Download Alterations in the functional capacity of albumin in patients with

LIVER FAILURE/CIRRHOSIS/PORTAL HYPERTENSION
Alterations in the Functional Capacity of Albumin in
Patients with Decompensated Cirrhosis Is Associated
with Increased Mortality
Rajiv Jalan,1 Kerstin Schnurr,2 Rajeshwar P. Mookerjee,1 Sambit Sen,1 Lisa Cheshire,1 Stephen Hodges,1
Vladimir Muravsky,2 Roger Williams,1 Gert Matthes,2,3 and Nathan A. Davies1
Albumin concentration is diminished in patients with liver failure. Albumin infusion improves
survival of cirrhotic patients with spontaneous bacterial peritonitis, and it is hypothesized that
this may be due in part to its detoxifying capabilities. The aim of this study was to perform
detailed quantitative and qualitative assessment of albumin function in patients with cirrhosis.
Healthy controls and patients with acute deterioration of cirrhosis requiring hospital admission
(n ⴝ 34) were included. Albumin function was assessed using affinity of the fatty acid binding
sites using a spin label (16 doxyl-stearate) titration and electron paramagnetic resonance spectroscopy and ischemia-modified albumin (IMA) was measured. Twenty-two patients developed
acute-on-chronic liver failure. Twelve were treated with the Molecular Adsorbents Recirculating
System (MARS) and 10 with standard medical therapy. For each parameter measured, the
patients’ albumin had reduced functional ability, which worsened with disease severity. Fifteen
patients died, and IMA, expressed as an albumin ratio (IMAR), was significantly higher in
nonsurvivors compared with survivors (P < 0.001; area under the receiver operating curve ⴝ
0.8). No change in the patients’ albumin function was observed following MARS therapy. A
significant negative correlation between IMAR and the fatty acid binding coefficients for sites 1
and 2 (P < 0.001 for both) was observed, indicating possible sites of association on the protein.
Conclusion: The results of this study suggests marked dysfunction of albumin function in advanced cirrhosis and provide further evidence for damage to the circulating albumin, which is not
reversed by MARS therapy. IMAR correlates with disease severity and may have prognostic use in
acute-on-chronic liver failure. (HEPATOLOGY 2009;50:555-564.)
See Editorial on Page 355
Abbreviations: ACLF, acute-on-chronic liver failure; AUROC, area under the
receiver operating curve; EPR, electron paramagnetic resonance; IMA, ischemiamodified albumin; IMAR, IMA/albumin ratio; MARS, Molecular Adsorbents Recirculating System; MELD, model of end-stage liver disease.
From the 1Liver Failure Group, Institute of Hepatology, University College
London, London, United Kingdom; 2MedInnovation GmbH, Wildau, Germany;
and the 3Institute of Transfusion Medicine, University Hospital Leipzig, Leipzig,
Germany.
Received October 28, 2008; accepted February 9, 2009.
Supported by The Seigmund Warburg Benevolent fund and by an unrestricted
grant from CSL Behring Grifols; and Talecirs; and BPL (for supplying Human
Serum Albumin); coordinated by the Plasma Proteins Therapeutic Association. This
work was undertaken at UCLH/UCL, who received a proportion of funding from
the Department of Health’s NIHR Biomedical Research Centres funding scheme.
The views expressed in this work are those of the authors and not necessarily those
of the Department of Health.
Address reprint requests to: Nathan Davies, Liver Failure Group, Institute of Hepatology, 69-75 Chenies Mews, University College London, London WC1E 6HX, United
Kingdom. E-mail: [email protected]; fax: (44)-2073800405.
Copyright © 2009 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.22913
Potential conflict of interest: Nothing to report.
A
lbumin is the major plasma protein and constitutes around 50% of the cell free protein in
healthy individuals. It is produced exclusively in
the liver, and therefore its concentration is reduced during
hepatic dysfunction.1 Following the Cochrane meta-analysis describing potential harmfully effect of albumin infusion in critically ill patients, there has been a reexamination of the use of albumin infusions for volume
replacement. However, the results of the recently published SAFE study have provided new data confirming the
safety of albumin infusion in critically ill patients.2,3
Liver failure results in multiple organ dysfunction, and
mortality rates without liver transplantation remain unacceptably high.4 However, recovery is associated with
complete reversal of multiorgan dysfunction. At present,
in patients with cirrhosis, albumin is used mainly to replenish the circulating volume. With increasing severity
of cirrhosis, there is a progressive increase in cardiac output, which is associated with a progressive reduction in
individual organ blood flow. This peculiar circulatory disturbance is thought to occur as a result of splanchnic
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JALAN ET AL.
