) 1582–1590
Male ddY mice (26–32 g) were purchased from the Shizuoka Agricultural
Cooperative Association for Laboratory Animals (Shizuoka, Japan), and weremaintained under conventional 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.

2.3. In vivo experiments
All proteins were radiolabeled with 111In using the bifunctional chelating
reagent DTPA anhydride according to the method of Hnatowich et al. [31], which
has been described in detail in a previous paper [32]. In previous works, wefound no significant differences in pharmacokinetic properties among thesealbumins, 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 modelfor the study of the pharmacokinetics of the HSAs. Mice received tail vein
injections of 111In-labelled proteins in saline, at a dose of 0.1 mg/kg and werehoused 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 the degradation products of 111In-labeledproteins using DTPA anhydride cannot easily pass through biologicalmembranes [33]. 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 and1585

plasma was obtained by centrifugation. After blood collection, the animals weresacrificed, organs were excised, rinsed with saline and weighed. The radioactivityof each blood and tissue sample was measured in a well-type NaI scintillation
counter (ARC-500, Aloka, Tokyo).
Pharmacokinetic analyses were performed as follows. The plasma 111In
radioactivity concentrations (Cp) were normalized with respect to the percentage
of injected dose and analyzed using the nonlinear least-square program MULTI[34]. The two-compartment model was fitted according to the Akaike
information criterion by Eq. (1).
Cp ¼ Aeat þ Bebt

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 the integration plot analysis. CLuptake
was calculated using Eq. (2).
CLuptake ¼

Xt =Ct
AUC0t =Ct

ð2Þwhere Xt is the tissue accumulation at time t, AUC0−t is the area under theplasma concentration time-curve from time 0 to t, and Ct is the plasma
concentration at time t. CLuptake was obtained from the slope of the plot of Xt/Ct
versus AUC0−t/Ct. We estimated the organ uptake clearances within a 30-min
period.

Table 1
Half-lives and organ uptake clearances of 111In-labeled variants and corresponding Alb A in miceHalf-life a(min)

Variant name (mutation)
Alb Malmö-95 (63 Asp → Asn)
Alb Tregasio (122 Val → Glu)Alb Hawkes Bay (177 Cys → Phe)Alb Herborn (240 Lys → Glu)
Alb Niigata (269 Asp → Gly)Alb Caserta (276 Lys → Asn)
Alb Canterbury (313 Lys → Asn)
Alb Brest (314 Asp → Val)Alb Roma (321 Glu → Lys)
Alb Sondrio (333 Glu → Lys)
Alb Parklands (365 Asp → His)
Alb Milano Slow (375 Asp → His)
Alb Kashmir (501 Glu → Lys)
Alb Maku (541 Lys → Glu)
Alb Church Bay (560 Lys → Glu)
Alb Verona (570 Glu → Lys)
Alb Milano Fast (573 Lys → Glu)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
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

259.4 ± 7.26
264.2 ± 6.14
250.3 ± 6.65⁎⁎
270.4 ± 1.23
176.3 ± 8.68⁎⁎
250.8 ± 5.58
265.8 ± 6.97
269.3 ± 4.51
269.7 ± 1.68
275.2 ± 4.68
254.8 ± 4.89⁎
269.3 ± 7.64
221.6 ± 4.65
231.6 ± 4.64
260.8 ± 7.64⁎
276.4 ± 5.46
269.4 ± 4.51
266.1 ± 3.65
251.1 ± 4.98
247.6 ± 7.89
267.3 ± 6.51
273.1 ± 4.49
268.7 ± 7.61
272.9 ± 1.43
273.2 ± 4.62
270.6 ± 5.48
245.9 ± 7.32
251.3 ± 6.51
270.3 ± 3.56⁎
276.3 ± 1.03
274.1 ± 3.98⁎
262.4 ± 4.14
263.9 ± 2.46
263.5 ± 6.45

Clearance (μL/h) a
Liver

Kidney53.14 ± 12.09⁎
79.72 ± 11.74
73.22 ± 18.70⁎
129.13 ± 11.87
494.40 ± 38.82⁎⁎
23.99 ± 2.68
132.45 ± 22.19⁎⁎
41.10 ± 5.37
14.12 ± 2.39
18.00 ± 3.27
45.86 ± 6.29⁎⁎
21.77 ± 5.03
119.37 ± 12.12
128.80 ± 29.97
35.48 ± 11.53
25.31 ± 6.10
56.18 ± 5.89⁎⁎
13.34 ± 2.91
44.72 ± 10.58
28.92 ± 8.71
30.99 ± 4.11⁎⁎
63.58 ± 6.54
43.88 ± 10.33⁎
17.57 ± 8.33
44.21 ± 6.34⁎
64.36 ± 9.46
144.86 ± 15.01⁎⁎
276.01 ± 47.95
13.37 ± 4.39
17.61 ± 8.08
99.12 ± 18.83
63.85 ± 11.72
119.41 ± 18.90
85.88 ± 11.35

