Blood Aging, Safety, and Transfusion: Capturing the “Radical” Menace

Article (PDF Available)inAntioxidants & Redox Signaling 14(9):1713-28 · October 2010with55 Reads
DOI: 10.1089/ars.2010.3447 · Source: PubMed
Abstract
Throughout their life span, circulating red blood cells (RBCs) transport oxygen (O(2)) primarily from the lungs to tissues and return with carbon dioxide (CO(2)) from respiring tissues for final elimination by lungs. This simplistic view of RBCs as O(2) transporter has changed in recent years as other gases, for example, nitric oxide (NO), and small molecules, such as adenosine triphosphate (ATP), have been shown to either be produced and/or carried by RBCs to perform other signaling and O(2) sensing functions. In spite of the numerous biochemical and metabolic changes occurring within RBCs during storage, prior to, and after transfusion, perturbations of RBC membrane are likely to affect blood flow in the microcirculation. Subsequent hemolysis due to storage conditions and/or hemolytic disorders may have some pathophysiological consequences as a result of the release of Hb. In this review, we show that evolution has provided a multitude of protection and intervention strategies against free Hb from "cradle" to "death"; from early biosynthesis to its final degradation and a lot more in between. Furthermore, some of the same naturally occurring protective mechanisms can potentially be employed to oxidatively inactivate this redox active protein and control its damaging side reactions when released outside of the RBC.
REVIEW ARTICLE
Blood Aging, Safety, and Transfusion:
Capturing the ‘Radical’ Menace
Paul W. Buehler, Elena Karnaukhova, Monique P. Gelderman, and Abdu I. Alayash
Abstract
Throughout their life span, circulating red blood cells (RBCs) transport oxygen (O
2
) primarily from the lungs to
tissues and return with carbon dioxide (CO
2
) from respiring tissues for final elimination by lungs. This simplistic
view of RBCs as O
2
transporter has changed in recent years as other gases, for example, nitric oxide (NO), and
small molecules, such as adenosine triphosphate (ATP), have been shown to either be produced and=or carried
by RBCs to perform other signaling and O
2
sensing functions. In spite of the numerous biochemical and
metabolic changes occurring within RBCs during storage, prior to, and after transfusion, perturbations of RBC
membrane are likely to affect blood flow in the microcirculation. Subsequent hemolysis due to storage conditions
and=or hemolytic disorders may have some pathophysiological consequences as a result of the release of Hb. In
this review, we show that evolution has provided a multitude of protection and intervention strate gies against
free Hb from ‘cradle’ to ‘death’’; from early biosynthesis to its final degradation and a lot more in between.
Furthermore, some of the same naturally occurring protective mechanisms can potentially be empl oyed to
oxidatively inactivate this redox active protein and control its damaging side reactions when released outside of
the RBC. Antioxid. Redox Signal. 14, 1713–1728.
Introduction
W
hen compared to other cells, human red blood
cells (RBCs) have modest but important metabolic
activities that include maintenance of cationic pumps and 2, 3-
diphosphoglycerate (2, 3-DPG) levels which are essential for
the allosteric modulation of O
2
binding to hemoglobin (Hb)
and the control of Hb oxidation. RBCs house a number of
reductive enzymes that keep Hb in the reduced functional
form and maintain membrane integrity. However, since RBCs
lack the ability to de novo synthesize and replenishes these
enzymes, an oxidant=antioxidant imbalance may occur as
these cells advance in age. RBCs may be stored for up to 42
days under controlled conditions before transfusion (Circular
of information for the use of human blood and blood com-
ponents. AABB, American Red Cross, America’s Blood Cen-
ters, and the Armed Services Blood Program. Bethesda, MD.
AABB.2009. http:== www.fda.gov=BiologicsBloodVaccines=
GuidanceComplianceRegulatoryInformation=Guidances =
Blood=default.htm, accessed on December 29, 2010) AABB
Circular, 2009). However, numerous changes occur in RBCs
during storage, collectively referred to as the ‘‘storage lesion’’
that can alter their biological function.
In an attempt to evaluate the efficacy of RBC transfusion, a
recent systematic review and meta-analysis was carried out
on 45 observational studies that involved 272,596 patients
(71). In 42 of the 45 studies, the risk of transfusion outweighed
the benefit. Overall, transfusion was associated with 70% in-
crease in death and 80% increase in risk of infection. The
question of why any rational physician would ever transfuse a
patient was raised in accompanying editorial to this paper
(26). However, a counter argument was made in favor of and
to the benefits of blood transfusion and was centered on the
fact that major progress has been made in improving blood
safety during the past 20 years and that the risk of human
immunodeficiency virus and hepatitis C has fallen to ap-
proximately 1 in 2–3 million (6).
According to the most recent transfusion-related fatalities
that were reported to the FDA, there was an increase in
transfusion-related acute lung injury (TRALI) fatalities from
RBCs. For the same blood products, fatal hemolytic transfu-
sion reactions seem to be declining, whereas reported fatali-
ties attributed to microbial infection remained the same.
Albeit the exact incidence of TRALI is not known, it has been
estimated that 1 in 5000 transfusions could demonstrate this
adverse outcome. TRALI has been the most common cause
Division of Hematology, Center for Biologics Evaluation and Research (CBER), U.S. Food and Drug Administration (FDA), Bethesda,
Maryland.
The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be
construed to represent any agency, determination, or policy.
ANTIOXIDANTS & REDOX SIGNALING
Volume 14, Number 9, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089=ars.2010.3447
1713
of transfusion related fatalities reported to the FDA since
2005 (http:==www.fda.gov=BiologicsBloodVaccines=Safety
Availability=ReportAProblem=TransfusionDonationFatalities=
ucm204763.htm, accessed on December 29, 2010). Many ef-
forts are currently being made to reduce the incidence of
TRALI. It was recently recommended that females should be
excluded as plasma donors during an international forum on
TRALI (111). In addition to the efforts to reduce TRALI, ef-
forts are also being made to reduce pathogens in RBC trans-
fusion products. Pathogen inacti vation in RBCs has been
studied and is still being investigated; however, there are no
licensed methods yet available (20, 57).