Fig. 1. A diagrammatic representation of the functional albumin
domains. BS-1, BS-2, BS-3, and BS-4 represent the functional fatty acid
binding sites; N terminus represents the Cu/Zn metal binding domain;
and Cys 34 represents the antioxidant property of albumin.
vasodilatation that culminates in a reduction of effective
arterial blood volume.5 In patients with cirrhosis, albumin infusion prevents the postparacentesis circulatory
disturbance in patients undergoing total abdominal paracentesis and reduces mortality in patients with spontaneous bacterial peritonitis.6 In a separate study, patients
with hepatorenal syndrome treated with Terlipresssin
plus albumin showed a significant improvement in outcome compared with patients treated with Terlipressin
alone.7 These effects of albumin were interpreted to be
due to its ability to produce adequate volume expansion,
thereby preserving renal circulation.6,7
Albumin has been shown to undertake a variety of
functions including fatty acid transport, metal chelation,
drug binding and anti-oxidant activity. These functions
are achieved through its antioxidant property provided by
the thiol moiety at cys-34 and several important binding
sites (Fig. 1).1 It is likely that it is this antioxidant and the
detoxification property of albumin that was associated
with improved outcome of stroke patients that received
albumin infusion compared with standard of care.8 Indeed, a recent study described functional disturbances in
the antioxidant function of albumin in patients with liver
failure.9 It is this functional property of albumin that has
been exploited by the detoxification systems based upon
the principles of albumin dialysis. One such device that is
commercially available is the Molecular Adsorbents Recirculating System (MARS) (Gambro, Lund, Sweden)
and studies using this device show beneficial pathophysiological and clinical effects particularly in reducing the
HEPATOLOGY, August 2009
severity of hepatic encephalopathy with small studies also
showing possible survival benefit in liver failure patients.10-12
However, at present, detailed functional characteristics
of the different binding sites in cirrhosis have not been
determined. The overall aim of this study was to perform
a detailed qualitative and quantitative assessment of the
functional capacity of albumin in patients with liver disease of varying severities and to determine whether these
alterations could be used as a marker of disease severity
and prognosis. We chose to use two methods of testing
albumin functionality. Electron paramagnetic resonance
(EPR) spectroscopy was used to perform detailed analyses
of the individual binding sites that are associated with the
main function of albumin. Ischemia-modified albumin,13
a test that measures the cobalt-binding capacity of albumin, was measured in the same cohort to determine
whether this simpler test could be used as a biomarker to
determine prognosis in patients with acute-on-chronic
liver failure (ACLF). In addition, we wanted to determine
whether dialysis against an albumin solution using the
MARS system has any ability to affect albumin functional
capacity in patients with ACLF.
Patients and Methods
All patients gave written informed consent, and the
study was approved by the local ethics committee.
Patients
Thirty-four patients with alcoholic cirrhosis defined
on clinical, radiological, biochemical, and/or histological
grounds were enrolled in the study. Included patients had
been admitted with acute decompensation of cirrhosis
manifested by increasing jaundice. Prophylactic antibiotics (cefotaxime) were prescribed following initial cultures
if there was a suspicion of infection, and stopped if subsequent cultures proved negative. Patients were excluded
if they were under 18 years of age or over 75 years of age,
had hyponatremia, had a hepatic/extrahepatic malignancy, had fewer than 3 days of post-gastrointestinal
bleeding, or if they received any immunomodulatory
therapy or albumin infusion prior to entry in the study.
The reference population of 80 healthy volunteer blood
donors (reference control [n ⫽ 80]) served as controls. All
patients received supportive therapy including nutrition
and vitamin supplementation according to local guidelines. Local treatment protocols were initiated for development of complications including, sepsis, hepatorenal
syndrome, and organ failure (full intensive care support),
as indicated.
Twelve of the 34 patients were included following correction of any electrolyte disturbances or hypovolemia
HEPATOLOGY, Vol. 50, No. 2, 2009
and 48 hours after initiation of specific therapy to treat the
precipitating event, and this group served as disease controls. The other 22 patients were included on the day they
were admitted to the intensive care unit with a diagnosis
of ACLF, which was defined as acute deterioration in liver
function over a period of 2 to 4 weeks, associated with a
precipitating event, leading to severe deterioration in clinical status, with jaundice and hepatic encephalopathy
(grade 2 or more) and/or hepatorenal syndrome, with a
sequential organ failure assessment score of 8 or more. Of
these 22 patients, 12 were treated with the molecular adsorbents recirculating system and 10 with standard supportive medical therapy. The selection for treatment with
MARS or standard of care was decided randomly as a part
of other clinical studies (unpublished data). Samples from
patients with cirrhosis without organ failure were collected from patients within 48 hours of admission, after
initiation of therapy for their precipitating event.