65.09 ± 18.83⁎
111.64 ± 18.18
71.42 ± 12.74
104.87 ± 24.94
374.08 ± 43.19⁎⁎
69.11 ± 11.67
137.86 ± 25.61
110.69 ± 12.94
83.69 ± 19.97
97.37 ± 23.26
203.48 ± 35.37⁎⁎
81.00 ± 13.51
331.98 ± 60.07⁎⁎
64.36 ± 20.01
102.87 ± 13.27
99.75 ± 16.32
117.48 ± 11.06⁎
83.49 ± 14.23
172.70 ± 40.37
169.51 ± 16.82
119.35 ± 14.93⁎
75.63 ± 20.15
47.34 ± 9.81
59.65 ± 22.73
76.48 ± 16.91⁎
138.53 ± 18.94
285.50 ± 44.74⁎⁎
90.25 ± 20.42
77.06 ± 16.01⁎
136.67 ± 30.62
128.12 ± 20.68
121.06 ± 16.44
218.05 ± 33.41⁎⁎
89.64 ± 10.02

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

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2.4. Analysis of experimental data
The effects of the single-residue mutations were evaluated by using thefollowing relationship:
Percent change ¼

ðResult for variantÞ  ðResult for Alb AÞ 100%
ðResult 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.

2.5. Statistical analysis
Statistical analyses were performed by using the Student t-test. A probability
value of P b 0.05 was considered to indicate statistical significance.

All genetic variants also have a modified liver and kidney
uptake clearance (Table 2). About half of the mutants havediminished liver uptake clearance, and about half of them have a
reduced kidney uptake. In general, the increases in organ uptake
clearance are more pronounced than the decreases. Thus, allpercent changes of organ uptake clearances of 100% or more areassociated with increases in uptake. For liver uptake it is Alb
Hawkes Bay (1961%), Roma (321%), Herborn (222%), Milano
Slow (150%) and Caserta (111%), and for kidney uptake it is
Alb Hawkes Bay (441%), Canterbury (416%), Maku (216%),
Caserta (151%) and Milano Fast (143%). However, no clear
correlation could be found between changes of protein net
charge and hydrophobicity, respectively, and liver and kidney
uptake clearance, respectively.

3. Results
3.1. Pharmacokinetic properties of HSA variants
Fig. 2 shows the time courses for radioactivity in mouseplasma after intravenous administration of 111 In-labeled
preparations of the variants and their corresponding Alb A.
For all 17 variants, the mutation affected the elimination of
HSA. In particular, Alb Hawkes Bay (C177F) showed rapid
elimination as compared with Alb A. However, in this example
the variant has undergone a major conformational change, see
below, because it has lost a disulfide bond [28]. Table 1 gives
the plasma half-lives, calculated by β-phase using the nonlinear
least-square program MULTI and Eq. (1), and liver and kidney
uptake clearances, determined by an integration plot analysis(Eq. (2)). As a control we have compared the pharmacokinetic
results obtained for endogenous Alb A (Table 1) with thoseobtained with commercial HSA (not illustrated), because bothtypes of preparations are assumed to represent the normal
protein. The average half-life for Alb A is 264.2 min. This value
is comparable to that determined for commercial HSA, i.e.,
268.2 ± 7.2 min (n = 6). The liver and kidney uptake clearances
for Alb A are on an average 64.64 μL/h and 100.2 μL/h,
respectively, whereas those for commercial HSA are 54.31 ±8.23 μL/h and 83.12 ± 5.43 μL/h, respectively. The slightly
lower values found for commercial HSA are most probably due
to differences in isolation and/or defatting procedures.
Table 2 shows the effects of the mutations on plasma halflives and organ uptake clearances as determined by Eq. (3). As
seen, 12 of the mutations diminished plasma half-life, i.e.,
negative percent changes, whereas one mutation had almost no
effect (Alb Milano Fast). By contrast, the remaining variants, allof which are +2 variants, had a prolonged half-life. Thesefindings are in accordance with those of Iwao et al. [9], who, byusing oxidized and recombinant mutants, observed that increasing the net negative charge of HSA increased its elimination from the circulation. The results of Table 2 also proposethat a change in hydrophobicity diminish plasma half-life. Thus,
Alb Malmö-95, Niigata, Caserta, Canterbury, Brest, Parklands
and Milano Slow all are more hydrophobic than Alb A, and all ofthem have negative values for the effect of mutation. Principally
the same was found to be the case for Alb Tregasio which is lesshydrophobic than its normal protein.