It was recently highlighted in the literature by several ret-
rospective analyses that older RBCs at time of transfusion in
certain patient groups may lead to adverse outcomes and
ultimately death (12, 58). In addition, it has also been reported
that transfusion of older RBCs promoted cancer progression
in an animal study (9). These reports on ‘older’ RBCs and
their deleterious effects raise the question ‘What is considered
an older RBC?’ Logically, one would be inclined to think that
these are cells near the end of their 42 day storage period.
Surprisingly, RBCs begin to undergo biochemical and phys-
iological changes early into storage, as in case of the reported
cancer study in rats, or 15 days and older for the retrospective
analyses in critically ill patients (9). Consequently, other ret-
rospective analyses have been performed to evaluate the
outcome of infusing older versus fresh RBCs into noncritically
ill patients (88, 109). The consensus is that there exists a cor-
relation between transfusing older blood and adverse events.
However, it should be taken into consideration that in pa-
tients receiving low volumes of blood, the age of the RBCs
may be negligible when compared to the severely injured or
critically ill patients who are transfused with large volumes.
In addition, the mechanism(s) underlying the toxicity of stored
RBCs must be understood to achieve safe storage limits.
Among the well-documented pathophysiologic conse-
quences of RBC storage are decreased deformability, im-
paired blood flow, impaired O
2
delivery to tissue, hemolysis,
and imbalance in nitric oxide (NO) homeostasis. Storage-
related changes in RBC membrane, specifically deformability
in human packed RBCs stored in CPDA-1 at 4–8C over a
4-week period have been investigated. It was shown that
human RBC deformability decreases significantly by 34%
after 4 weeks of storage. However, metabolic rejuvenation
restored RBC deformability to control levels (fresh RBCs) (28).
Several recent investigations have focused on the effects of
storage conditions on O
2
delivery, including the direct mea-
surements of local perfusion and microvascular O
2
distribu-
tion when 28-day stored RBCs are introduced into anemic
normovolemic hemodiluted animals. Circulation of stored
RBCs in these hemodiluted animals resulted in significantly
malperfused and under oxygenated microvasculature that
was not detectable at the systemic level (107).
Hemolysis, a measure of RBC destruction, is an important
risk factor as RBCs age and in the case of hemolytic anemias.
Hemolysis-associated pulmonary hypertension, transfusion-
associated lung Injury, post-perfusion renal and cerebral
dysfunction, morbidity and mortality of stroma-free Hb-
based blood substitutes, and aged blood associated mortality
in trauma are well-documented consequences of decom-
partmentalization of Hb (86). Despite efforts to better under-
stand the storage of RBC, these measures remain the most
useful predictors of RBC survival and function (86). Multiple
biochemical components of RBC storage-induced changes
were recently quantified, including hemolysis. It was shown
that cell-free Hb in stored blood increases steadily in the
medium reaching approximately 0.02 mM at the end of the 6
week storage and reaches to approximately 1 mM immedi-
ately after infusion in circulation, consistent with other pre-
vious reports (16). Activated leukocytes could be a source for
oxygen free radicals and their potential contribution towards
RBC hemolysis, alloimmunization, and febrile reactions, and
this has led to discussions about universal leukocyte reduc-
tion. Currently, in the United States, a leukocyte reduced
blood component is defined as a component containing less
than 510
6
residual donor leukocytes per final product (13).
A recent investigation on the effects of RBCs after pro-
longed storage in a mouse transfusion model showed that
transfusion of stored RBCs (14 days), or washed stored RBCs,
increases plasma non-transferrin bound iron, produces acute
tissue iron deposition, and initiates inflammation and en-
dotoxinemia. In contrast, the transfusion of fresh RBCs, or
transfusion of stored RBC-derived supernatant, ghosts, or
stroma-free lysates, does not produce these effects (48).
The impact of RBC storage on NO homeostasis is less clear
but was more visible in recent scientific and popular press.
One line of investigation promoting the physiological role of
intraerythrocytic s-nitrosolthiol Hb (SNO) at Cysb93 has been
proposed to play a critical role in maintaining RBCs ability to
dilate blood vessel, thus maintaining blood flow (90). Ac-
cordingly, stored blood and after the first few hours after
collection undergoes a gradual loss of its ability to release NO
from Cysb93 and that stored blood must be compensated for
the loss in NO before infusion. Another alternative source for
NO in blood that involves Hb is nitrite. In this case, it has been
suggested, based in large part on in vitro observations, that Hb
enzymatically reduces nitrite to NO which could also be used
to rejuvenate old RBCs (69). The role of NO or its metabolites
in restoring NO homeostasis, preventing hemolysis-induced
vasculopathy as well as its therapeutic applications remains,
however, largely speculative. NO is an autacoid molecule that
acts largely in the immediate microenvironment, and its long
term and global vascular reach may have been overestimated
(112).
Relevant biochemical, structural, and functional changes
related to RBC storage is multifactoral and complex in nature.
Impact of storage on Hb oxygenation and NO homeostasis is
likely to be transient and uncertain of significance. In contrast,
perturbations of RBC membrane are likely to adversely affect
blood flow in the microcirculation. More importantly, Hb loss
due to storage and or induced hemolysis may represent a
more serious safety problem that need to be fully understood
and controlled.
The focus of this article is to review recent advances in
transfusion practices, with a particular focus on the role of free
Hb released from aged RBCs and=or during pathological
events leading to hemolysis. The role of natural protective
pathways operative in the early genesis of Hb formation as
well as the role of endogenous protective mechanisms in
controlling the redox toxicity associated with free Hb during
RBC circulation are reviewed. It is hoped that lessons can be
drawn from these naturally occurring and effective antioxi-
dant mechanisms in the design of future interventions to
control Hb toxicity.
