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ALBUMIN, BILIRUBIN, AND ACTIVATEDCARBON: NEW EDGES OF AN OLD TRIANGLE
Veronika V. Sarnatskaya,1,* W. Edward Lindup,2 Paul Walther,3
Vitaly N. Maslenny,1 Larisa A. Yushko,1 Alexej S. Sidorenko,1
Andrey V. Nikolaev,3 and Vladimir G. Nikolaev1
1Department of Artificial Organs, R. E. Kavetsky
Institute of Experimental Pathology, Oncology and Radiobiology
of the National Academy of Sciences, 45, Vasilkovskaya Street,
Kiev, 03022, Ukraine2Department of Pharmacology and Therapeutics, University of
Liverpool, P.O. Box 147, Liverpool L69, UK3Sektion Elektronenmikroskopie Universitat Ulm,
Albert-Einstein-Allee II, D-89069 Ulm, Germany
ABSTRACT
The problem of interaction of human serum albumin (HSA),
unconjugated bilirubin (UB) and high porosity activated HSGD
carbons is investigated in this study. The decrease of UB to HSA
molecular ratio by more than 300 times was demonstrated while
the batch experiments in HSA–UB admixtures after contact with
HSGD. HSGD carbons express extremely high activity for the
removal of UB from HSA containing solutions (more than 100 mg
of UB per 1 g of activated carbon). Ex-tempore albumin-coating of
* Corresponding author. E-mail: [email protected]
113
ART. CELLS, BLOOD SUBS., AND IMMOB. BIOTECH., 30(2), 113–126 (2002)
Copyright D 2002 by Marcel Dekker, Inc. www.dekker.com
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carbon surface decreases adsorbent capacity by bilirubin on 21%.
At the same time ex-tempore albumin-coating of HSGD carbon
surface as well as blood citratization prevent platelet and leuko-
cytes loss and clotting inside of the column. Pharmacopoeia solu-
tion of HSA containing acetyl-tryptophan or octanoate used for
albumin-coating of HSGD adsorbents, becomes ligand-free and
rather more active in complexing with protein-bound substances.
Combination of albumin-coated HSGD carbon as haemosorbent
with HSA ligand-free solution as a transfusion media seems a new
prospective modality of the extracorporeal removal of protein-
bound toxins.
Key Words: Liver failure; Albumin-coated adsorbents; Bilirubin
INTRODUCTION
It is known that activated charcoal easily adsorbs human serum albumin
(HSA). Adsorptive capacities of charcoals can range from several up to hund-
reds of mg of protein per gram of carbonic matrix depending on its structure and
albumin concentration.[1,2] At the same time, activated charcoals of appropriate
structure possess the high enough capacity and good kinetic properties in respect
to UB mixed with albumin solution.[3] UB is a very suitable marker for
evaluation of adsorbents capacities to remove albumin-bound substances
because of its high association constant (primary site assoc. const� 108 M � 1)
with this protein. So, if the adsorbent activity by bilirubin is sufficient, other
protein-associated markers of hepatic insufficiency like phenols, bile acids, free
fatty acids, mercaptans etc. could also be removed easily.[4]
In this study in-vitro experiments are used to answer some important
questions concerning interaction between albumin and bilirubin adsorption onto
the surface of the highly activated carbon HSGD which was developed in our lab
especially for removal of strongly protein bound substances. In parallel, prob-
lems of adsorbent haemocompatibility and ex-tempore preparation of ligand-free
HSA solution are discussed.
MATERIALS AND METHODS
Chemicals
The following analytically pure reagents were used: crystalline human
serum albumin (fraction V, Mr 66.500 essentially fatty acid-free, bilirubin,
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sodium caprylate, salicylic acid, phenol, sodium salt of deoxycholic acid (Sigma
Chemical, Poole, UK), HSGD (HemoSorbent Granulated Deliganding, IEPOR,
Kiev, Ukraine). All other reagents were of analytical grade and used without
additional purification.