3.2. Relationships between structure, stability andpharmacokinetic properties of HSA variants
Previously, we have studied the effects of the single-residue
substitutions on the α-helical content and thermal stability of
Table 2
Percent changes of half-life and organ uptake clearances of HSA variants in mice
Variant name(mutation)

Halflife(%)

Liverclearance(%)

Kidneyclearance(%)

α-helical
content a
(%)

ΔHv a
(%)

Alb Malmö-95(63 Asp → Asn)
Alb Tregasio(122 Val → Glu)Alb Hawkes Bay(177 Cys → Phe)Alb Herborn(240 Lys → Glu)
Alb Niigata(269 Asp → Gly)Alb Caserta(276 Lys → Asn)
Alb Canterbury(313 Lys → Asn)
Alb Brest(314 Asp → Val)Alb Roma(321 Glu → Lys)
Alb Sondrio(333 Glu → Lys)
Alb Parklands(365 Asp → His)
Alb Milano Slow(375 Asp → His)
Alb Kashmir(501 Glu → Lys)
Alb Maku(541 Lys → Glu)
Alb Church Bay(560 Lys → Glu)
Alb Verona(570 Glu → Lys)
Alb Milano Fast
(573 Lys → Glu)

− 1.82−33.34− 41.69− 9.68− 46.71− 7.43−43.30− 31.89

2.02

9.76

− 29.70

1960.58441.31− 10.00− 8.53− 1.30222.2624.54− 4.12− 21.59− 2.00−21.56− 14.05− 3.92− 18.83− 5.38110.68151.21

0.68

5.74− 4.32−7.32

415.82

1.47

3.28− 5.64

40.17

3.13

1.57

10.53

1.24

321.2640.70− 1.77

11.31

1.41

54.66

1.88

− 1.57− 12.14− 2.12−51.2657.81− 3.1329.19− 1.54149.77− 20.63− 5.30− 32.230.96−31.30− 44.79

0.37− 0.60− 2.15−47.52

216.360.40− 20.76− 2.17−24.12− 43.62

1.18

5.28

4.46

55.24

5.83

2.55

36.24

0.17

39.03

143.24− 2.95− 16.64a
The values for α-helical content and ΔHv are taken from Kragh-Hansen
et al. [11].

Y. Iwao et al. / Biochimica et Biophysica Acta 1774 (2007) 1582–1590

albumin [11], and the results of that study are included in Table 2.
The α-helical contents were estimated from far-UV intrinsic
circular dichroism spectra by using the ellipticity values at
222 nm. The effect of the mutations on the reversible thermal
denaturation (25–85 °C) was also monitored by circular dichroism at 222 nm. These additional data allow for a more detailed
comparison between molecular characteristics and pharmacokinetic properties. Fig. 3 gives the correlations between mutationinduced changes in α-helical content and pharmacokinetic
parameters. For the mutations in domain I and III, i.e., positions
63–177 and 501–573, respectively (Fig. 1), the same tendencies
between the data were observed; therefore they have beengathered in Fig. 3A–C. For these mutations, an increase in αhelical content implies increased plasma half-lives, but decreased liver and kidney uptake clearances. Surprisingly, mutations in domain II, i.e., positions 240–375 (Fig. 1), have theopposite effects. In these cases, an increase in α-helical content
results in decreased plasma half-lives (Fig. 3D) but in an
increment of kidney uptake clearances (Fig. 3F). By contrast,
there is no correlation between changes in α-helical content andliver uptake clearances (Fig. 3E).