1714 BUEHLER ET AL.
Current Transfusion Practices Using Stored
Red Blood Cells
At present, the blood supply is safer than any time in the
history of transfusion in the United States and is considered to
be among the safest in the world (29). RBCs can be stored and
transfused when stored in an approved anticoagulant and=or
additive solution for up to 42 days and stored under refrig-
eration (1–6C) (Circular of information for the use of human
blood and blood components. AABB, American Red Cross,
America’s Blood Centers, and the Armed Services Blood
Program. Bethesda, MD. AABB. 2009. http:==www.fda.gov=
BiologicsBloodVaccines=GuidanceCom plianceRegulatory
Information=Guidances=default.htm and AABB’s Technical
Manual. 16
th
edition (93)). When collected in a sterile or
‘closed’ system, the storage requirements and expiration
dates of the RBCs vary significantly when compared to being
collected or processe d in an ‘open’ system. Table 1 shows
selected RBC components with their respective storage con-
ditions and shelf lives (93). Due to the limited allowable
storage period, it is not surprising that the RBC inventory is
subject to recurring highs and lows. Both scenarios are of
concern, because a periodic shortage is potentially life
threatening. On the other hand, when the inventory is high,
blood products may be lost due to outdating. In addition to
storing RBCs under refrigeration, they may be stored for 10
years when store d frozen in compliance with Federal regu-
lations and AABB guidelines (47).
Approximately 40 years ago, frozen RBCs were used for
transfusion. At the time, it was anticipated that with concur-
rent advances in technology their use would increase, how-
ever this did not materialize (87). At present, active research
continues to focus on extending the shelf life of RBCs without
compromising their integrity and effectiveness. A few exam-
ples of ongoing investigations are the storage of RBCs under
anaerobic conditions and the creation of universal donor
RBCs using either conventionally collected RBCs or progeni-
tor cell types that can be driven into mature RBCs (39).
As previously mentioned, RBCs undergo biochemical and
physiological changes during storage. It has been investigated
and well documented that some of these changes are revers-
ible, such as 2,3-DPG levels. The function of 2,3-DPG is to
stabilize deoxyHb in order to allow RBCs to deliver O
2
at
increased levels of tissue PO
2
. A rapid decline of 2,3-DPG
occurs after 7 days of storage. However, when RBCs are
transfused, the 2,3-DPG levels are restored in patients within a
day (up to 48 h) of transfusion (108). This is illustrated in
Figure 1. Nevertheless, questions remain regarding changes
to the RBC that may occur during storage and are not restored
after transfusion, for example, cellular deformability. When
do such changes occur? Do they occur during the first 14 days
of storage or after 30 days? Moreover, it should be noted that
the distribution of the RBC age in the circulation is presumed
to be approximately Gaussian, with an average centered at
their circulatory half life. Therefore, ‘fresh’ blood has a frac-
tion of RBCs at the end of their cycle, which may contribute to
Hb levels in the circulation early on.
A loss of cellular deformability and intravascular hemoly-
sis or inflammation could contribute to clinical adverse events
when RBCs reach critical storage durations (43, 48). Thus,
biochemical and physiological changes as shown in Figure 1
are an inevitable occurrence during RBC storage. Therefore, at
the end of storage, there will be RBCs that are able to carry and
deliver O
2
and there will be RBCs that will no longer be able to
carry out this function. We have recently shown that during
routine cold storage of AS-5 preserved RBCs, the RBCs re-
tained their ability to carry oxygen as they age. Using equi-
librium and rapid mixing kinetic measurements, we showed
for the first time that cells as old as 42 days of storage largely
preserved their in vitro interactions with oxygen. Other tested
parameters such as 2,3-DPG, ATP etc. decreased, whereas
percent hemolysis, extra cellular lactate levels, etc. increased
during the 42 days of storage as has been well documented in
the literature (40).
Storage Preservation, Hemoglobin Oxidation,
and Their Impact on Red Blood Cell Survival
With a few exceptions, the encapsulation of Hb within the
RBC in the animal kingdom can be viewed as nature’s re-
sponse to the toxicity of Hb, while ensuring O
2
delivery to
critical organs and tissues. The RBC therefore not only pro-
tects Hb from the body’s proteolytic degradative activities but
it also protects the body from Hb redox toxicity and provides
an efficient vehicle for O
2
sensing and transport. The impact of
Hb loss from RBCs during intravascular hemolysis in several
hemolytic conditions and its contribution to human diseases
is well established and has recently been reviewed (see (96) for
review).
Lesser known human health complications have been re-
ported with the transfusion of old RBCs that can be attributed
specifically to the oxidation of Hb and subsequent loss of the
Table 1. Maximal Storage Periods for Selected Red Blood Cell (RBC) Components Under Refrigeration
(1–6C) According to the AABB Technical Manual, 16
th
Edition
Red Blood Cell Components:
a
Storage Period:
21 Days or 28 Days 35 Days 42 Days
RBCs, 21 Days: ACD; CPDA-1 AS-1;AS-3;
Leukocytes reduced CPD;CP2D AS-5
RBCs, irradiated Original expiration or 28 days from
date of irradiation, whichever is sooner
Apheresis RBCs, CPDA-1 AS-1;AS-3;
Leukocytes reduced AS-5
a
Frozen RBC components, deglycerolized and rejuvenated RBCs are not included in this table. Consult the AABB Technical Manual, 16
th
Edition, Table 9-1 for a complete list of all blood components. ACD, acid-citrate-dextrose; AS, additive solution; CPD, citrate-phosphate-
dextrose; CP2D, citrate-phosphate-dextrose-dextrose; CPDA-1, citrate-phosphate-dextrose-adenine.