Adsorptive Treatment
Albumin solution (30 mg/ml) for batch and perfusion experiments was
prepared from pharmaceutical human serum albumin (100 mg/ml) for intra-
venous infusion. Albumin–bilirubin solutions (0.2 mg/ml) were made using
crystallized nonconjugated bilirubin. The desired weight of bilirubin was
initially dissolved in a minimal amount of 0.1 M sodium hydroxide and the
final volume was adjusted by solution of albumin in sodium phosphate buffer.
Batch experiments were carried out by shaking 50 mg carbon HSGD with
6 ml bilirubin–albumin solution for 120 minutes at room temperature. The
assays for initial and equilibrium concentrations of bilirubin solution were done
according to the commercial protocol (605-S Sigma Diagnostics) or by protocols
described in the literature.[5] Solution concentration of albumin was determined
by spectrophotometer.[6] Albumin and bilirubin concentrations were measured
thrice and an average result was calculated.
The perfusion experiments were carried out in three parallel micro-
columns with independent micro-pump channels and with a common inlet
solution by pumping albumin–bilirubin solution at a rate of 1.25 ml/min through
10 cm3 micro-columns (with a height-to-diameter ration 7:2 cm) packed with
HSGD adsorbent. Samples were taken from the outlet of all three columns at
15, 30, 60, 90, 120, 180 and 240 minutes of perfusion.
Albumin coating of HSGD adsorbent was made by perfusion of fatty
acids free albumin solution of HSA (100 ml) through the micro column at a rate
of 1 ml/min.
The working protocol included 10 healthy donors (5 males and 5 females)
aged 35–53 years. The whole blood was obtained in the amount of 410 ml per
person and divided into 135 ml and 275 ml portions. The first portion was
mixed with 27 ml of standard ACD solution, and the second one was mixed
with 50 ml of 0.9% sodium chloride solution containing 2000 IU of Fraxipar-
ine. The heparinized fresh blood divided in 2 equal portions was immediately
pumped through micro-columns containing 500 mg of albumin-coated and
uncoated HSGD. The perfusion was carried out at a rate of 1.25 ml/min for 2
hours. The portion of blood stabilised by ACD solution was pumped through
uncoated HSGD at a similar rate for 2 hours. The drop of pressure at the inlet
and outlet of micro-column was measured by an EMT-035 electromanometer
(Elema-Shonander, Sweden).
ALBUMIN, BILIRUBIN, AND ACTIVATED CARBON 115
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Leukocyte and platelet counts were performed by standard microscopic
methods described elsewhere.[9]
Differential Scanning Microcalorimetry (DSMC)
The melting thermograms of defatted albumin preloaded by bilirubin and
purified albumin were recorded before and after adsorptive treatment on a
DASM-4 microcalorimeter (Biopribor, Puschino, Russia) at a scanning rate of
1 �C/min. The working concentration of albumin was 5.0 mg/ml. The temperature
maximum T1 and T2 of the components were obtained by mathematical de-
convolution[7] of the melting curve. Preliminary experiments with threefold re-
peated measurements of albumin thermal denaturation demonstrated that a
deviation of temperature maximum of more than 0.5 �C coincided with a
significant level of p < 0.01.
Flow Microcalorimetry (FMC)
The temperature effects of albumin coupling with different marker ligands
(sodium octanoate, salicylic acid, phenol red, deoxycholic acid (sodium salt) in
equimolar concentrations were measured by a flow microcalorimeter 2277
Thermal Activity Monitor (LKB, Bromma, Sweden) at ambient temperature.
The enthalpies of complexing were calculated.[8]
Table 1. Changes in the Concentrations of Nonconjugated Bilirubin and Albumin After 2
Hours of Contact with Activated Carbon in Batch Experiment (Bilirubin–Albumin
Solution—6 mL, HSGD—0.05 g; n = 5; M ± SD)
Conditions
Substance Before After 2 Hours
Nonconjugated bilirubin, mg/dL 17.65 ± 1.04 0.38 ± 0.08
Albumin, g/L 27.38 ± 0.26 17.82 ± 0.18
Figure 1. First two pictures (A, B) were done in low resolution mode to demonstrate
shape parameters of granules of HSGD carbons. Granule in the middle of the first picture
was taken for high magnification examination. Three last pictures (C, D, E) are con-
sequent magnification zooms of the same area on the granule. Scale bars are provided in
the bottom right corner of each picture. Numbers correspond to the length of the entire
scale bars.
