Nephrol Dial Transplant (2008) 23: 2304–2310
doi: 10.1093/ndt/gfn002
Advance Access publication 5 April 2008

Original Article

Influences of haemodialysis on the binding sites of human serum
albumin: possibility of an efficacious administration plan usingbinding inhibition
Toyotaka Nishio1,2 , Norito Takamura3 , Ryuuichi Nishii4 , Jin Tokunaga3 , Mitsuyoshi Yoshimoto1 andKeiichi Kawai1,51Kanazawa University Graduate School of Medical Science, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, 2 Nova Pharmacy,
1170-1 Shimowada, Yamato, Kanagawa 242-0015, 3 School of Pharmaceutical Sciences, Kyushu University of Health and Welfare,
1714-1 Yoshino, Nobeoka 882-8508, 4 Research Institute, Shiga Medical Center, 5-4-30 Moriyama, Moriyama-City, Shiga 524-8524
and 5 University of Fukui Biomedical Imaging Research Center, 23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida, Fukui 910-1193,
Japan

Abstract
Background. We have studied the possibility that low-dose
treatment utilizing the inhibition that may occur between
two drugs at the same site of human serum albumin (HSA)
improves the pharmacological effects. The purpose is toelucidate the differences in the binding capacities of sites I
and II of HSA between pre-haemodialysis (HD) and postHD in patients with end-stage renal disease.
Methods. We evaluated free fractions of site probes, 14 Cwarfarin (site I) and 14 C-diazepam (site II), by ultrafiltration in serum between pre-HD and post-HD. To investigate effects on the binding capacities of HSA sites,
free fractions of site probes were calculated from the radioactivities measured with a liquid scintillation counter.
Endogenous uraemic toxins, 3-carboxy-4-methyl-5-propyl2-furanpropionate (CMPF), indoxyl sulphate (IS) and hippurate (HA), were determined by HPLC. Free fatty acid
(FFA) as an endogenous substance was determined with anautomatic multi-item simultaneous analyser.
Results. The concentrations of HSA and FFA increased
significantly (post-HD/pre-HD ratio: 1.18 ± 0.10, 5.46 ±4.91), the concentrations of IS and HA decreased significantly (post-HD/pre-HD ratio: 0.69 ± 0.10, 0.33 ± 0.15)
and CMPF concentrations did not alter significantly (postHD/pre-HD ratio: 0.97 ± 0.12, P = 0.471). The free fractions of 14 C-warfarin decreased in all 14 patients at site
I at post-HD compared to pre-HD (post-HD/pre-HD ratio: 0.59 ± 0.13). The free fractions of 14 C-diazepam atsite II remarkably decreased in 10 of 14 patients (postHD/pre-HD ratio: 0.61 ± 0.17) and unexpectedly increased
in 4 (post-HD/pre-HD ratio: 1.08 ± 0.06) post-HD compared to pre-HD. In these four patients, when we investiCorrespondence and offprint requests to: Norito Takamura, Second Department of Clinical Pharmacy, School of Pharmaceutical Sciences,
Kyushu University of Health and Welfare, 1714-1 Yoshino, Nobeoka,
Miyazaki 882-8508, Japan. Tel: +81-982-23-5537; Fax: +81-982-235539; E-mail: noritotaka@phoenix.ac.jp

gated the influences of these variation factors on the reduction of the binding capacities of site II, [FFA]/[HSA]
increased significantly post-HD, compared to pre-HD(post-HD/pre-HD ratio: 6.91 ± 6.58). ([FFA]/[HSA] ratios of the 4 patients were from 1.22 to 3.55, the highest forthe 14 patients post-HD, but the ratios of the other 10 were
below 1.2 post-HD.)
Conclusion. The binding capacity of site II was unexpectedly decremented by the effects of the remarkable elevationof FFA. Therefore, monitoring the binding capacity of site
II in HD is important for patients with end-stage renal disease in the efficacious administration plan using the binding
inhibition of HSA.
Keywords: binding site; free fatty acid; haemodialysis;
human serum albumin; protein binding