BLOOD AGING AND TRANSFUSION 1715
protein from the RBCs (54). This is due in large part to the
presence of several mechanisms within human RBCs that
presumably control the spontaneous oxidation of the iron
center of Hb. The redox hiatus within RBCs in the face of the
continuous reversible binding of O
2
to Hb is brought about in
large part by active antioxidative enzymatic machinery
within these cells. When ferrous (oxy) Hb is spontaneously
oxidized (auto-oxidation) or by chemically induced oxidation,
the latter is recycled back to the ferrous functional form so that
in the steady state the amount of intracellular metHb is kept
below 1%. The metHb is reduced by NADH-cytochromeb5-
metHb reductase. In addition, reduction can be done by
several dependant metHb reductases and direct reduction by
intracellular ascorbate and glutathione. The buildup of O
2
and H
2
O
2
as Hb undergoes auto-oxidation are controlled by
the abundant enzymes, superoxide dismutase (O
2
scaven-
ger) and catalase (H
2
O
2
scavenger) within the RBCs. In ad-
dition to catalase, glutathione peroxidase1 (GPX1) and
peroxiredoxin (Prx) II are also involved in H
2
O
2
elimination
within the RBC (18).
However, in spite of these natural antioxidative and pro-
tective mechanisms operating within RBCs, it has been shown
that young RBCs contain more potassium and sodium, have a
greater cell volume, and have a lower density than older cells.
Immature RBCs appear to be more resistant to hemolysis in
hypotonic media than older cells (18). A progressive decrease
in activities of several enzymes of the glycolytic pathway and
of the hexose monophosphate shunt pathway and in con-
centrations of organic phosphate esters is associated with
aging of mature mammalian RBCs. Although removal
mechanisms of senescent RBCs from circulation exist, the
survival of RBCs in vivo may still be determined by the sta-
bility the enzyme proteins that are required for maintenance
of the reduced functional state of Hb (18).
Unlike the well-documented Hb-mediated oxidative reac-
tions and subsequent oxidative injuries in vivo, little is known
about the extent of Hb oxidation during ex vivo storage of
RBCs. The formation of cross-links between Hb and mem-
brane proteins, a process known to be initiated by Hb oxi-
dation, and oxidative changes that accompany this process
were recently demonstrated in RBCs subjected to prolonged
hypothermic storage (59).
A number of possible scenarios in which Hb oxidation can
be implicated in the storage lesion was recently suggested
(54). First, in packed RBC units which contain 42–80 g of Hb,
the concentration of Hb that is clinically functional may be
reduced over time because of storage lesions, especially in
units reaching their maximum shelf life (49). Second, cell se-
nescence and changes in RBC reducing power (i.e., depletion
of endogenous antioxidants) can accelerate the rate of Hb
auto-oxidation and elevate the concentration of intracellular
ROS. Third, the level of molecular O
2
within the RBC unit that
is available for redox reactions can also be reduced (113) [For
recent discussion of auto-oxidation reactions of Hb and its
relationship to oxygen, see (91)].
Finally, the storing vehicle, including the blood bag and the
additive solutions, may also alter the Hb oxidative state (113).
Recent studies confirm the role of Hb oxidation in promoting
storage lesions (59). RBCs preserved in citrate–phosphate
dextrose–adenine storage solution units for up to 6 weeks
suffered oxidative injury characterized by the attachment of
denatured Hb, presumably hemichromes, to membrane
phospholipids and cytoskeleton proteins, such as spectrin.
They also reported that traces of denatured Hb were pr es-
ent in microparticles released from the cell membrane
throughout storage (60). The incid enc e of Hb-induced
membrane damage increased as a function of storage period,
reaching significant levels of Hb-membrane adducts after
35 days.
Extracellular Hb oxidation and quality of RBC relative to
preoperative blood salvage has recently been examined. De-
spite washing, extracellular Hb concentrations remained high
(up to 0.7 g=l in a given blood bag) and was associated with a
decrease of haptoglobin (Hp) in patients, despite a concomitant
inflammatory syndrome. Accordingly, it was recommended
that hemolysis must be limited during preoperative blood
salvage in order to prevent exposure to oxidized Hb and its
metabolites that may trigger cellular injury (43).
FIG. 1. Biochemical and physi-
ological changes of aging RBCs.
RBCs can be stored in an ap-
proved additive solution and
transfused up to 42 days when
stored at 1–6C. During the 42-
day storage period, biochemical
and physiological changes occur
(e.g., a rapid degradation of 2,3-
DPG). However, 2,3-DPG is re-
stored in patients after transfusion
(108). In each RBC unit there are
RBCs present of different ages (at
different stages of senescence) and
therefore, during the 42-day stor-
age period, different rates of
changes will occur. Overall, it
seems that some of the other ob-
served changes such as a decrease in pH and ATP seem to occur gradually over the 42-day storage period. This suggests that
the majority of RBCs in a unit after weeks of storage still have relatively normal biochemical properties and account for a
normal in vivo survival after transfusion (70).
1716 BUEHLER ET AL.
Potential Mechanisms and the Origin
of Hb Oxidation Within RBCs
Recently, Rifkind and his team (75) reported a set of ex-
periments that shed some light on the role and origin of Hb
oxidation within RBCs and how this may potentially be re-
lated to the process of aging. They showed that ROS gener-
ated in the cytosol are normally neutralized by abundant
antioxidant enzymes. However, H
2
O
2
generated by the
membrane-bound Hb is not accessible to the cytosolic anti-
oxidants and reacts to generate fluorescent heme degradation
products in vitro. For the first time, these studies established
a pathway for oxidative stress associated with Hb auto-
oxidation, despite the extensive antioxidant system in RBCs.
These byproducts of Hb oxidation and oxidative chemistry
that occur at the membrane surface may be released from the
RBC to affect nearby tissues and=or react with the RBC
membrane, altering RBC function and possibly contributing
to the removal of RBCs from circulation (75).