1587

The effect of the mutations on albumin stability was examined in terms of heat stability, which was quantified bydetermining van't Hoff enthalpies (ΔHv-values) (Table 2). Fig. 4
shows that no correlation was found between the percent
changes in ΔHv and any of the pharmacokinetic parameters.
Principally the same observations were made when pooling all
the results for domains I–III; in that case the P-values were in therange 0.79–0.97 (not shown).
4. Discussion
From a clinical point of view it would be beneficial, if protein
engineering could result in the production of HSA with aprolonged half-life in the circulation. In addition, because of its
half-life of 19 days in humans, its ease of synthesis and its known
structure albumin is an attractive candidate for use in recombinant fusion proteins. These proteins combine a therapeuticprotein (that would otherwise be rapidly cleared) and a plasma
protein with a slow clearance in a single polypeptide chain.
Furthermore, HSA has been proposed as a carrier in drugdelivery systems. Also in the two latter types of examples it

Fig. 3. Relationships between percent change of mutation-induced alterations of α-helical content and percent changes of half-life (A and D), liver uptake clearance(B and E) and kidney uptake clearance (C and F).

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Y. Iwao et al. / Biochimica et Biophysica Acta 1774 (2007) 1582–1590

Fig. 4. Relationships between percent change of mutation-induced alterations of ΔHv and percent changes of half-life (A and D), liver uptake clearance (B and E) andkidney uptake clearance (C and F).

would be advantageous to be able to modify the plasma half-lifeof the protein product. Therefore, in the present study, weinvestigated the half-life and organ uptakes of a series of genetic
HSA variants. Since the isoforms have known single-residue
mutations, we tried to identify molecular characteristics responsible for any modified pharmacokinetics.
The present variants have been detected by electrophoresis
performed under native conditions. Therefore, their changes incharge are located on the surface of the protein, where they are
exposed to the solvent. The only exception is Alb Hawkes Bay
which rather is detected due to major conformational changes
caused by the loss of the disulfide bond between Cys 168 and
Cys 177 [28]. Thus, the amino acid substitutions affect thehydrophilic/hydrophobic characteristics of accessible regions of
the protein. It should be mentioned that we did not quantify
these effects experimentally. Instead, we used the knowledge ofthe amino acid substitutions, and when doing that we found that
pronounced decreases in net charge (i.e., + 2 variants) resulted
in increased plasma half-lives. By contrast, increases in hydrophobicity implied shorter half-lives (Tables 1 and 2). Theseobservations are in full accordance with those of Iwao et al. [9],who studied the effects of oxidation and recombinant mutations.
Furthermore, Sheffield et al. [35], who used rabbit serum
albumin, found that the recombinant mutant D494N, but not the
charge-neutral mutant V14T, had a shorter half-life than thewild-type protein.

In addition to change in net charge and surface hydrophobicity we considered whether the modified plasma half-lives
could be correlated to other molecular parameters. Therefore, in Fig. 3A and D the half-lives have been related to changes
in α-helical content, which in this case were caused by singleresidue substitutions; the results showed a good correlation
between the two parameters (P = 0.1). However, surprisingly, theeffect depended on the location of the molecular change, becausechanges in α-helical content taking place in domains I and III
showed a positive correlation to changes in half-life, whereaschanges situated in domain II had the opposite effect. It can also
be seen that most changes in α-helical content resulted in fasterelimination from the circulation. The changes in half-life werealso compared to mutation-induced changes in heat stability;
quantified by using ΔHv. However, in this case there was nocorrelation between the two parameters (Fig. 4A and D).
Iwao et al. [9] found that the diminished half-lives of their
HSA preparations were accompanied by increased liver uptake
clearances. Nakajou et al. [36] reported that a recombinantmutant of HSA in which the lysine residues in positions 199, 439
and 525 all had been substituted for alanine disappeared fasterthan the wild-type protein from the circulation. The authors
suggested a faster than normal loss of the mutant to extravascularspaces. However, the triple-residue mutant also exhibited
increased liver and kidney clearances. For being able to explain,at least in part, the modified plasma half-lives of the present