Introduction
After absorption, drugs bind to serum proteins, althoughsome differences exist in the degree of binding strength.
Most high-affinity drugs bind to human serum albumin(HSA) [1] which accounts for 60% of serum proteins [2].
HSA has two major drug-binding sites, namely, sites I
and II [3]. The drugs that bind to site I are warfarin andfurosemide, while those that bind to site II are diazepam
and diclofenac [4]. When two drugs bind to a common site,
competitive inhibition may occur. As a result, the binding
affinity of the inhibited drug decreases and the free concentration of the drug associated with the pharmacologicaleffects increases. It was reported that the distribution of adrug to a target tissue is increased by the inhibitory effectof a drug binding to HSA [5]. An inhibitor that reduces thebinding capacity of each site should possess the following
properties: (1) a potent inhibitor of the protein binding ofC The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Alteration of binding capacities of site II by haemodialysis

drugs; (2) when administered in large doses, its plasma concentration reaches high levels and (3) it is extremely safe
and suitable for prolonged administration [6]. If we utilize
the inhibition of the protein binding of drugs, the low-dose
drugs could have a sufficient pharmacological effect.
We have demonstrated that bucolome improves the diuretic effect of furosemide. Bucolome (K = 1.5 × 106 M−1 ),an inhibitor which reduces the binding capacity of site I,elevates the concentration of free furosemide (K = 2.0 ×
105 M−1 ) bound to the same site [6,7]. Figure 1 shows the
distribution process of free furosemide by the inhibitory
effect of bucolome. Furosemide binds to site I of HSA
without bucolome in state 1. The binding of furosemide isinhibited by bucolome in state 2 (furosemide is distributed
from vein to tissue). Consequently, an administration planthat utilizes the artificial decrease in the binding capacity of
HSA sites with the coadministration of an inhibitor would
have an efficacious pharmacological effect.
Since there has been a report that the binding affinityof a drug is changed by haemodialysis (HD) [8,9], we
propose that it is important, rather than utilizing the artificial alteration of the binding affinity caused by an inhibitor, to instead utilize the spontaneous alteration of thebinding affinity produced by HD. The present study was
therefore undertaken to investigate the difference in thebinding capacities of site I and site II of HSA between preHD and post-HD in patients with end-stage renal disease(ESRD).

Materials and methods
Samples (subjects)
The protocol was approved in advance by the Ethics Committee of Fujimoto Hayasuzu Hospital. The subjects were
12 males and 2 females, 20–74 (54.9 ± 16.0) years old, on
HD for 4–116 (33.3 ± 8.3) months (Table 1). The causeof ESRD was diabetic nephropathy in eight patients, IgA
nephropathy in one patient, Wegener’s granulomatosis in
one patient, purpuric nephropathy in one patient and unknown aetiology in three patients. The serum was extracted
before and after HD from 14 renal failure patients, who participated as outpatients on HD. We referred to the clinicallaboratory test results for the concentration of HSA of eachpatient.
The conditions of the haemodialysis sessions were as
follows. All patients who were diagnosed with chronic renal failure (i.e. ESRD) and had a permanent arterial-venous
fistula on the forearm underwent the usual haemodialysis
using a polysulfone membrane (TORAYSULFONE TS-M,
Toray Medical, Japan) with a duration of 4–4.5 h. The
dialyzer surface area was 2.0 m2 and replacement fluid(LYMPAKTA3, Nipro, Japan) was delivered after themembrane was inserted into the venous limb of the circuit.
Blood was pumped through the membrane at a rate between
2.0 and 4.0 mL/kg/min. The dialysate was delivered countercurrently to the blood flow using a volumetric pump.
Ultradiafiltrate flow was set at a rate of between 500 and1300 mL/h. Heparin was administered as an anticoagulant
throughout the procedure, and aspirin, aspirin-dialuminate,ticlopidine or warfarin was used as an antiplatelet agent.

