Human Serum Albumin and its N-Terminal Tetrapeptide
(DAHK) Block Oxidant-Induced Neuronal Death
Elizabeth T. Gum, MS; Raymond A. Swanson, MD; Conrad Alano, PhD; Jialing Liu, PhD;
Shwuhuey Hong, BS; Philip R. Weinstein, MD; S. Scott Panter, PhD
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Background and Purpose—Studies using animal models of stroke have shown that human serum albumin (HSA)
significantly ameliorates cerebral ischemic injury after both transient and permanent ischemia, even when administered
after the onset of ischemia or reperfusion. The mechanism of this effect remains uncertain, and prior studies suggest both
indirect hemodynamic and direct cytoprotective effects. HSA is a potent antioxidant, in part because of its strong
copper-binding capacity. Here we examined the effect of HSA on oxidant-induced neuronal death in a cortical cell
culture system.
Methods—Murine cortical cultures were exposed to oxidative stress generated by hydrogen peroxide and by a mixture of
copper plus ascorbic acid. We examined the ability of HSA and a tetrapeptide occupying its N-terminus (DAHK) to
prevent neuronal death after these challenges.
Results—H2O2 and CuCl2/ascorbic acid were used at concentrations that, in the absence of HSA, killed ⬎90% of the
neurons. HSA provided complete protection at a concentration of 37.5 ␮mol/L and 50% protection at 3.75 ␮mol/L. The
copper-binding tetrapeptide DAHK had nearly identical potency and efficacy. HSA and DAHK were also equally
effective in preventing neuronal death induced by CuCl2/ascorbic acid.
Conclusions—HSA has potent antioxidant properties, probably due to binding of copper and other transition metals. HSA
extravasation into ischemic brain may provide neuroprotection by limiting metal-catalyzed oxidant stress. The
tetrapeptide DAHK may be an effective, small-molecular-weight alternative to HSA as a therapeutic agent for stroke.
(Stroke. 2004;35:590-595.)
Key Words: albumins 䡲 antioxidants 䡲 copper 䡲 ischemia 䡲 neurons
A
recent series of studies has explored the efficacy of
human serum albumin (HSA) as a therapeutic agent in
experimental models of stroke.1–7 HSA was administered
intravenously to treat permanent or transient cerebral ischemia and was more effective in transient ischemia. HSA
significantly reduced infarct size, edema, and sodium accumulation and improved neurological outcome, even when
administered up to 2 hours after onset of ischemia. While
these studies demonstrated significant efficacy for HSA in the
treatment of stroke, the mechanism for this robust neuroprotection remains undetermined. A number of possible mechanisms have been examined, including the effect of HSA on
local cerebral perfusion, blood-brain barrier disruption, systemic fatty acid responses, and microvascular patency. While
many of these mechanisms probably contribute, none appear
robust enough to account for the large neuroprotective effect
of HSA.2–5,8,9
HSA is a unique molecule. It maintains colloidal osmotic
pressure in the vasculature and has a number of important
functional properties.10 It strongly binds fatty acids, some
drugs, and drug metabolites, and it has a number of cation and
anion binding sites.8,10 HSA is also a potent antioxidant,
acting both as a free radical scavenger and as a chelator of
transition metals and heme.10 A metal binding site on HSA,
the 4 –amino acid terminal sequence of the molecule, is
unique to human albumin and may play a prominent role in
its neuroprotective effects.11–15
The 4 amino acids occupying the N-terminus of HSA—
aspartate, alanine, histidine, and lysine (DAHK)— constitute
a relatively high-affinity binding site for a number of cations,
specifically nickel, cobalt, and copper.11 In recent series of
reports, it was demonstrated that a synthetic peptide composed of these 4 amino acids could inhibit copper-induced
oxidative DNA double-strand breaks and telomere shortening
in cell cultures.12 It was also determined that DAHK prevented lipid oxidation in a copper-catalyzed oxidant system.13
The protective effects of DAHK may also be due in part to the
fact that, in addition to acting as a copper chelator, the
copper-DAHK complex is a potent superoxide dismutase
mimetic, thereby increasing the antioxidant potential of the
Received July 23, 2003; final revision received September 24, 2003; accepted October 14, 2003.
From the Departments of Neurology (E.T.G., R.A.S., C.A.) and Neurosurgery (J.L., S.H., P.R.W., S.S.P.), San Francisco Veterans Affairs Medical
Center and University of California at San Francisco.
Correspondence to Dr S. Scott Panter, Veterans Affairs Medical Center, Neurology (127), 4150 Clement St, San Francisco, CA 94121. E-mail
[email protected]
© 2004 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
DOI: 10.1161/01.STR.0000110790.05859.DA
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Human Serum Albumin Blocks Oxidative Neuronal Death
591
tetrapeptide or HSA itself.13 It was recently demonstrated that
a novel synthetic analogue of DAHK could improve recovery
of rat hearts from an ischemia/reperfusion insult,16 suggesting
that this approach may similarly be useful in the treatment of
stroke.