ALBUMIN, BILIRUBIN, AND ACTIVATED CARBON 117
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Scanning Electron Microscopy (SEM)
Due to the well defined shape of HSGD granules, electron microscopy of
the granules was relatively straightforward. Some number of granules were
placed into a plastic Petri dish. The support plate, with double-sided sticky tape,
was lightly pressed into granules crowd by taped side from above. Later, to
granules which stuck on the tape the special conductive silver paste was applied
from the side in order to provide better mechanical stability and high electron
beam delivered charge removal rate. In the result the sides of granules available
for electron microscopy examination were not covered by any agent or metal or
subjected to any extra processing after rinsing and drying. Thus it is highly
likely that observed surface properties such as microporous structure are not
artefact of preparation method. The scanning electron microscope used was a
Hitachi S-5200 in-lens field emission; the accelerating voltage was 4 kV. Images
were recorded using the secondary electron signal.
RESULTS
Figure 1 describes an SEM picture of the adsorbent external surface. Under
the small magnification spherical adsorbent granules look solid and smooth
enough. But under the higher magnifications one can see that the adsor-
bent surface consists of a great number of open pores. Calculations (omitted)
Figure 2. Melting curves of HSA: (A) defatted HSA; (B) loaded with unconjugated
bilirubin up to bilirubin–albumin molecular ratio 0.74; (C) purified onto HSGD carbons
up to molecular ratio 0.0024.
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give the part of volume of these granules occupied with carbon itself not ex-
ceeding 5–10% of granules total volume.
Table 1 reflects the fact that HSGD carbon activity adsorbs both UB and
albumin from its mixture. Under the particular conditions used here the bilirubin
concentration drop was 97.8% and the albumin concentration drop was 34.9%
with adsorbent capacity for albumin 1147 mg/g and for bilirubin—20.7 mg/g.
The bilirubin–albumin molecular ratio after adsorptive treatment was dimin-
ished from 0.74 to 0.0024, or by approximately 310 times. A high level of
purification of residual (unadsorbed) albumin was also demonstrated by marked
changes in HSA melting curves, which after treatment approached the melting
curve of analytically pure defatted albumin (Figure 2).
Table 2. Average Results of 3 Microcolumn Experiments with Albumin-Coated Activated
Carbon and Saline Rinsing of 0.5 g HSGD Charcoal (HSA Inlet Concentration—81 g/L,
Perfusion Rate—1.25 ml/min)
Time (min)
15 30 45 60 75
Albumin Concentration, g/L
Albumin-coated Outlet 75.8 80.0 81.0 81.0 81.0Pool 48.7 64.8 70.0 72.8 74.6
Rinsing Outlet 0.701 0.492 0.472 0.534 0.539Pool 11.5 6.2 3.6 3.0 2.6
Figure 3. Average outlet bilirubin concentration curves for uncoated (5) and albumin-
coated (.) HSGD carbon.