2305

The dosage of heparin, the body weight between preand post-HD and the ultrafiltration volume are shown in
Table 1. The medications were mainly antihypertensives(beta-blockers, calcium channel blockers, furosemide,ACE-inhibitors, ARBs), phosphate binders, CaCO3 ,potassium-binders, proton-pump-inhibitors, H2 -blockers,
insulin, hypoglycaemics and vitamin D supplements.
Protein binding study
Materials. Warfarin and diazepam radiolabelled with 14 C,[α-14 C-benzyl] warfarin (56 mCi/mmol) and [2-14 C] diazepam (55 mCi/mmol) were purchased from Amersham
Biosciences (UK).
We obtained 3-Carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) as a pure substance from Cayman Chemical(Ann Arbor, Michigan, USA). Indoxyl sulphate (IS) andhippurate (HA) were purchased as pure substances from
Nacalai Tesque (Kyoto, Japan). All other chemicals were ofanalytical grade.
Measurement of free fraction using ultra-filtration. To investigate the variations in the binding of sites I and II of
HSA between pre-HD and post-HD, 14 C-warfarin or 14 Cdiazepam was added to the serum. The free fractions ofradiolabelled site probes, 14 C-warfarin and 14 C-diazepam,
in the serum were calculated by the ultrafiltration method
and the following processes: The solution of a radiolabelled
site-probe (14 µL) and saline solution (14 µL) was added
to 700 µL of serum. Aliquots (10 µL) of the solution wereplaced in vials. The radioactivity of each solution before filtration was measured. Ultrafiltration experiments were performed using Minicent-10 centrifugal filter devices (Tosoh,
Japan). A 200-µL volume of the solution was placed in
the upper compartment. It was ultra-filtered at 3000 rpm
for 10 min at room temperature using the centrifuge. A
10-µL solution isolated from the filtrate was counted as
the radioactivity of the solution after filtration. The radioactivity of both solutions before filtration ([RAS ]) and after
filtration ([RAF ]) was measured with a liquid scintillation
counter (LSC-5000, Aloka, Japan) after adding scintillation
fluid (ACSII, Amersham, UK) to scintillation vials.
The free fraction of the radiolabelled site probe was calculated as the per cent ratio of the activities of the solutions
before and after filtration using the following equation [10]:Free fraction (%) = [RAF ]/[RAS ] × 100
Endogenous substances assay and evaluation
Uraemic toxins assay. After 0.6 mL of acetonitrile wasadded to 0.3 mL of serum, mixed and churned for 10 min
at room temperature, the mixed solution was centrifuged
to remove protein at 3500 rpm for 10 min at room temperature. Then, 0.45 mL of the supernatant was separated
after processing to remove protein. The concentrations ofuraemic toxins, CMPF, IS and HA [11] in the serum were
determined by HPLC (a system consisting of an LC-6AUV detector, Shimadzu; RP-18 Column, Waters) at a flowrate of 1 mL/min, using acetonitrile/0.2 M buffer of acetic
acid/acetic acid (80:120:1 v/v) for CMPF and acetonitrile/0.2 M buffer of acetic acid (10:90 v/v) for IS and2306

T. Nishio et al.

Fig. 1. Process of distribution of free furosemide by inhibitory effect of bucolome. State 1: binding of furosemide to serum albumin in the absence of
bucolome. State 2: inhibition of furosemide by bucolome (increase of distribution to tissue).
Table 1. Patients on haemodialysis
Patient

Age

Sex(years)Duration of HD(months)

1
2
3
4

70
57
50
54

M
F
M
M

24
116
30
56

5
6
7
8
9

70
62
28
74
65

M
M
M
F
M

33
17
28
20
40

10
11
12
13

39
59
66
54

M
M
M
M

18
58
12
4

14

20

M

10

Mean
SD
Range

54.9
16.0
20–74

Underlying
diseasea

Diabetes
IgA nephropathy
Diabetes
Wegener’s
granulomatosis
Diabetes
Purpuric nephropathy
Diabetes
Diabetes
Secondaryhyperparathyroidism
Congenital myopathy
Diabetes
Diabetes
Hypertensive
intracerebral haemorrhage
Diabetes