Extensive data exist in the literature which suggest that
membranechangesseeninRBCsaremostlylikelyduetohe-
moglobin oxidation, rapid heme loss, and then uptake of heme
into the membrane (67, 95). However, most recent data point out
that the role of the membrane in the formation of H
2
O
2
and the
resultant heme degradation products are result of noncystolic
oxidation of Hb, as depicted in Figure 2, based on the proposal
by Rifkind et al. (75). The primary event that triggers this reaction
cascade begins with the binding of Hb to the cytoplasmic end of
band 3 on the RBC membrane. Since deoxyHb has a higher
affinity for band 3 (98) than oxyHb, hypoxic conditions found in
the microcirculation will favor binding to the membrane. Partial
oxygenation of Hb that enhances binding to the membrane also
dramatically increases the rate of Hb auto-oxidation (92). Auto-
oxidation of Hb bound to the membrane produces a pool of
H
2
O
2
that is relatively inaccessible to catalase, a cytoplasmic
enzyme. This pool of H
2
O
2
will increase under hypoxic condi-
tions with the increased binding of Hb to the membrane. Al-
though H
2
O
2
in the region of the membrane can be removed
by peroxiredoxin and GPX (68), the activity of this enzyme is
dependent on the reducing power of NADPH=NADH, which
declines with age of cells and for various pathological condi-
tions. Under these conditions, the H
2
O
2
reacts with nearby Hb
to initiate a redox cycle in which ferric =ferryl Hb and possibly
its radical protein, resulting in a self-destructive cycle that leads
to the formation heme degradation products before it diffuses
into the cytoplasm to react with catalase.
The clinical consequences of excessive amounts of free Hb
in the circulation have recently been described (96). Hb acts as
a NO scavenger (30, 81) and, once released from RBCs, it will
immediately react with NO, depleting plasma levels of this
important signaling and vasodilator that has been shown to
affect regulation of blood flow, smooth muscle responses, and
intravascular thrombosis. This has clearly been documented
with free Hb in animals (19) and also when chemically or
genetically modified Hbs known as blood substitutes infused
in patients experiencing blood loss (101).
Extracellular Hb can rapidly oxidize to metHb due to the
process of auto-oxidation, reactions with cellular oxidants, and
through its interactions with NO (4). Accumulation of metHb
and possibly other oxidative byproducts can be enhanced if en-
dogenous mechanisms to eliminate it, such as Hp-mediated
clearance, are exhausted (19). MetHb and its denatured products,
such as heme, have been shown to intensify the inflammation
response of vessel endothelial cells and to promote atheroscle-
rosis through the oxidation of low-density lipoproteins.
However, the pathological effects of blood storage on
endothelial function have been in recent years attributed
solely to disruption of NO homeostasis (41, 64). NO is an
FIG. 2. Mechanisms and ori-
gin of Hb oxidation within
RBC. Proposed biochemical and
oxidative changes that stored
RBCs can undergo with time. In
young RBCs (left), antioxidative
enzymes can control reactive
oxygen species (ROS) resulting
from Hb auto-oxidation and ox-
idative reactions in the cytosol.
As RBC advances in age (right),
its reductive capacity is reduced
with time. Enhanced oxidative
reactions occurring at the mem-
brane surface of the RBC that are
unhindered by cystolic reductive
enzymes will perpetuate oxida-
tive changes sustained by Hb’s
radical chemistry, leading to the
formation of highly reactive and
damaging ferryl radical species
(75). Hb can be carried to the
outside of RBCs by released mi-
croparticles that ultimately lead
to free Hb into plasma (60).
(To see this illustration in color
the reader is referred to the web
version of this article at www
.liebertonline.com=ars).
BLOOD AGING AND TRANSFUSION 1717
important signaling as well as a vasodilator diatomic gas
produced by the vascular system (30, 81). The reaction is
primarily with the heme group that can be completed within
a few seconds with a profound consequence (i.e., blood
vessel constriction and elevation in both systematic and
pulmonary blood pressures: approximate mean arterial
blood pressure changes ranges between 15 and 30 mmHg).
However, blood pressure elevations seen after infusion of
free Hb ‘blood substitutes’ in both animals and humans
appear to follow a predictable path that can return to normal
within 2 hours.
It has been argued recently that reduced NO bioavailabil-
ity, as a result of free Hb in plasma, can augment thrombosis,
microcirculatory perturbations, or injury in patients with a
compromised vascular system. Accordingly, NO therapies in
the form of NO donors or by inhibition of NO synthetic
pathways or by modifying Hb Hb=RBCs to become a source
of NO have been advocated (41).
Similar approaches in which Sildenafil was given to pa-
tients with sickle cell anemia to control pulmonary blood
pressure triggered by free Hb has been, however, very dis-
appointing and these trials had to be stopped prematurely
because of the increased frequency of pain crises in these
patients (25). Bunn and a number of researchers, who are
actively involved in sickle cell disease research and manage-
ment, came up recently against clinical trails that were de-
signed to increase the bioavailability of NO for sickle cell
patients with clinical manifestations which were related to
plasma Hb. They further argued that NO levels are either not
crucial or are only one of many factors that influence the
pathophysiology of sickle cell disease (25).
In order to fully appreciate the complexity and the multi-
tude of naturally occurring mechanisms that are designed to
protect against Hb toxicity within the human body, it is im-
portant that a good understanding of the synthesis, regula-
tion, and protection against the radical chemistry originating
from the heme in its free and complexed forms is reviewed
within the following sections. It is hoped that a lesson or two
can be learned from these naturally occurring antioxidant
mechanisms that can be applied therapeutically to safely
controlling free Hb in hemolytic disorders and=or when
found in storage lesions.
Enzymatic Activities of Hemoglobin:
Hemoglobin as a Radical Enzyme
Hb is one of the most studied and characterized hemo-
proteins. While the function of Hb is primarily to carry O
2
from the lungs to tissues, our understanding of the physio-
logical function of Hb has changed over the past decade. Be-
cause of the catalytic nature of some of these newly reported
reactions, Hb has been given the title ‘honorary enzyme’ with
radical enzymatic and pseudoenzymatic activities and be-
cause of its toxicity; in some cases, Hb has been referred to as
the ‘rogue’ enzyme. These enzymatic activities include nitric
oxide dioxygenase, nitrite reductase, and peroxidase=
pseudoenzymatic were recently reviewed by Reeder (89).
Since the discovery in the 1980s that a diatomic gas such as
NO is the endothelial-derived relaxing factor (EDRF), there
has been an explosion in research carried out and published
on the physiological role of NO and the many biological
molecules that interact, carry, or destroy it.
NO reacts avidly with RBC’s oxyHb (HbO
2
) and muscle
myoglobin (MbO
2
) to form stoichiometric nitrate (NO
3
) and
ferric (Met) Hb or Mb (Eq.1) (31).