ALBUMIN, BILIRUBIN, AND ACTIVATED CARBON 119
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In Table 2 one can see that during albumin-coating the outlet concen-
tration of HSA becomes equal to the inlet concentration after 45 min of
perfusion (adsorbent saturation). At this time the column retains approximately
7.9% of pumped albumin, or 1186 mg of protein per gram of adsorbent. During
the rinsing of the column with saline a zero outlet concentration of albumin
could not be reached by 75 min of perfusion but, from a comparison of the outlet
and pool concentration data, one can conclude that the main albumin release
takes place during the first 15 minutes. Total protein release consisted of 3.28%
of previously pumped albumin or 492.4 mg per gram of adsorbent. Conse-
Table 3. Albumin Consumption with Uncoated and Albumin-Coated HSGD Carbon
(Average Results from 3 Microcolumn Experiments; HSA Inlet Concentration—34.3 g/L)
Time (min)
15 30 60 90 120 180 240
Albumin Concentration, g/L
Uncoated Outlet 20.7 31.9 33.2 33.2 33.8 33.8 33.8Pool 8.93 18.4 26.5 28.8 29.9 31.6 32.7
Albumin-coated Outlet 34.3 34.1 34.1 34.1 33.8 33.8 33.8Pool 25.5 29.7 31.3 32.4 32.4 32.7 32.7
Figure 4. Mean flow resistance (DP) of microcolumns during the perfusion with fresh
donor blood.
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quently, the HSA portion, more or less strongly fixed onto the HSGD carbon
surface amounted to nearly 5.6% of the perfused protein, or 693 mg per gram.
Figure 3 shows the curves of outlet concentrations of UB for columns
containing uncoated or albumin-coated HSGD carbon. Both columns exhibit
extremely high activity for removal of UB from the albumin–bilirubin mixture,
but adsorptive capacity of albumin-coated adsorbent is 21.4% less than that of
uncoated one (80 mg of UB per gram of adsorbent versus 63 mg/g, respectively).
These data are actually for 240 min of perfusion time but at the end of the
experiment both columns still preserved essential adsorptive capacity. Linear
approximation of the outlet curves up to the inlet level of bilirubin concentration
(dotted lines) gave estimates of the complete capacity of uncoated and albumin-
coated carbon as 113 mg/g and 88 mg/g, respectively.
Difference in the outlet HSA concentration for albumin-coated and
uncoated HSGD (Table 3) was evident at the beginning of the experiment and
became negligible with the increase of the amount of perfused protein as the dry
Table 4. Evaluation of Statistical Significance of the Difference in Leukocytes and
Thrombocytes Count Between Inlet and Outlet of the Microcolumns HSGD Carbon
Perfusing with the Fresh Donor Blood for 120 Min
Uncoated HSGD,
heparinization
Leukocytes Significant (p < 0.01) 35–50% drop
in 30 and 45 min time-periods
Thrombocytes Significant (p < 0.01) 25–40% drop
in 30 and 45 min time-periods
Albumin-coated HSGD, Leukocytes Non-significant in all time-periods
heparinization Thrombocytes Non-significant in all time-periods
Uncoated HSGD, Leukocytes Non-significant in all time-periods
citratization Thrombocytes Non-significant in all time-periods
Figure 5. Melting curves of officinal HSA solution for transfusions before (A) and after
(B) its purification onto HSGD carbons in the regimen, similar to the adsorbent coating
with albumin. (C) melting curves of defatted HSA, ‘‘Sigma,’’ USA.
ALBUMIN, BILIRUBIN, AND ACTIVATED CARBON 121
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weight of latter exceeds the weight of sorbent in 257 times at the end of
perfusion. At the same time, albumin-coating strongly influenced the microcol-
umn flow resistance in the case of the heparinized human blood in-vitro per-
fusion. The rapid increase of flow resistance and ‘‘caking’’ of uncoated HSGD
carbon (Figure 4) coincided with the remarkable consumption of platelets and
leukocytes inside the microcolumn (Table 4).
In the case of albumin-coating of the adsorbent or citratization of blood
there were no platelets and white blood cells loss observed and an increase of
microcolumn flow resistance with time was very slow (Figure 4, Table 4).
If an official (pharmacopeial) solution of HSA for i.v. transfusion was
pumped through the column the melting curve of HSA at the outlet looks quite
different from its initial shape and very similar to the melting curve of the pure
defatted HSA (Figure 5).