33.3
8.3
4–116

Heparindosage (Unit)

Body weight (kg)

Ultrafilteration
volume (mL)

Pre-HD

Post-HD

post/pre ratio

5000
5000
4000
3000

56.3
46.2
61.7
54.6

55.1
43.6
59.8
51.2

0.98
0.94
0.97
0.94

2100
3200
2300
4200

3000
4000
4000
4000
4000

50.3
61.2
71.2
50.0
72.1

47.1
57.8
68.2
48.5
69.6

0.94
0.94
0.96
0.97
0.97

3930
4100
3400
1600
2800

4000
4000
1500
3000

46.3
61.8
53.0
66.3

43.8
58.6
51.0
63.6

0.95
0.95
0.96
0.96

2900
3900
3000
3200

5000

54.8

52.5

0.96

2800

3821.4952.9
1500–5000

0.48
0.77
46.2–72.1

1.13
0.93
43.6–69.6

0.96
0.01
0.94–0.98

3102.1773.041600–4200

a All patients had end-stage renal disease.

HA as the mobile phase. A UV monitor was used to assay
for CMPF, IS and HA at a UV wavelength of 261 nm, 285
nm and 240 nm, respectively.
Free fatty acid assay. The concentration of free fatty acid
(FFA) was determined with an automatic multi-item simultaneous analyser, COBAS INTEGRA 400 plus (Roche
Diagnostics, Switzerland).
Evaluation of endogenous substances. In general, themagnitude of the binding inhibition capacity of the HSA

site is dependent on the ratio of the inhibitor concentration
to a mole of HSA [12]. Accordingly, the procedure of evaluation by the ratio of [endogenous substance]/[HSA] wasutilized since the level of inhibition of IS, HA and FFA
was different between pre-HD and post-HD. The moleculeweight of HSA was set as 66 500 [13,14].
Statistical analysis
Statistical significance was evaluated using Student’s paired
t-test to analyse differences between two groups, pre-HD

Alteration of binding capacities of site II by haemodialysis

Fig. 2. Free fractions of 14 C-warfarin (A) and 14 C-diazepam (B) pre-HDand post-HD. (A) Lines are for expressing a variation in two values offree fractions of 14 C-warfarin pre-HD and post-HD. (B) Lines are forexpressing a variation in two values of free fractions of 14 C-diazepam
pre-HD and post-HD. Thick lines mean that the rate post-HD was higher
than that pre-HD.

and post-HD. The P value for statistical significance wasset at <0.05. Data are expressed as mean ± S.D.

Results
Alterations in the binding capacities of sites I and II
between pre-HD and post-HD
To evaluate the binding affinity of HSA sites I and II, respectively, the free fractions of 14 C-warfarin (site I) and 14 Cdiazepam (site II) as site probes were calculated. Increases
in the free fractions of 14 C-warfarin and 14 C-diazepam indicate decreases in the binding capacities of HSA sites I and
II. The decrease in the free fraction indicates an increase in
the binding capacity of the site. Figure 2A shows that thefree fractions of 14 C-warfarin in all 14 patients decreased
post-HD compared to pre-HD (post-HD/pre-HD ratio: 0.59
± 0.13). The free fractions of 14 C-diazepam in 10 of 14 patients remarkably decreased post-HD compared to pre-HD(post-HD/pre-HD ratio: 0.61 ± 0.17), and the free fractionsof 14 C-diazepam in 4 patients (nos. 3, 7, 13, 14) increased
post-HD (post-HD/pre-HD ratio: 1.08 ± 0.06). In these four
patients, HSA concentration post-HD was about 1.2 times
as much as pre-HD (Figure 2B).

2307

Fig. 3. The variation of 14 C-diazepam binding of HSA concentration between pre-HD and post-HD. Lines indicate each patient whose
14 C-diazepam free fraction increased post-HD. : experimental values
pre-HD, : experimental values post-HD.