Study Design
Peripheral venous blood was aseptically collected into
pyrogen-free tubes (BD Vacutainer Lithium-Heparin [60
U per tube]; BD, Plymouth, UK) at the time of inclusion
into the study and used for routine biochemistry, markers
of oxidative stress, and albumin functional capacity. For
harvesting plasma, blood was placed immediately on ice.
After centrifugation, plasma was aliquoted into cryotubes
(Corning Inc., Corning B.V., Netherlands) and stored at
⫺80°C until further analysis. Bilirubin, albumin, liver
function tests, coagulation parameters, full blood count,
and c-reactive protein were routinely performed. model of
end-stage liver disease (MELD)14 and Pugh score15 were
calculated. The patients were followed prospectively over
a period of 90 days. In the patients that developed ACLF,
plasma samples were collected again 7 days after inclusion.
Measurement of Albumin Function
The functional characteristics of albumin were measured using two techniques. The molecular structure is
shown in Fig. 1 which also depicts the binding sites studied.
Electron Paramagnetic Resonance. The functional
capacity of the albumin binding sites were measured using
a spin label and electron paramagnetic resonance spectroscopy as described.16,17 There are six well-known binding sites for fatty acids on the albumin molecule, three
with high and three with lower affinity, that were examined in this study. The spin label used for albumin 16doxyl stearic acid (Sigma-Aldrich GmbH, Germany) was
added to plasma samples in defined concentrations. Ethanol was used as a polar reagent (99% puriss, USPXXII,
JALAN ET AL.
557
Merck KG, Germany), increasing aliquots of which were
added to the labeled plasma samples, which were then
incubated at 37°C for 10 minutes before the EPR spectra
were recorded (MMS, MedInnovation GmbH, Germany). Analysis of the recorded spectra, using MMS software, provided information on albumin conformation
and binding properties. Two different situations were
considered: (1) in vitro conditions, which included noncompetitive examination of fatty acid adsorption, transport capacity, and fatty acid unloading; and (2) a
competitive test that simulated in vivo conditions in
which albumin loading, transport and unloading were
studied for the functional parameters of albumin quality
as transport drug in competitive situations. Using EPR
spectroscopy, albumin conformational modifications can
be determined from its interaction with other ligands.
The EPR spectra-based parameters (KB1, binding coefficient, and N1, capacity of high-affinity sites; KB2, binding
coefficient, and N2, capacity of secondary binding sites;
RTE, real transport efficiency; DTE, detoxification efficiency) allow conclusions to be made as to the extent of
maintained transport of fatty acids, drugs, bilirubin, metabolites, biomarkers, and so forth. In addition, it is possible to assess the detoxification function of measured
albumin.16,17
Ischemia-Modified Albumin. Ischemia-modified albumin (IMA) is identified using an assay to determine the
ability of the protein to chelate cobalt. There are several
sites on the protein that have the ability to bind metals,
though most interest to date has focused on the N-terminal region.13,18 IMA was determined according to the cobalt-binding assay method of Bhagavan et al.13 Briefly,
plasma was incubated with a cobalt chloride (1 g/L, 10
minutes) and dithiothreitol (1.5 g/L, 2 minutes) before
dilution in saline prior to measurement at 470 nm in a
spectrophotometer (Agilent 8453 Diode Array, Agilent,
UK). IMA was calculated from the difference between
samples measured with and without dithiothreitol.
Plasma albumin concentration was determined using an
automated COBAS Integra biochemical analyzer (Roche
Diagnostics, UK).
Markers of Oxidative Stress
Malondialdehyde. Malondialdehyde (MDA) was determined using a modified thiobarbituric acid reactive
substances assay described by Lapenna et al.19 wherein the
major interfering/oxidizable component in the plasma is
inhibited by addition of sodium sulphate.