We hypothesized that the antioxidant properties of this
4 –amino acid terminus of HSA might explain, in part, its
neuroprotective effects after ischemia/reperfusion injury in
brain. To test this hypothesis, we utilized HSA and DAHK in
a neuronal cell culture model of oxidative injury. Both HSA
and DAHK were neuroprotective in cell culture models of
hydrogen peroxide– and copper ascorbate–induced
neurotoxicity.
Materials and Methods
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HSA was obtained from Centeon L.L.C. as a 25% solution, and
DAHK was obtained from Genemed Synthesis Incorporated. All
other reagents were obtained from Sigma-Aldrich, except where
otherwise noted. Adventitious metals were removed from HSA by
applying it to a column of iminodiacetic acid attached to cross-linked
polystyrene, after which it was reconcentrated to a 25% solution.
Cell Cultures
The studies were conducted in accordance with National Institutes of
Health guidelines and with the use of protocols approved by the local
institutional committee on animal studies. Mice were anesthetized
with isoflurane before being killed for harvesting of brains. Cortical
cell cultures were prepared in a 2-step process, which has previously
been described in detail.17,18 Astrocytes were plated at 1.5⫻105 cells
per well. Neurons were plated on the astrocyte layers at a density of
6⫻105 cells per well.
Experimental Procedures
Experiments were begun by replacing the culture medium with a
balanced salt solution (BSS), as previously described.17,18 The pH of
the BSS was adjusted to pH 7.2 and during equilibration in a 5% CO2
atmosphere. Osmolarity was measured with a Wescor vapor pressure
osmometer and adjusted with H2O or NaCl when necessary to
achieve 280 to 320 mOsm. Test compounds were prepared as ⫻100
stock solutions in distilled deionized water and were diluted to
working concentrations in BSS before use. The test compounds were
added to the cultures in BSS, and the cultures were then replaced in
the 37°C, 5% CO2 incubator. In studies in which H2O2 was used, the
medium was replaced with BSS after 60 minutes of H2O2 exposure.
All other treatment combinations were maintained for 20 to 22 hours.
Control wells received only medium exchanges. In each experiment,
all comparisons were made with the use of sister cultures derived
from single plating.
Assessment of Neuronal Survival
Neuronal survival was assessed by the propidium iodide exclusion
method.18,19 Propidium iodide was added at 0.03 mg/mL to each
well. Dead (fluorescent) and live (nonfluorescent) neurons were
counted in 4 optical fields chosen randomly in each well, with the
use of a Nikon fluorescence microscope with phase-contrast optics.
Neurons were easily distinguished from the underlying astrocyte
layer by their phase-bright, process-bearing morphology (Figure 3).
Results from each well were expressed as percent neuronal survival,
calculated as (live cells⫻100)/(live cells⫹dead cells). In a subset of
the experiments, cell counts were also performed by a second
observer in a blinded fashion to exclude observer bias.
Statistical Analysis
Data are presented as mean⫾SEM. Statistical significance was
assessed with the use of 1-way ANOVA followed by the Dunnett
post hoc test for multiple comparisons against a control group.
Differences were considered significant at P⬍0.05.
Figure 1. Hydrogen peroxide produced neuronal death in a
dose-dependent manner. The cultures were treated with H2O2
for 60 minutes. Twenty-four hours after treatment, neuronal
death was assessed by propidium iodide staining. n⫽16 to 20,
pooled from 5 independent experiments. **P⬍0.001.
Results
To determine the antioxidant capacity of HSA and DAHK,
we first used H2O2 to generate oxidative stress. Cortical
cultures were exposed for 1 hour to a range of H2O2
concentrations, and neuronal survival was assessed by propidium iodide staining after an additional 22 to 24 hours in
BSS. The resulting concentration-response curve showed a
threshold effect on neuronal survival (Figure 1), similar to
previous reports.18,20 Exposure to 100 ␮mol/L caused nearcomplete neuronal death, and this concentration was used for
all subsequent studies.
We evaluated the efficacy of HSA as a neuroprotective
agent by adding various concentrations of HSA to the culture
simultaneously with H2O2. HSA had a dose-dependent neuroprotective effect, with neuronal death reduced to a level
comparable to control conditions at concentrations of ⱖ15
␮mol/L (Figure 2A). To determine whether the chelating
tetrapeptide DAHK would also prevent neuronal death resulting from H2O2, cultures were incubated with 100 ␮mol/L
H2O2 in the presence of a range of DAHK concentrations.