In general, drug binding affinity is increased at sites I
and II of HSA because waste substances in the blood areremoved by HD and the blood is purified [8,15]; however,the binding capacities of HSA site II in the four patients(nos. 3, 7, 13, 14) decreased post-HD. To elucidate the factors in the decreases of the binding capacities in these four
patients, we investigated the effects of endogenous substances on HSA binding of 14 C-diazepam (14 C-diazepam
free fraction).
Alterations in concentrations of HSA and endogenous
substances
Table 2 shows the alterations in concentrations of HSA
and endogenous substances. The concentration of HSA wassignificantly increased post-HD compared to pre-HD. Thismeans that pre-HD serum was haemoconcentrated postHD, i.e. indicating that the number of binding sites increased. In uraemic toxins (CMPF, IS and HA) and FFA,
which have high binding affinities to HSA sites, the concentration of CMPF was not significantly altered but theconcentrations of IS and HA were significantly reduced,
and the concentration of FFA was significantly increased,
post-HD compared to pre-HD. Therefore, we investigated

Table 2. Concentrations of HSA, CMPF, IS, HA and FFA (n = 14)

HSA
(µM)
CMPF (µM)
IS
(µM)
HA
(µM)
FFA (µEq/L)

Pre-HD patients (a)Range

Post-HD patients (b)Range

Ratio (b)/(a)Range

Statistics (a) versus (b)554.2 ± 77.8
78.4 ± 24.1
160.4 ± 69.0
282.6 ± 341.0
251.9 ± 359.3

436.1–706.8
27.5–112.5
47.7–278.7
20.1–1395.5
43.9–1344.0

656.3 ± 105.9
76.2 ± 25.6
108.8 ± 42.5
78.5 ± 89.1
721.6 ± 528.6

451.1–887.2
27.5–112.5
33.0–188.8
16.1–372.3
189.2–1939.7

1.18 ± 1.10
0.97 ± 0.12
0.69 ± 0.10
0.33 ± 0.15
5.46 ± 4.91

1.03–1.45
0.67–1.09
0.53–0.84
0.16–0.80
0.78–20.28

P = 0.000006 < 0.00001
P = 0.47, N.S.
P = 0.00007 < 0.0001
P = 0.0099 < 0.01
P = 0.002 < 0.005

Each value represents the mean ± S.D. (n = 14). Statistical analysis was performed with paired Student’s t-test. N.S.: not significantly different.

2308

T. Nishio et al.

Fig. 4. Inhibition of uraemic toxins at site II of HSA; IS (A) and HA (B), 14 C-diazepam binding of HSA pre-HD and post-HD. (A) Lines indicate eachpatient whose 14 C-diazepam free fraction increased post-HD. : experimental values (ratios of [IS]/[HSA]) pre-HD, : experimental values (ratios of[IS]/[HSA]) post-HD. (B) Lines indicate each patient whose 14 C-diazepam free fraction increased post-HD. : experimental values (ratios of [HA]/[HSA ]) pre-HD, : experimental values (ratios of [HA]/[HSA]) post-HD.

whether the effects on the binding capacity of HSA site II
were due to HSA, IS, HA and FFA, which showed a significant difference in concentration.
Effects of concentrations of HSA and endogenous
substances on binding capacity of site II
Effects of HSA concentrations. To evaluate the effects of
HSA concentrations on the binding capacity of site II between pre-HD and post-HD, the relationship between the
14
C-diazepam free fraction and HSA concentration was investigated (Figure 3). The HSA concentrations increased
about 100 µM post-HD as compared to pre-HD in the four
patients. HSA concentrations post-HD were about 1.2 times
those pre-HD, indicating an increase in the binding capacityof site II; however, the free fraction of 14 C-diazepam wasincreased in the four patients. It was suggested that the driving force of the decrement of the binding capacity of site
II by other factors was greater than the driving force of theincrement of the binding capacity of site II by the increasein HSA concentration. To evaluate the influences of IS, HA
and FFA on the inhibitory effects on HSA site II, the relationship between the 14 C-diazepam free fraction and eachof [IS]/[HSA], [HA]/[HSA] and [FFA]/[HSA] was investigated (see ‘Evaluation of endogenous substances’ underthe ‘Materials and methods’ section).
Effects of uraemic toxin concentrations. To evaluate theeffects of the uraemic toxins, IS and HA on the binding capacity of site II between pre-HD and post-HD, therelationship between the 14 C-diazepam free fraction and[IS]/[HSA] and [HA]/[HSA] was investigated. [IS]/[HSA]
and [HA]/[HSA] decreased post-HD compared to pre-HD
because IS and HA were removed from the blood by HD[16]. However, the 14 C-diazepam free fraction increased inFig. 5. Inhibition of FFA to 14 C-diazepam on site II of HSA pre-HD andpost-HD. Lines indicate each patient whose 14 C-diazepam free fraction
increased post-HD. : experimental values (ratios of [FFA]/[HSA]) preHD, : experimental values (ratios of [FFA]/[HSA]) post-HD.