F2 Isoprostanes. F2 isoprostanes (free 8-isoprostane
F2␣) was assayed with a commercial EIA kit (Cayman
Chemical, Ann Arbor, MI) according to the manufacturer’s instructions and as described.20 Briefly, 200 ␮L
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JALAN ET AL.
plasma was deproteinized with 600 ␮L ethanol containing 3H-prostaglandin E2 as an internal standard to account for losses. After centrifugation, the supernatant was
reduced to near dryness, and 2 mL acetic acid was added
and applied to a preconditioned C18 SPE cartridge (Waters, Milford, MA). The column was washed with water,
dried with nitrogen, and eluted with high-performance
liquid chromatography– grade hexane. The prostanoid
fraction was eluted with 5 mL ethylacetate containing 1%
methanol, eluant reduced to dryness and reconstituted in
450 ␮L of EIA buffer, 100 ␮L being used to determine
recovery of 3H-PGE2 and 50 ␮L added to the EIA plate
with isoprostane tracer and antibody. Isoprostane levels
were determined by reference to authentic standards and
corrected for losses.
Statistical Analysis
All the data were described as mean and standard error.
Differences between groups were calculated using an independent samples t test for the normally distributed data
and the Mann-Whitney test for data not normally distributed. Correlations between variables were calculated using linear regression. Survival analysis was performed
using the Kaplan-Meier method and the log-rank test was
used to test statistical significance.
Results
Patients
The main causes leading up to acute decompensation
in the patients were infection (n ⫽ 18), superimposed
alcoholic hepatitis (n ⫽ 11), and variceal bleeding (n ⫽
5). Twenty-two patients proceeded to deterioration in
their clinical condition requiring prolonged hospital admission and supportive care in a high-dependency/intensive care unit and were considered to have ACLF. Of
these, 12 were treated with MARS and the rest with standard of care. No clinical or biochemical differences were
observed between the patients treated with MARS compared with the group treated with standard medical care.
In the MARS-treated patients, there was a reduction in
bilirubin (P ⬍ 0.01) and creatinine (P ⬍ 0.05) concentrations at day 7, which resulted in a reduction in MELD
score. However, although this would suggest an improvement in outcome, survival remained unaffected, with
eight of the 12 patients dying in the MARS group and 7 of
the 10 patients dying in the group treated with standard
of care (Table 1). The cause of death was multiorgan
failure in both groups.
Albumin Function
Electron Paramagnetic Resonance. Albumin functionality is characterized by four groups of EPR spectrum
HEPATOLOGY, August 2009
Table 1. Patient Characteristics
Age
Sex
MELD score
Child-Pugh score
SOFA score
Albumin (g/L)
Bilirubin (␮mol/L)
INR
Creatinine (␮mol/L)
MDA (␮mol/L)
F2-Isoprostanes (pg/mL)
In-hospital mortality
Cirrhosis, No Organ
Dysfunction
(n ⴝ 12)
ACLF Treated
with MARS
(n ⴝ 12)
ACLF Treated
with SMT
(n ⴝ 10)
47 ⫾ 3.4
10 M, 2 F
9.3 ⫾ 2*
7.9 ⫾ 1.3†
5.5 ⫾ 1.2†
37 ⫾ 4†
83 ⫾ 9‡
1.2 ⫾ 0.2‡
73 ⫾ 8‡
3.3 ⫾ 2.1†
267 ⫾ 32†
None
47 ⫾ 4.4
9 M, 3 F
19.7 ⫾ 3
11.4 ⫾ 1.5
9.3 ⫾ 2.5
26 ⫾ 1.7
428 ⫾ 57
1.8 ⫾ 0.2
146 ⫾ 41
7.4 ⫾ 1.9
423 ⫾ 39
8 patients
48 (2.9)
8 M, 2 F
20 ⫾ 3
12.2 ⫾ 1.4
9.4 ⫾ 1.7
25 ⫾ 1.3
323 ⫾ 47
1.8 ⫾ 0.1
179 ⫾ 67
7.7 ⫾ 2.3
489 ⫾ 28
7 patients
Data are expressed as the mean ⫾ standard error.
Abbreviations: F, female; INR, international normalized ratio; M, male; SMT,
standard medical therapy; SOFA, sequential organ failure assessment.
*P ⬍ 0.05, †P ⬍ 0.01, ‡P ⬍ 0.001 for the cirrhosis, no organ dysfunction
group versus the ACLF groups.
parameters: Albumin functionality is characterized by
three groups of EPR spectrum parameters: (1) capacity of
binding sites and strength of fatty acid holding; (2) global
parameters of albumin transport (transport, loading, and
unloading of fatty acids); and (3) functional parameters of
albumin quality in regard to its ability to uptake and hold
toxic substances produced via metabolism. The EPR data
of the healthy subjects were used as reference values. All
healthy volunteers showed normal albumin conformation
and transport functionality. Examination of the data obtained from the 80 control subjects showed no significant
differences in any measured value for either age or sex
(Fig. 2). For each parameter measured, patients with both
stable cirrhosis and those with ACLF were significantly
worse compared with the healthy volunteers.