NO þ HbFe
II
- O
2
! HbFe
III
þ NO
3
(Eq: 1)
This reaction is critical to human physiology affecting NO
metabolism, signaling, and toxicity. This reaction also ham-
pered the application of cell-free Hb, developed as blood
substitutes and has been reported to be responsible for pul-
monary hypertension in patients with sickle cell anemia. Free
Hb, unlike RBCs, reaches NO production sites (i.e., the vas-
cular endothelium quite readily and reacts rapidly with NO).
Nitrite (NO
2
) can react with deoxyHb (HbFe
II
) to form
NO and ferric Hb according to Equation 2.
NO
2
þ HbFe
II
þ H
þ
! NO þ HbFe
III
þ OH
(Eq: 2)
The NO formed in this reaction can then bind to another
deoxyHb to form NO heme-bound nitrosylHb. Several oxi-
dative intermediates, including ferrylHb, have been involved
in these reactions (83). Accordingly, nitrite, a naturally oc-
curring circulating small molecule, can be readily converted
to NO inducing vasodilatation under hypoxic conditions. Hb
as a functional nitrite reductase can therefore be of potential
therapeutic values in sickle cell disease and other cardiovas-
cular indications (89).
Binding and release of NO has been suggested to be under
allosteric control of Hb, because the reactivity of the signal
amino acid, Cys b93, and that this reaction is conformation
dependant according to this thesis. The allosteric transition in
Hb from the tense (T) state (deoxygenated) to R (relaxed)
oxygenated state promotes the release of an NO group from
Hb’s hemes to its thiols (Cysb93) forming Hb-Cys b93 NO
(SNO-Hb).
Hb[Fe
II
NO] - Cysb93 þ 2O
2
! Hb[Fe
II
O
2
]
- Cysb93 - SNO þ O
2
þ H
þ
(Eq: 3)
Accordingly, O
2
can serve as an electron acceptor resulting
in O
2
production. RBC SNO-Hb has been suggested to
contribute to RBCs principal function, O
2
delivery via regu-
lation of blood flow, and therefore the RBC can be harnessed
for therapeutic purposes, including the reversal of RBC aging
(103).
The pseudoperoxidase activities of Hb and myoglobin
(Mb) have been under intense investigation for centuries
in vitro, and recent evidence is accumulating that these reac-
tions do occur in vivo with some serious consequences (89). It
has been known for some time that Hb can react with H
2
O
2
resulting in a complex redox chemical reactions. First, H
2
O
2
oxidizes ferrous Hb to generate the higher oxidation state of
the protein, (ferryl) ðHb
IV
¼ O
2
) and when reacting with the
ferric protein, a protein-based cation radical (
Hb
IV
¼ O
2
)is
formed:
Hb
II
þ H
2
O
2
! Hb
IV
¼ O
2
þ H
2
O (Eq: 4)
Hb
II
þ Hb
IV
¼ O
2
þ H
þ
! 2Hb
III
þ OH
(Eq: 5)
Hb
III
þ H
2
O
2
!
Hb
IV
¼ O
2
þ H
2
O (Eq: 6)
1718 BUEHLER ET AL.
Lessons Learned from Cell-Free Hemoglobin
Developed as Blood Substitutes
Many laboratories, over many years, have searched for ways
to prepare safe and effective cell-free Hb that will perform
similar functions outside RBCs. For use as blood substitutes,
also commonly known as cell-free Hb-based O
2
carriers
(HBOCs), Hb must be capable of cooperative O
2
uptake and
delivery with appropriate transition from relaxed R (oxy)- and
tense (T) –(deoxy) state affinities, and of maintaining reasonably
long functionality in circulation without adverse side effects.
Intracellular O
2
carriers prevail in the vertebrate kingdom,
due to the advantages derived from packaging reductants
and allosteric effectors together with Hb within RBCs. In the
absence of this packaging, HBOCs as decompartmentalized
Hbs are more readily lost to the circulation by renal filtration,
and lack the mechanisms to maintain the reduced functional
heme. More recently it has been shown that packaging Hbs
within red blood cells also helps avoid NO scavenging and
concomitant increases in blood pressure (66).
Chemically and=or genetically altered HBOCs have so far
demonstrated efficacy in proof of concept preclinical studies
and reasonable risk in toxicology studies involving normal
animals. However, HBOCs have not yet demonstrated a fa-
vorable risk to benefit ratio in human clinical trials (for a more
recent review of the subject, see Ref. 3). It remains to be seen if
the reduced allosteric properties of these HBOCs make them
ineffective as O
2
carriers in vivo. The physiological conse-
quence of the reduced cooperativity and reduced Bohr and
chloride effects of the HBOCs could be significant in affecting
tissue acid–base balance, O
2
delivery to tissues, and CO
2
transport to the lungs.
The reduced allosteric responses and enhanced rates of
auto-oxidation exhibited by the HBOCs make them prob-
lematic for use in an extracellular environment. The auto-
oxidation process is a source for nonfunctional metHb, as well
as reactive oxygen species (O
2
and H
2
O
2
). Recent research
shows that auto-oxidation of Hb can also be a source of highly
reactive ferryl heme and heme degradation products (76).
Nitrite-induced oxidation of air-equilibrated HBOCs can also
be a source of metHb and ferryl Hb (83). Cellular toxicity
attributed to ferryl heme includes promotion of lipid perox-
idation, lactate dehydrogenase release, and DNA fragmen-
tation, phenomena recognized as markers of cell injury and
death by apoptosis and necrosis (27). The globin-based ferryl
Hb radical was also detected in whole normal blood (105).
In vivo studies from our laboratory and others have indeed
shown that unhindered oxidation reactions and ferric Hb ac-
cumulation occur in animals from which blood was exchanged
transfused with HBOCs (22). These reactions occur at much
higher rates in animals such as guinea pigs that lack endoge-
nous reductive mechanisms, such as ascorbic acid, as opposed
to animals that are enzymatically capable of producing ascor-
bate (22). Indirect EPR measurements of ferryl radicals in rab-
bits infused with HBOCs were reported recently (32). Subtle
oxidative changes at the amino acids levels in proteins after
infusion of HBOCs or stroma-free Hb were recently identified
by more sensitive mass spectrometric methods (53).