spite of decreases in [IS]/[HSA] (post-HD/pre-HD ratio:
0.61 ± 0.03) and [HA]/[HSA] (post-HD/pre-HD ratio:
0.40 ± 0.21) in four patients (Figure 4A and B). It wassuggested that the driving force of the decrement of thebinding capacity of HSA site II by other factors was greaterthan that of the decrement of the binding capacity of site II
by the decrease of IS and HA.
Effects of FFA concentrations on the binding capacity ofsite II. To evaluate the effects of FFA on the binding capacity of site II between pre-HD and post-HD,

Alteration of binding capacities of site II by haemodialysis

2309

Fig. 6. In the four patients (nos. 3, 7, 13, 14), the mechanism of inhibitions pre-HD and post-HD. The allosteric and competitive inhibition: the allostericinhibition mechanism of 14 C-diazepam bound to HSA site II by FFA. FFA binds to the primary site of FFA on HSA. FFA bound to the primary siteof FFA on HSA provided an allosteric inhibition on 14 C-diazepam bound to site II (the secondary site of FFA). The competitive inhibition mechanismof FFA for 14 C-diazepam bound to HSA site II. FFA inhibits competitively at site II and displaces 14 C-diazepam from site II (direct process). Cascadeeffect: the cascade inhibition mechanism of FFA and uraemic toxins to 14 C-diazepam at HSA site II. Each FFA displaces uraemic toxins at HSA site II
which is the secondary binding site of FFA allosterically and competitively. The displaced uraemic toxins competitively inhibit diazepam bound earlier
to another HSA site II. Then, diazepam is displaced by competitive inhibition of the uraemic toxins. White arrows: allosteric inhibition at site II. Thincurved arrows: competitive inhibition at site II. Broken curved arrows: displacement or desorption from site II by inhibition.

the relationship between the 14 C-diazepam free fractions
and [FFA]/[HSA] was investigated. The free fraction of
14
C-diazepam increased, and [FFA]/[HSA] also increased
remarkably in the four patients (post-HD/pre-HD ratio:
6.91 ± 6.58). Each ratio of [FFA]/[HSA] post-HD wasover 1.2 in the four patients (range of [FFA]/[HSA] ratio:
1.22–3.55) (Figure 5).
From the result above, it was suggested that the increaseof the FFA concentration mainly contributed to the decrement of the binding capacities of site II in the four patients.

Discussion
When we estimated the binding capacities of sites I and II of
HSA between pre-HD and post-HD, we found that, in all 14
patients post-HD, the binding capacities of site I increased
and, unexpectedly, the binding capacities of site II did notincrease. The binding capacities of site II in 4 of 14 patientsdecreased post-HD. It was suggested that the factors in theincrease of the binding capacities of sites I and II were theincrease in HSA concentration by haemoconcentration andthe decrease in IS and HA concentration by HD. However,the cause of the decrease in the binding capacity of site II atpost-HD was not the increment of the HSA concentrationor the decrease in the IS and HA concentrations, but rather
the remarkable increase in the FFA concentration in the four
patients. The inhibitory effects of FFA were considered to
exceed the promoting effect on the binding capacity of HSA
site II. The inhibition mechanism of FFA for the unique reduction of the binding capacity of site II is discussed below.