Transport Efficiency. Albumin transport function
parameters were evaluated following the model of a threestep transport process for fatty acids. This incorporates
substrate sorption and binding parameters in which albumin from healthy subjects demonstrate high binding constants for substrate uptake. The second phase is transfer of
the bound substrates into the circulatory system. Finally,
the albumin should be able to demonstrate the ability to
release the substrate to target objects. Substrate delivery
from albumin requires albumin receptor or albumin surface interaction with the cell membrane; in this process,
the albumin substrate binding constant is controlled by
hydrophobic interactions at the target binding site. In the
EPR studies, the local albumin membrane delivery operations were simulated using ethanol for modification of
these hydrophobic interactions. Overall, the albumin
transport efficiency in ACLF patients was found to be
only about 10% of the normal functional ability of albu-
HEPATOLOGY, Vol. 50, No. 2, 2009
JALAN ET AL.
559
Within these measures, cirrhotic patients were found
to have significantly lower (P ⬍ 0.001, KB1 and P ⬍
0.001, KB2) fatty acid binding capability compared with
healthy controls. This ability was then further reduced in
patients with acute decompensation. Albumin detoxification efficiency for uptake of toxins was evaluated to show
the fixation of toxins and a minimal dissociation rate of
albumin as the ratio of albumin sorption capability compared with its delivery efficiency. In this measure it was
found that healthy controls had a significantly better calculated detoxification capacity compared with stable cirrhotic subjects (P ⬍ 0.001), which was in turn
significantly higher than that of ACLF patients (P ⬍
0.05). The detoxification efficiency was significantly
lower in nonsurvivors compared with survivors (P ⬍
0.007) (Table 2). The detoxification efficiency correlated
closely with transport efficiency (r ⫽ 0.93; P ⬍ 0.0001).
Effective Albumin Concentration. From these results, it was possible to derive an effective albumin concentration that revealed that cirrhotic subjects had
significantly lower functionality than healthy controls
(P ⬍ 0.001). This functionality was again found to be
reduced further in the ACLF group compared with the
cirrhotic, no organ dysfunction group (P ⬍ 0.05) (Table 3).
Ischemia Modified Albumin. It was not possible to
differentiate between groups using the IMA results due to
the wide variance in the values obtained. However, when
the IMA values were considered relative to the patients’
Fig. 2. Functional alterations in albumin obtained from the reference
population of healthy volunteers. (A) Relationship between age and real
transport efficiency (RTE). (B) Relationship between age and detoxification efficiency (DTE). In each case the lines indicate the line of best fit
(linear regression) for the data, and the line was not found to be
significantly different from zero. (C) The measurements of albumin functionality were sex-independent (black bars, males; white bars, females).
min from healthy volunteers (Table 2). These values were
found to be significantly lower in cirrhotic patients compared with healthy controls (P ⬍ 0.001 in each case) and
were further reduced in ACLF subjects. Transport efficiency was significantly lower in nonsurvivors compared
with survivors (P ⬍ 0.05).
Detoxification Efficiency. Albumin screening parameters were evaluated using concentration parameters
of free fatty acid and 16-DS bound to albumin. The measured binding capacity was significantly reduced in subjects with liver disease compared with healthy controls
(P ⬍ 0.05). This finding demonstrates that the threedimensional protein structure of the albumin is affected
in patients with liver disease.
Table 2. Functional Albumin Parameters in Different Patient
Groups
Measure
EPR spectroscopy
Binding coefficient (highaffinity KB1)
Number of labels (highaffinity site, N1)
Binding coefficient (lowaffinity KB2)
Number of labels (lowaffinity site, N2)
Calculated real transport
efficiency
Calculated detoxification
efficiency
Effective albumin
IMA
IMAR
Healthy
Cirrhosis,
No Organ
Dysfunction
(n ⴝ 12)
ACLF
(n ⴝ 22)
97.4 (15.6)
27.5 (5.6)*
17.9 (3.5)*
3.1 (0.1)
2.4 (0.1)
2.5 (0.9)
58.3 (11)
20.1 (3.1)*
14.1 (1.8)*,†
2.9 (0.2)
3.1 (0.1)
2.6 (0.1)*,†
75 (6.9)
27.5 (5.1)*
14.3 (1.9)*,†
120 (33)
50.26 (3.02)
0.69 (0.034)
0.010 (0.001)
28.2 (5.3)*
25.6 (3.1)*
0.64 (0.022)
0.021 (0.001)
11.6 (2.6)*,‡
20.98 (2.2)*
0.64 (0.017)
0.03 (0.002)*,‡
Data are expressed as the mean (standard error). See text for explanations
regarding the description of the various binding sites.