Protection Against Hemoglobin from Cradle to the Grave
Figure 3 illustrates the diverse and complex physiological
pathways that are deployed in the mammal system to control
Hb oxidative reactions and the proteins that have been spe-
cifically designed to lessen the toxicities associated with the
Hb and the by products of Hb oxidative reactions, for exam-
ple, heme (discussed here) and iron (discussed elsewhere, see
(13) for review). These control mechanisms span from early
erythropoiesis to heme degradation in macrophages and
many other pathways as outlined in Figure 3.
Alpha hemoglobin stabilizing protein
Toxicity of Hb molecule is driven in large part by its redox
active heme prosthetic group. However, even at the very early
erythropoietic developmental stages and when heme is in-
corporated into its respective a or b chains, the a chain in
particular is less stable than its b chain counterpart of the
protein. In erythroid cells, ROS react with aHb, causing its
breakdown and precipitation, and also damage other cellular
constituents. Both a and b globin gene loci are located on
chromosome 16 and 11 which produce a-and b-globin mRNA
and a- and b-globin polypeptides, respectively. These two
combine to form a Hb dimer and when combined a full
functional Hb tetramer is formed. The intrinsic instability of
the a chain has been recognized for some time. However, it
was only recently that this process was shown to require a
helper or chaperon protein, the alpha Hb stabilizing protein
(AHSP). This protein now we know plays a critical role in
stabilizing the a subunit of Hb molecule.
In vivo evidence confirming the critical role of AHSP came
from studies reported in mice where the ASHP gene was
completely ablated, that is, the ability of ASHP protein-coding
was removed to ensure complete loss of protein expression in
homozygous-null (AHSP=) animals. In these animals,
mild hemolytic anemia with shortened RBCs survival was
observed in which excess Heinz bodies, indicative of dena-
tured Hb, were also found. Moreover, AHSP=RBCs pro-
duced excessive ROS, and exhibited oxidative damage to
endogenous proteins, contrary to heterozygous (AHSPþ=).
RBCs were normal in number, appearances, and life span
(110). More recent work showed that AHSP also act a mo-
lecular chaperone to stabilize nascent a-globin for the final
HbA assembly. It promotes native folding of the apo-a-globin
and its assembly into aHb in solution and stabilized a pool of
free aHb (115).
The three-dimensional structure of AHSP was determined
based on studies that utilized resonance spectroscopy and
X-ray crystallography (Fig. 4) (36). AHSP forms an elongated
three a-helix bundle fold. The binding of aHb was determined
to be at the C-terminus of helix 1, the loop connecting helices 1
and 2, and the N-terminal part of helix 2. These studies also
revealed that AHSP complexes with aHb in the same region as
one of the two b chains of HbA at the a
1
b
1
interface region
which is located directly opposite to the aHb heme pocket.
The proposed AHSP-mediated stabilization of aHb have
been described by a series of elegant experiments performed
by Weiss and his group (for review, see (74)). The a-stabilizing
protein minimizes the deleterious effects of the free a-subunit
by limiting its prooxidant activity. AHSP binds to oxy-a-Hb,
alters the structure of the heme, and induces rapid auto-
oxidation, which generates H
2
O
2
. This is followed by the
formation of a Fe-III bis-histidyl complex that prevents further
redox cycling and the formation of the ferryl subunit. This
hemichrome form of a-Hb is resistant to further oxidation and
BLOOD AGING AND TRANSFUSION 1719
heme loss, because of the sixth coordinate position of the
heme iron is occupied and unable to generate ROS (36). It was
recently reported that the recovery of this inert form of aHb
subunit can be accomplished by further reduction to the fer-
rous functional form prior to it binding to its subunit coun-
terpart, the b chains of HbA (116).
Similar to AHSP-induced changes within a subunit of Hb
detailed above, the reorganization of the heme pocket region
into a hexacoordinated configuration via the distal-proximal
histidines was also found in naturally occurring neuroglobin
(found in neurons) and cytoglobin (expressed in all tissues). It
is interesting to note that Hp, similar to AHSP binds to a
specific region on the protein close to the heme pocket.
However, in the presence of oxidants and unlike ASHP which
structurally reorganizes the heme pocket, Hp accomplishes
redox stability by short circuiting the emerging and damaging
radicals from the heme (Cooper et al., unpublished studies).
AHSP–aHb interactions described thus far may have clin-
ical and biological relevance in b-thalassemia. AHSP has been
reported to act as a genetic modifier in this disease. The loss of
AHSP may worsen b-thalassemia, most likely by destabilizing
excess free aHb, apo-a-globin, or likely both. AHSP also
provide a selective advantage for the survival of red cells,
especially when there are excess of either a-orb-globin
present. Moreover, because of its effects on preventing a-
globin denaturation, AHSP may also provide an additional
selective advantage to the RBC under conditions of oxidative
stress that may lead to hemolysis.