These studies showed a potent, dose-dependent effect of
DAHK on H2O2-induced neuronal death (Figure 2B). Photomicrographs showing the effect of HSA on H2O2-induced
neuronal death are shown in Figure 3.
We tested the ability of HSA to protect neurons against the
mixture of 25 ␮mol/L CuCl2 and 50 ␮mol/L ascorbic acid
(Figure 4), which in oxygenated solutions generates oxygenderived free radical species.21,22 In the absence of HSA, this
exposure killed ⬎95% of the neurons, but in the presence of
150 ␮mol/L HSA neuronal death was reduced to control
values. HSA has a high-affinity binding site for copper and
other transition metals at its N-terminus in the form of a
DAHK tetrapeptide.21–23 Synthetic DAHK tetrapeptide alone
also completely blocked Cu/ascorbic acid– dependent neurotoxicity when added at concentrations of ⱖ50 ␮mol/L,
although the potency of DAHK was ⬎100-fold greater than
HSA when the concentrations of both were expressed as
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Figure 3. Photomicrographs show the protective effects of HSA
on H2O2 neurotoxicity. The cultures were treated simultaneously
with 100 ␮mol/L H2O2 plus various concentrations of HSA for 60
minutes. Twenty-four hours after treatment, the photomicrographs were taken with combined fluorescence and phasecontrast optics. Dead neurons were identified by propidium
iodide fluorescence.
Figure 2. A, HSA decreased H2O2-induced neuronal death. The
cultures were simultaneously treated with 100 ␮mol/L H2O2 plus
various concentrations of HSA for 60 minutes. Twenty-four
hours after treatment, neuronal death was assessed by propidium iodide staining. n⫽24 to 30, pooled from 6 independent
experiments. **P⬍0.01. B, DAHK decreased H2O2-induced neuronal death. Cultures were exposed to H2O2 plus various concentrations of DAHK for 60 minutes. Neuronal death was
assessed 24 hours after treatment. n⫽16 to 20, pooled from 4
independent experiments. **P⬍0.01.
percent weight/volume. H2O2 requires interaction with a
transition metal to produce reactive oxygen species.24
The antioxidant effect of HSA could, in principle, occur in
either the extracellular or the intracellular compartments,
since HSA can be internalized by neurons under some
conditions.25 As a possible way to determine the compartment
in which the neuroprotective effect of HSA was exerted, we
assessed the ability of HSA to prevent N-methyl-D-aspartate
(NMDA)–induced neuronal death, a process that is mediated
in part by intracellular production of oxygen free radicals and
is blocked by cell-permeant oxygen free radical scavengers.26,27 HSA had no effect on NMDA neurotoxicity. This
suggests that the action of HSA occurs in the extracellular
space (Figure 5).
To test whether it is the DAHK moiety of HSA that is
primarily responsible for its antioxidant effects, we compared
HSA with several other proteins that do not contain this
tetrapeptide at the N-terminus: lactalbumin, ␥-globulin, bovine serum albumin, and casein. Somewhat surprisingly, each
of these proteins also protected neurons against H2O2 toxicity
with potencies roughly similar to that of HSA (Figure 6).
Discussion
These results show that the neurotoxic oxidant stress induced
by either hydrogen peroxide or copper/ascorbic acid can be
blocked by HSA and by a tetrapeptide that is the same
sequence as the N-terminus of HSA. These results may be
relevant to the recent series of articles that describe the
protective effect of HSA in animal models of stroke and
traumatic brain injury.2–5,9
In vivo, the concentration of HSA in cerebrospinal fluid is
approximately 3.7 ␮mol/L.28 This concentration of HSA
produced an approximately 40% reduction in H2O2-induced
neuronal death under the conditions used in the cell culture
studies described here. Additional HSA may enter the brain
from the plasma compartment after stroke through an open
blood-brain barrier.25 Since the concentration of HSA in
serum is approximately 588 ␮mol/L,29 even a small movement of serum proteins across the blood-brain barrier could
substantially raise HSA concentrations in the extracellular
space surrounding postischemic neurons and increase resistance to oxygen free radicals in the extracellular space.
Gum et al
Human Serum Albumin Blocks Oxidative Neuronal Death
593
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Figure 4. DAHK decreased CuCl2/ascorbic acid–induced neuronal death in a dose-dependent manner. Cultures were exposed
to CuCl2/ascorbic acid plus various concentrations of DAHK or
HSA for 60 minutes. Neuronal death was assessed 24 hours
after treatment. HSA and DAHK produced similar protection
when expressed as micromoles per liter. n⫽16 to 20, pooled
from 4 independent experiments. **P⬍0.01.
For the purpose of testing the neuroprotective effects of
HSA and DAHK in cultured neurons, 2 different models of
oxidant-mediated neuronal injury were developed. The first
uses hydrogen peroxide as the stressor, and the second uses a
copper/ascorbic acid– driven free radical– generating system.