*P ⬍ 0.001 compared with healthy volunteers; †P ⬍ 0.05, ‡P ⬍ 0.01
compared with cirrhosis, no organ dysfunction group.
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JALAN ET AL.
HEPATOLOGY, August 2009
Table 3. Characteristics of Survivors versus NonSurvivors
Factor
MELD score
Child-Pugh score
SOFA score
Real transport
efficiency
Detoxification efficiency
IMAR
Survivors
(n ⴝ 19)
Nonsurvivors
(n ⴝ 15)
P Value
AUROC
13.1 (1.5)
9.4 (0.9)
7.1 (2.3)
19.5 (2.2)
11 (0.4)
9.5 (1.2)
⬍0.05
0.3
⬍0.01
0.76
0.66
0.74
26.2 (4)
22.5 (4.4)
0.020 (0.001)
14.9 (2.7)
12.2 (2.9)
0.033 (0.003)
⬍0.05
⬍0.01
⬍0.01
0.65
0.72
0.80
Data are expressed as mean (standard error).
Abbreviation: SOFA, sequential organ failure assessment.
plasma albumin levels, significant differences were observed. The IMA/albumin ratio (IMAR) was significantly
higher in ACLF patients (P ⬍ 0.05, Table 2). Regression
analyses comparing the binding constants determined using the spin label studies and the measured IMAR scores
found a significant relationship at both binding sites 1 and
2. Similar significance levels were found between the
IMAR and the log10 derivative values from BS1 and BS2
(IMAR versus logKB1, r ⫽ 0.6; IMAR versus logKB2, r ⫽
0.62; P ⬍ 0.001 for both) (Fig. 3), indicating a relationship between the inability of cobalt to bind to the protein
and the function of these sites.
Higher IMAR correlated closely with the MELD score
(r ⫽ 0.81; P ⬍ 0.007) (Fig. 4) but not Child-Pugh score.
IMAR levels were significantly higher in nonsurvivors
compared with survivors (area under the receiver operating curve [AUROC]: 0.8) (Fig. 5). With a cutoff ratio
value of 0.02, the sensitivity and specificity were 83% and
67% respectively. Kaplan-Meier analysis confirmed increased mortality in the group with an IMAR ⬎0.02;
log-rank: P ⫽ 0.03) (Fig. 6).
during the study period, this difference was not statistically significant.
Discussion
This study provides the first detailed description of the
qualitative and quantitative reduction in albumin function in patients with liver disease. The data indicate that
the ability of albumin to transport protein-bound substances and drugs and act as detoxification agent is severely compromised in patients with cirrhosis, and is
further reduced in patients with ACLF. Additionally, our
data suggest that the severity of the functional abnormalities of albumin function measured by IMAR may be a
useful biomarker to determine survival of patients with
acute decompensation of cirrhosis, though this will be
dependent on an appropriately powered validation study.
From the pathophysiologic perspective, the increase in
IMAR levels with disease severity provides evidence of
protein modification, which in keeping with the published literature has been shown to be associated with
severity of oxidative stress.21,22
The use of EPR spectra provides data regarding
changes in fatty acid binding of serum albumin under
modulation of medium hydrophilicity and allows conclusions for transport and detoxification capacity of this integral plasma protein.16,17,21 The efficiency of the
Markers of Oxidative Stress
MDA and F2 isoprostanes were significantly higher in
the ACLF group compared with the group that did not
develop organ failure (P ⬍ 0.05 and P ⬍ 0.01, respectively) (Table 1). MDA was insignificantly higher in nonsurvivors, but F2 isoprostanes were significantly greater in
nonsurvivors compared with survivors (P ⬍ 0.05). IMAR
correlated significantly with MDA (r ⫽ 0.67; P ⬍ 0.01)
and F2 isoprostanes (r ⫽ 0.6; P ⬍ 0.05), but the relationship with the detoxification efficiency and transport efficiency were statistically insignificant.
Effect of MARS Therapy on Albumin Function
No significant differences in albumin function were
found between days 0 and 7 for the ACLF patients in
either the SMT or MARS groups (Fig. 7). Although it
appears that in the SMT group the effective albumin concentration and detoxification efficiency measures improve
Fig. 3. There was good correlation between the binding coefficients
(KB1 and KB2) at binding sites 1 and 2 with IMAR.