Table 5 describes the difference in transport abilities of the official and
purified HSA in relation to four marker ligands for the main binding sites of
albumin. Table 5 shows that pharmacopeial albumin after exposure to HSGD
carbon exhibited enhanced binding towards each of the ligands. This is demon-
strated by the enhancement of the appropriate microcalorimetric parameter Hc
(enthalpy of complexing). This purified preparation in its complex-forming
abilities was very close to analytically pure ‘‘Sigma’’ fatty free HSA (Table 5).
DISCUSSION
Batch experiments with HSGD carbon demonstrated very good adsorption
of UB from defatted HSA solution. (Table 1). In such experiments it is difficult to
compare different capacities of adsorbents because of different levels of residual
(equilibrium) concentration of adsorbed substances. However, despite a very low
final concentration of UB, capacity of HSGD adsorbent (20.1 mg/g) is definitely
higher than that of both conventional synthetic hemosorbent (SCN, 0.8–1.1 mg/
g) and of previous version of HSGD carbon (5–15 mg/g).[4]
Table 5. Comparation of Flow Microcalorimetry Data (DHc, kcal/mole) Reflecting
Complex-Forming Abilities of Main Binding Sites of Different Samples of HAS
Ligands
Pharmacopeial
HSA for i.v.
Transfusion
Pharmacopeial
HSA After the Contact
with HSGD Carbon
Fatty Acid
Free HSA
Sodium octanoate 0 36.9 ± 0.52 37.8 ± 0.46
Phenol 11.3 ± 0.30 27.6 ± 0.34 28.3 ± 0.36
Sodium salicylate 15.86 ± 0.60 24.8 ± 0.57 24.0 ± 0.60
Sodium deoxycholate � 2.6 ± 0.30 � 16.5 ± 0.44 � 15.9 ± 0.35
SARNATSKAYA ET AL.122
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Because of relatively high amount of adsorbent in comparison with the
amount of protein (50 mg of carbon against 164 mg of albumin) albumin loss
was also apparent (35%). Anyway, the ligand-carrier molecular ratio was di-
minished by more than 300 times, which reflects the high quality of purification
of residual 65% of albumin from tightly (association constants of the order of
108 M � 1)protein-bound ligand. The results of differential scanning microcalo-
rimetry also demonstrated that the molecular conformation of this purified albu-
min was very similar to that of a Sigma fatty acid-free standard.[10]
In a dynamic experiment with microcolumns and high (81 g/l) concen-
tration of HSA the saturation of HSGD carbon occurred after 30–45 minutes of
perfusion. The protein–sorbent ratio weight in these experiments was 152 or 46
times higher than in the previous case. So, the proportion of protein retained
with the adsorbent consisted of only 7.9%. After rinsing with saline solution the
weakly bound fraction of albumin molecules was washed out mainly in the first
15 minutes and the residual amount of protein consisted of 693 mg per gram of
HSGD adsorbent. This was a significant amount in relation to the weight of
adsorbent, so such an adsorptive system could be defined as protein-carbonic,
but not purely carbonic.