Both oxidants were used under conditions that, in the absence
of HSA or DAHK, killed nearly 100% of the neurons in the
cultures. Copper-ascorbic acid– driven stress involves a transition metal, and it is almost certain that the hydrogen
Figure 6. Other proteins also decreased H2O2-induced neuronal
death. The cocultures were exposed simultaneously to 100
␮mol/L H2O2 plus lactalbumin, ␥-globulin, bovine serum albumin, and casein for 60 minutes. Neuronal death was determined
24 hours later. n⫽12, pooled from 4 independent experiments.
**P⬍0.01.
Figure 5. HSA did not decrease NMDA-induced excitotoxicity.
Cocultures were exposed to 3 different concentrations of NMDA
for 20 minutes in the presence of 37.5 ␮mol/L HSA, which was
added 1 hour before the NMDA. At 20 minutes, medium was
removed and replaced with fresh medium plus 37.5 ␮mol/L
HSA. Neuronal death was determined 24 hours later. n⫽16,
pooled from 4 independent experiments. **P⬍0.01.
peroxide neurotoxicity also requires transition metals. In the
absence of metals, hydrogen peroxide is extremely stable,
with a calculated half-life for its uncatalyzed unimolecular
homolysis at 30°C of 1011 years.24 H2O2 interaction with
organic molecules requires transition metals, and chelation of
transition metals prevents this interaction.
Transition metals (in particular, iron and copper) are
capable of cycling between their reduced and oxidized states
and, in the process, generating an electron that can create a
free radical. In many cases, transition metals themselves may
be bound by a lipid, protein, or nucleic acid molecule, and a
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February 2004
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free radical causes site-specific damage at or near its binding
site.21–23 It is this type of activity that is most likely responsible for copper-dependent cell death in culture. If copper
and/or iron is decompartmentalized by ischemia/reperfusion,
the neurotoxic effects may be accelerated, and the presence of
HSA or its N-terminal tetrapeptide may prevent its binding to
sites where it could contribute to cellular injury. Alternatively, ambient, normal levels of transition metals in the
extracellular space may have no deleterious effects under
normal conditions but may become highly deleterious in the
presence of H2O2 formed by the superoxide generated during
ischemia/reperfusion.
The N-terminus DAHK of HSA is not the only amino acid
sequence that can bind transition metals or copper specifically.30 –33 In the present studies DAHK was found to be
equipotent with HSA in preventing H2O2 or copper/ascorbic
acid neurotoxicity, despite the fact that HSA is 126-fold
larger than DAHK. This suggests that the DAHK N-terminal
tetrapeptide is the primary locus of HSA antioxidant activity.
However, the other proteins examined had effects similar to
those of HSA, despite absence of the DAHK domain. It is
possible that relatively weak interactions between sulfhydryl
groups and/or amino acids (particularly tryptophan or histidine34,35) on these proteins and transition metals produce a
chelating effect that, in aggregate, is comparable to that
achieved with HSA or DAHK. The relatively high-affinity,
nonspecific binding of copper to proteins has been previously
reported to inhibit its capacity to generate hydroxyl radicals,34,35 which may explain in part the neuroprotective
effects of proteins other than HSA. Regardless of the mechanism of this effect, these results suggest that other proteins
may also contribute to brain antioxidant effects during bloodbrain barrier breakdown. This possibility has not been tested
in vivo.
In summary, we have demonstrated that HSA and its
N-terminal tetrapeptide DAHK can block oxidant-driven
neuronal injury produced with the use of 2 different oxidantgenerating systems: hydrogen peroxide and copper/ascorbic
acid. The fact that the peptide and HSA can block the
neurotoxicity of the latter generating system in a stoichiometric fashion implies that they are both binding copper and can
stop its redox cycling. The efficacy of DAHK in the hydrogen
peroxide– driven system suggests that copper is also involved
in oxidant-driven neurotoxicity in vitro and may be involved
in tissue injury after ischemia and reperfusion in vivo. DAHK
may be a useful alternative to HSA for the treatment of
stroke.
Acknowledgments
This work was supported by a Department of Veterans Affairs REAP
grant and National Institutes of Health grant RO1-HL53040 (to Dr
Panter). We gratefully acknowledge the technical assistance of
Angelo Zegna and the advice of Dr Weihai Ying.
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Human Serum Albumin and its N-Terminal Tetrapeptide (DAHK) Block
Oxidant-Induced Neuronal Death
Elizabeth T. Gum, Raymond A. Swanson, Conrad Alano, Jialing Liu, Shwuhuey Hong, Philip
R. Weinstein and S. Scott Panter
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Stroke. 2004;35:590-595; originally published online January 15, 2004;
doi: 10.1161/01.STR.0000110790.05859.DA
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