HEPATOLOGY, Vol. 50, No. 2, 2009
Fig. 4. Relationship between IMAR and MELD score. Regression
analysis yielded r2 ⫽ 0.65 and P ⬍ 0.007.
albumin transport system depends on two factors. In the
case of decreased transport function and detoxification
efficiency, it signifies modification of the albumin molecule. This may be due to the binding/accumulation of low
molecular hydrophobic compounds (toxins, drugs, and so
forth) or permanent modifications to the albumin that
have occurred, rendering the protein incapable of normal
function. This loss of function is highlighted by the reduction in the measured binding constant (KB) for the
spin label observed in cirrhotic patients. This finding indicates a reduced ability of the albumin to bind and hold
the materials to be transported within the body. Coupled
with a decreased binding capacity (number of labels per
albumin molecule), this indicates that these materials are
Fig. 5. Receiver operator curve analysis of IMAR scores to examine its
predictive use for assessing mortality (AUROC ⫽ 0.8).
JALAN ET AL.
561
Fig. 6. Kaplan-Meier analysis of outcome based on a cutoff value of
0.02, as determined from the AUROC analysis shown in Fig. 4. A
significant difference in survival was observed (P ⬍ 0.03).
more likely to exist as a free component within the vasculature with the ability to react arbitrarily rather than being
delivered to a specific site. This is most pertinent with
regard to albumin-bound drugs and toxin removal. The
reduction in detoxification efficiency is especially noteworthy, indicating that the albumin in patients with cirrhosis is incapable of binding and removing waste
products for metabolism or excretion effectively. There is
Fig. 7. Difference in functional binding capacity determined by spin
label binding studies at days 0 and 7 in groups treated either with MARS
or standard medical therapy (SMT). (A) Calculated detoxification efficiency. (B) Real transport efficiency. (C) Measured binding capacity at
BS1. (D) Calculated effective albumin concentration. No significant
difference was found between treatment groups or between day 0 and 7
in either cohort.
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already evidence to suggest that the plasma antioxidant
status of cirrhotic patients is reduced compared with
healthy controls.9,23 Because albumin is the dominant
plasma protein and is known to exhibit antioxidant properties, this lack of functional ability to both remove toxins
and prevent oxidant stress damage suggests that further
decompensation may occur more readily.
It is interesting to note that the initial results from
the IMA studies failed to show any differences between
groups. However, once the IMA values were normalized to the plasma albumin concentration, a clear distinction between the patients with ACLF compared
with those who did not have organ failure became apparent. In the currently licensed use of this assay, as a
rule-out test for cardiac ischemia, most patients would
be expected to have normal albumin levels. This is not
true for patients with liver disease in whom a progressive decrease in albumin concentration is associated
with reduced hepatic function, which may be reduced
further with bleeding or sepsis.24 A reduced albumin
level will immediately result in a lower capacity to bind
cobalt, yielding an artificially high false positive IMA
score; however, this provides no information to the
degree of albumin damage or oxidative stress load.
Hence, a normalbuminemic subject with significant
albumin damage would have a similarly high IMA
score compared with a hypoalbuminemic individual,
even if the albumin present was functioning normally
(e.g., following venesection). It is only by expressing
the IMA per unit albumin that an understanding of the
amount of albumin dysfunction can be determined. By
normalizing the measured IMA score to the plasma
albumin concentration in this way, a relative indicator
of albumin damage, and hence the environment of
oxidative stress, can be obtained. We would therefore
suggest that IMA should be expressed relative to the
albumin concentration (IMAR) to minimize the interference from reduced albumin level. Using this rationale, our studies revealed that IMAR levels predicted
mortality of patients with acute decompensation of cirrhosis accurately with an AUROC of 0.8. Though it is
likely that a patient with liver disease is likely to have
reduced albumin levels, our study indicates that when
this is coupled with ongoing albumin damage and expressed as a ratio, the prognostic indication is less favorable. This observation must be taken in light of the
fact that it is a relatively small study and would require
confirmation in larger studies. These observations do,
however, indicate alternative mechanisms to explain
the proven value of albumin infusion in patients with
cirrhosis with spontaneous bacterial peritonitis6 and
also where albumin formed part of a combination ther-
HEPATOLOGY, August 2009
apy for hepatorenal syndrome.7 According to the classical hypothesis, the benefit of albumin in these
situations is thought to be due to expansion of the
circulating plasma volume, thereby ensuring adequate
renal perfusion. The results of this study indicate that it
is likely that additional benefit may have been conferred by the enhancement of the transport and detoxification function of albumin contributing to the
improved outcome in these patients.1
Though the exact mechanism/stress mediator leading to the formation of IMA could be varied, in the
laboratory the effect can be consistently repeated via
exposure of albumin to hydroxyl radicals.18 The significance of this assay in determining periods of ischemia
is evident in that it has recently been licensed by the
U.S. Food and Drug Administration as a rule-out test
for cardiac ischemia.25 Though the presence of IMA is
not diagnostic of a cardiac infarct, as any regional ischemia has the potential to generate IMA, its absence
indicates that it is most unlikely and patients with a low
IMA score can be discharged. The loss of metal chelation function, as evidenced by increasing IMA, would
contribute to an environment of continued oxidative
stress. As proteins become damaged and release metal
ions into the system, where they become redox active,
they contribute to radical formation by Fenton chemistry processes.26,27 Albumin ordinarily provides a firstline defense against these reactions by binding and
removing metal ions from the plasma, an ability that
appears to be deficient in this patient cohort.