Y. Iwao et al. / Biochimica et Biophysica Acta 1774 (2007) 1582–1590

genetic variants we also investigated their liver and kidney
uptake clearances (Table 1). No clear correlation could be foundbetween organ uptakes and changes in protein charge or hydrophobicity, respectively. With respect to the effect of changes in
α-helical content the domains again behaved differently. Forchanges in domains I and III good correlations were found(P = 0.1–0.2) between increases in percent change of α-helicalcontent and decreases in the change of liver (Fig. 3B) and kidney(Fig. 3C) uptakes. For domain II changes, a good correlation(P = 0.1) was observed for kidney uptake (Fig. 3F), whereas nocorrelation was registered between the molecular alterations andchanges in liver uptake clearance (Fig. 3E). As with the plasma
half-lives, no correlations were found between changes in organ
uptakes and changes in ΔHv (Fig. 4B, C, E and F).
Uptake of HSA by liver and kidneys is mainly due to thepresence of cell membrane receptors which recognize theprotein and then internalize it by endocytosis. Hepatocytes andthe nonparenchymal cells of the liver are involved in galactosyl
receptor-mediated and mannose receptor-mediated endocytosis,
respectively. Due to the specificities of these receptors, it is not
likely that the effects of the present single-residue mutations onliver uptake clearance can be explained by one of these mechanisms. The liver also possesses receptors for rapid uptake ofoxidized albumin and albumin with advanced glycation end
products. Whether small molecular changes such as singleresidue mutations can initiate endocytosis by scavenger receptors such as gp18 or gp30 is at present only speculative. On theother hand, liver uptake by adsorptive endocytosis could be
influenced by the amino acid substitutions, because this type of
uptake is dependent on the net charge of the protein.
Normally, glomerular filtration of HSA in the kidneys isfollowed by its return into the venous circulation without
degradation (the albumin retrieval pathway). However, a small
fraction is degraded in proximal tubular cells most probably after
uptake by the endocytic receptors megalin and cubulin. Whether
genetic modification of HSA results in increased glomerular
filtration and increased uptake by this receptor-complex remains
to be clarified. It should be noted that no radioactivity was
detected in the urine during the present experimental time. An
alternative explanation for the increased uptake of the albumin
isoforms by the kidney could be uptake by tubular receptors foradvanced glycation end products (RAGE-receptors). As with thescavenger receptors of the liver, it is not known whether singleresidue mutations of HSA can initiate uptake by these receptors.
Fujino et al. [37] have found that oxidized bovine serum
albumin, in contrast to the native protein, can be cleaved byoxidized protein hydrolase. Because this endopeptidase is foundin the blood, it could partly hydrolyse some of the genetic
variants in the mouse circulation and thereby render them more
exposed to organ uptake. The enzyme selectively recognizeshydrophobic regions in its substrate. Therefore, this mechanism can be especially relevant for the genetic variants withincreased hydrophobicity, which all have shorter plasma halflives (Tables 1 and 2).
More recently, another type of endocytosis of HSA has been
identified in virtually all nucleated cells which results in reuse of
the protein [38]. After pinocytosis, albumin binds intracellularly

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and in a pH-dependent manner to the receptor FcRn. Thereby theprotein is diverted from the lysosomal degradation pathway andexocytosed back to the circulation in an intact form extending its
plasma half-life. Chaudhury et al. [39] have proposed that the
intracellular binding of HSA to FcRn is caused by interaction(s)between histidine residue(s) in the receptor and histidine
residues in domain III of albumin. By contrast, Andersen et al.
[40] suggested that FcRn interacts with negatively charged andsurface exposed residues on domain III of HSA. Thus, especiallygenetic variants with domain III substitutions could have modified plasma half-lives due to a modified HSA-FcRn recycling
process.
In conclusion, the plasma half-life of HSA can be modified bysingle-residue mutations on its surface. No clear relation exists
with respect to type of mutation, but + 2 variants increase thehalf-life, whereas increased hydrophobicity decreases it.
Changes in the proteins α-helical content have a positive effecton the half-life, if they take place in domain I or III, else they
have a negative effect (domain II). By contrast, no correlation
was found between the half-lives and changes in ΔHv representing thermal stability. All mutations modified liver and kidney uptake clearances, and good correlations were found whenrelating liver (partly) and kidney uptakes to changes in α-helicalcontent. No correlations were found to type of mutation, changes
in charge or hydrophobicity or to changes in ΔHv. Organ uptakes
are brought about by different types of endocytosis with different
characteristics. Some of these lead to protein destruction in thelysosomes, whereas interaction with FcRn results in recycling of
HSA. The relatively few correlations between molecular albumin parameters and organ uptakes could be due to different
effects of the mutations on the various forms of endocytosis.
However, substitutions of domain III of HSA could modify
binding to FcRn and thereby alter its recycling. Although several
details with respect to organ uptakes still have to be shed light on,
the present information should be useful when designing recombinant HSA mutants with a modified plasma half-life.
Acknowledgements
This work was supported, in part, by Grants-in-Aid for
Scientific Research from the Ministry of Education, Science,Sports and Culture of Japan (14370759) and by Fonden af 1870.
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