Inhibition mechanisms of FFA for the reduction of thebinding capacity of site II
In general, the heparin used in HD activates lipase in the
blood and increases the concentration of FFA remarkably[17–19]. It has been reported that the binding capacity ofsite II was decreased by increasing FFA concentration [20].
From these reports, there is no doubt that the increment of
FFA contributed to the reduction of site II binding capacity.
In the four patients (nos. 3, 7, 13, 14), it was assumed
that FFA competitively inhibited 14 C-diazepam bound tosite II of HSA, because the region of site II coincides withthe secondary binding site of FFA on HSA and the binding
constant K value of the secondary site for FFA, 107 M−1[21] is greater than that of diazepam, 3.8 × 105 M−1 [22].
Therefore, when the ratio [FFA]/[HSA] increases, FFA,
which retains high binding affinity to HSA, also inhibitssite II (the secondary site of FFA), to which 14 C-diazepam
bound directly [23]. Thus, the inhibitory mechanism postHD was considered to add not only allosteric inhibition(weak) but competitive inhibition (strong) [8], since the ratio [FFA]/[HSA] in three of the four patients (nos. 3, 7, 14)
was <0.5 pre-HD and >1.2 post-HD. The binding capacity of site II post-HD in comparison with that of pre-HD
is remarkably influenced by the change of these allostericand competitive inhibitions. Because the [FFA]/[HSA] ratio of one (no. 13) of the four patients pre-HD was high at
2.98, the binding inhibition of FFA at site II was considered
to contribute not only to allosteric inhibition but also to
the competitive inhibition. The effect of competitive inhibition in site II post-HD would be therefore greater than its

2310

inhibition pre-HD, since the ratio [FFA]/[HSA] increased
to 3.55 post-HD. In those cases, if uraemic toxins bound tosite II when competitive inhibition of site II was produced
by increasing FFA remarkably, the uraemic toxins would
be inhibited by increasing FFA and would bind to the other
HSA site II, possibly displacing 14 C-diazepam competitively. Thus, it was suggested to be a good possibility thatthe mechanism of the synergistic inhibitory ‘cascade effect’
was produced by the chain inhibition of FFA and uraemic
toxins at site II [8].We considered that unexpected reduction of the binding affinity of site II in the four patients wasproduced by adding the allosteric inhibition to competitive
inhibition of FFA and by the ‘cascade effect’ of FFA anduraemic toxins (see Figure 6).
Strategy of administration
In dosage plans that utilize binding inhibition, we consider
that if a drug binding with high affinity to site I is administered pre-HD, the free fraction of the drug can beincreased. On the other hand, since a drug binding to site
II is sometimes inhibited by increasing FFA remarkably
post-HD, we consider that a drug binding to site II of HSA
should be medicated with the optimum timing. A drug that
could utilize the drug binding inhibition of HSA for effective administration plans should possess the properties ofpotent protein binding, with a small volume of distribution
and high distribution to the target tissue. It is expected thata drug with these properties can increment distribution to
the target tissue by utilizing the binding inhibition on sites
I and II of HSA by HD. One drug that can be used for
efficacious administration using binding inhibition is ketoprofen, which is used as an analgetic and antiphlogistic
for the pain and inflammation of arthrosteitis in HD patients, as an example. It has been reported that this drug
binds to site II of HSA strongly and is greatly inhibited byincreasing uraemic toxins and FFA in HD patients. Therefore, we consider that ketoprofen may be highly distributed
to inflammatory tissue by using the inhibition of protein
binding.
In conclusion, this study elucidated that a significant increase in FFA sometimes reduces the binding capacity of
HSA site II post-HD compared to pre-HD. Consequently,
we conclude that monitoring the binding capacity of site IIof HSA between pre-HD and post-HD is clinically important for patients with ESRD in planning efficacious administration utilizing the binding inhibition of HSA.
Conflict of interest statement. None declared.

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Received for publication: 11.5.07
Accepted in revised form: 31.12.07