Of further interest is the observed relationship between the calculated binding constant (KB) of binding
sites 1 and 2 and the IMAR score. Previous research
into the areas of albumin affected to result in increased
IMA have focused on the metal binding region at the N
terminus of the protein. However, a recent study28 has
suggested that although cobalt may bind at this site
transiently, its electronic configuration is better suited
to binding at the fatty acid binding sites. We observed
a significant relationship between the IMAR and the
functionality of these sites, supporting the hypothesis
that this is the binding location (Fig. 3). This finding
suggests that IMAR may be a more useful indicator of
albumin function than previously thought, providing
insight into the normal functional capacity of this important, ubiquitous protein.
MARS is an extracorporeal albumin dialysis device
that has been shown to be of value as a detoxification
tool with evidence that it can effectively remove protein-bound substances from the circulation and has
been shown to be useful for the treatment of hepatic
encephalopathy and in patients with severe cholestasis
HEPATOLOGY, Vol. 50, No. 2, 2009
to possibly improve survival.4,10-12 Current hypotheses
suggest that the removal of excess protein-bound toxins
in the extracorporeal circuit is likely to regenerate the
native albumin, thereby enhancing its functional capacity so that it may be able to transport and detoxify
more toxins. Contrary to this existing hypothesis, albumin dialysis using MARS did not improve the measured functional capacity of the patient’s circulating
albumin. This indicates that although MARS therapy
is capable of removing albumin-bound materials (e.g.,
bilirubin) it remains unable to regenerate the functional characteristics of the native albumin. Similarly,
no significant changes in IMAR levels were observed
following MARS therapy (data not shown), indicating
that the amount of functionally damaged albumin remained elevated after treatment. Furthermore, the observed improvement in the biochemistry values and the
prognostic scores merely reflect removal of bilirubin
and creatinine, and possibly other metabolic toxins,
indicating that albumin dialysis in its current form can
be used to treat the symptoms of the condition, but
does not address the underlying functional impairment
of the patients’ albumin. An explanation for the lack of
improvement in the functional capacity of albumin
may be that the disease process irreversibly damages the
plasma albumin. The nature of such damage has yet to
be defined, though interruption of the disulphide
bridges within albumin would lead to a breakdown of
the globular structure and may prevent binding site
access. It should also be considered that the transport
properties of the albumin used as dialysate in the
MARS system may be significantly lower than in
healthy subjects in vivo. In a separate study, it was
found that the detoxification efficacy of commercial
albumins is only 20% to 40% of the native protein.17
In conclusion, the results of this study clearly indicate that the functional ability of albumin in cirrhosis is
severely compromised, which further worsens in liver
failure. In addition to these functional disturbances,
the albumin concentration was markedly reduced,
which most likely further compounds the overall functional capacity of albumin. This loss of albumin function and an increase in IMAR levels, indicating protein
modification, was associated with poor survival.
Whether this loss of albumin function is merely a consequence of liver failure or is indeed of pathogenic
significance needs to be answered in future studies.
Though albumin dialysis has been shown to remove
albumin-bound toxins, it does not restore the functional ability of the patient albumin. These findings
should be considered in the development of new types
of albumin dialysis systems and for the use of albumin
JALAN ET AL.
563
for dialysis with improved transport and detoxification
capacity. Furthermore, these findings argue for further
studies of albumin biology in cirrhosis, giving consideration to the use of albumin infusion not for fluid
replacement, but as an agent to increase detoxification
capacity.
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