The above-described ex-tempore albumin-coating diminished the adsorp-
tive capacity of HSGD carbons by UB by at least 20%. It is a typical situation for
all kinds of coating, regardless of its chemical nature and origin of the polymers
used, and derives from polymer-induced enhancement of diffusion resistance on
the border between liquid and solid phases.[11] At the same time, a decrease in the
capacity of HSGD adsorbents by bilirubin could not be explained by the com-
petitive adsorption of another ligand, because the protein used for the preparation
of the bilirubin–HSA mixture was highly purified and fatty-acid-free. These
results contradict some previous data[12] where the authors found that pretreatment
of adsorbents by albumin enhanced the adsorptive capacity of activated carbons
towards direct and indirect bilirubin. This disagreement could be explained by the
difference between the adsorbents that were used: if adsorbent has a small capacity
for bilirubin removal from albumin solutions, adsorption of pure albumin onto the
surface of a less-effective carbonic adsorbent can overcome the negative effect of
the coating by this biopolymer. Nevertheless in our experiment HSGD carbon
demonstrated an impressive adsorptive capacity of 113 mg/g for uncoated and 80
mg/g for albumin-coated adsorbent. Extrapolation of these data to real plasma-
perfusion conditions, would mean that 10 grams of HSGD carbon would be
enough for removal of 600–800 mg of bilirubin per session. This amount of
adsorbent could be compared with the 875 g of adsorptive materials, recently used
by Italian authors to achieve the similar bilirubin removal rate.[13]
However, plasmoperfusion is not the only use for HSGD adsorbent. One
can see that (Figure 4, Table 4) albumin-coated grains of this carbon demon-
strate stable hydrodynamics and good haemocompatibility properties during in
vitro contact with whole human blood. Uncoated carbon starts to initiate the
ALBUMIN, BILIRUBIN, AND ACTIVATED CARBON 123
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clotting of heparinized blood and catch blood cells after 30 minutes of perfusion,
which is quite expectable taking into account the rough microstructure of the
granules’ external surface (Figure 1). Thus microcolumn flow resistance rapidly
goes up and plugging occurs between 40 and 45 minutes of perfusion. In total,
these findings are in good agreement with the classical data of Chang,[14] who
demonstrated first the remarkable role of albumin-coating of charcoal surface in
the prevention of blood cell trauma and column plugging. At the same time,
citratization of blood gives pressure drop results similar to albumin-coated
adsorbent, and enhances some other parameters of adsorbent haemocompati-
bility. This coincides with result of [15] also obtained much earlier.
Nevertheless, albumin-coating plus heparinization remains very attractive
treatment modality, especially taking into account the expanding role of massive
albumin transfusion in the treatment of hepatic coma and some complications of
ascites.[16] The use of conventional pharmacopeial solution of HSA for intra-
venous transfusion instead of analytically pure defatted albumin for ex-tempore
coating of HSGD carbon leads to drastic changes in properties of pharmacopeial
HSA solution because of the effective removal of thermostabilizers (octonoate,
n-acetyl-tryptophan) and some natural hydrophobic ligands by adsorbent. The
molecular conformation of this albumin and its complex-forming abilities come
close to those of defatted analytically pure protein or liquid protein adsorbent
Albomax.[17]
Transfusion of such ‘‘activated’’ HSA before or during extracorporeal
sessions could be useful for attracting hydrophobic toxins from the tissue
compartment into the blood stream and to make adsorptive treatment more
effective.[18] If HSA transfusion is used in encephalopathic patients, one should
remember that the caprylate (octanoate) anion is traditionally described as an
important encephalotoxin[19] and consequently octanoate-free albumin solution
is preferable for the treatment of hepatic coma and precoma. Nothing will be
lost, however, if a part of the adsorptive capacity of adsorbent is used for
octanoate removal from the HSA transfusion solution, because this small deficit
of column efficacy should be compensated for by the enhancement of complex-
forming activity of transfused HSA. So, a combination of albumin-coated HSGD
adsorbent with the transfusion of purified albumin onto this adsorbent looks to
be an attractive new modality of the treatment of some end-stage hepatic
diseases and its complications.
CONCLUSIONS
1. Deligandizating granulated hemosorbents HSGD express extremely
high activity (100 mg/g) for the removal of unconjugated bilirubin
from albumin solution.
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2. Ex-tempore albumin-coating of carbon surface decreases adsorbent
capacity by bilirubin on 21%.
3. Ex-tempore albumin-coating of HSGD carbon surface as well as
blood citratization prevent platelet and leukocytes loss and clotting
inside of the column.
4. Conventional (pharmacopeial) solution of HSA used for albumin-
coating of HSGD sorbents, becomes octanoate-free and rather more
active in complexing with protein-bound ligands.
5. Combination of albumin-coated HSGD carbon with ligand-free solution
of HSA seems to be a new prospective modality of extracorporeal
biochemical correction in patients with hepatic insufficiency.
ABBREVIATIONS
HSGD haemosorbent granulated deliganding, new generation of activated
carbons
HAS human serum albumin
UB unconjugated bilirubin
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