Haptoglobin
The primary mammalian Hb binding proteins circulating in
plasma are Hp-a
2
–sialoglycoproteins made up of Hp-a and
Hp-b globin chains and collectively termed Hp. While all
mammalian species possess Hp protein or the gene(s) to ex-
press it, only humans are known to express phenotypically
differing forms originating from two gene variants (Hp1 and
Hp2). Differences occur only in gene variants controlling for
Hp-a globin chains and therefore the designation is dependent
onthepresenceofHp-a
1
or Hp-a
2
globin chains resulting in Hp
1-1 (a
1
a
1
), 2-1 (a
2
a
1
), and 2-2 (a
2
a
2
) (62). Hp-a globin chains are
primarily involved in disulfide bond formation with Hp-a
1
FIG. 3. Protection of he-
moglobin from cradle to the
grave. Detoxification systems
throughout the different com-
partments in human physi-
ology that provide protection
against Hb=heme=iron are
outlined. These processes and
key proteins that trigger these
activities are indicated within
each compartment. Alpha he-
moglobin stabilizing protein
(AHSP) provides protection
against oxidative damage to a
subunit during early erythro-
poiesis. Haptoglobin (Hp) and
CD163 receptors on macro-
phages coordinate Hb dimers
clearance when Hb is released
from aging RBCs or during
hemolysis. Once Hb dimers
are cleared by macrophages,
heme oxygenase (HO) de-
grades heme released from
Hb into iron, bilirubin, and
carbon monoxide (CO). When
heme is released into plasma,
several proteins with different
affinities to heme collectively
bind and clear heme from
circulation. Some of these
proteins that are reviewed
here include: hemopexin
(HPX), high and low density
proteins (HDL=LDL), albu-
min, and a
1
- microglobulin
(a
1
-M). Transport, storage,
and metabolism of iron, the
by product of heme degra-
dation is briefly outlined here
and described fully elsewhere
(see (14) for review).
1720 BUEHLER ET AL.
chains forming single disulfide bonds with an adjacent Hp-a
chain, and each physically is associated with one Hp-b globin
chain. Hp-a
2
chains differ in that they can form two disulfide
bonds and are therefore polymeric mixtures with each Hp-a
globin associated with one Hp-b globin chain (84). The Hp-b
chains are involved in the binding of Hb dimers with contact
sites previously identified in both Hb-a and Hb-b globin chains
(55, 114). Recent studies with chemically crosslinked and po-
lymerized Hbs have demonstrated the critical role of exposed
Hb-a chains in Hp 1-1 and Hp 2-2 binding (23). The binding of
Hp 1-1 to human dimeric Hb is reported to occur rapidly
(k ¼ 5.5 x 10
5
M
1
sec
1
) with high affinity and a very small
dissociation rate constant, while Hp 2-1 and Hp 2-2 bind Hb
dimers more slowly (77). Hp 1-1 is therefore considered to be
the most efficient Hb plasma binding protein and can remove
Hb in a ratio of 1:1 (Hp 1–1:Hb). Together Hps circulate as
mixtures in human plasma in a approximate concentration
range from 30 to 200 mg=dl (99). Most mammalian species
possess only Hp 1-1 at varying plasma concentrations, only
certain nonhuman primates and humans posses additional
phenotypes, suggesting a need for evolutionary divergence in
non-human primates and humans.
Hp is often termed an ‘antioxidant protein’ based on two
critical properties. First, Hp binds free Hb and rapidly directs
it toward downstream clearance pathways, preventing tissue
distribution. Second, Hp can protect bound Hb’s damaging
influences in peroxidative environments such that heme em-
anated radical generation is prevented from causing damage
to the Hb protein and the surrounding environment. Previous
in vitro studies have shown that H
2
O
2
induces Hb protein
damage is initiated by amino acid oxidation and later heme
pocket, a helical structural damage, protein–protein and
protein–heme crosslinking with increased H
2
O
2
exposure
(55). Similar effects are observed in vivo within various tissue
compartments with exposure to extracellular Hb (21). How-
ever, the oxidative effects cause by H
2
O
2
are attenuated once
the Hb–Hp complex is formed, suggesting a unique antioxi-
dant role of Hp in addition to role as Hb binding protein.
The effective removal of Hb by Hp 1-1 from circulation has
been associated with reduced risk long-term vascular-related
sequela from numerous pathological conditions having he-
molytic components (65). As a result, exogenous administra-
tion of Hp used as a Hb scavenging therapeutic could be
particularly useful in the event of mild to moderate hemolytic
conditions once low levels of endogenous Hp are saturated.
Macrophage scavenger receptor (CD163)
Once extracellular Hb complexes with Hp in circulation,
the complex is rapidly cleared by monocyte=macrophage cell
surface cysteine rich scavenger receptors identified as CD163
(61). Clearance can take place within the circulation or in the
liver; however, the process appears to be saturable and de-
signed to accommodate removal of only complex within a
limited but not well-defined concentration range (34). Recent
work revealed that when Hb–Hp complex was administered
to guinea pigs and dogs at increasing concentrations, the
circulating half-life increased by approximately 50-fold, while
the nonrenal clearance was decreased by a similar magnitude
(19). Therefore, after saturation of CD163, clearance of the
complex is dictated by the pharmacokinetics of Hp. In-
dependent of Hp complex formation, Hb can interact directly
with CD163 with a low affinity (Kd approximately 400 fold
less than Hb–Hp) (80). The process is dependant on direct
interaction with N-terminal amino acids of Hb’s b globin
chain and may be a relevant clearance pathway for extracel-
lular Hb in persons with anhaptoglobinemia (97).
The interaction and uptake of either Hb–Hp or Hb via the
CD163 receptor into monocytes=macrophages leads to the
breakdown of heme via heme oxygenase (HO). This cascade is
the essential next step leading to iron and heme detoxification.
Heme oxygenase
The microsomal heme oxygenase system consists of two
primary isoforms including HO-1, HO-2, and a third isoform,
HO-3, of little known human relevance. HO-1 is the inducible
while HO-2 is the constitutive isoform in mammalian species
and both play a critical role in heme metabolism. The stability
of heme containing proteins plays a critical role in HO-1
regulation (11) and this process of heme breakdown is initi-
ated by heme in the following sequence:
(1) heme þ O
2
þ NADPH ? Fe
II
þ carbon monoxide (CO)þ
biliverdin (iron release)
(2) biliverdin ? bilirubin (heme catabolism)
(3) Fe
II
? ferritin ? hemosiderin (tissue iron storage)
(4) Fe
III
? transferrin (plasma iron binding and transport)
In reaction (1), the process is enzymatically controlled by
the microsomal heme oxygenase system made up of HO and
NADPH-P450 reductase, while reaction (2) is controlled en-
zymatically by biliverdin reductase. The sequestration and
storage of Fe
II
in process (3) following reaction (1) occurs
FIG. 4. Crystal structure of alpha-hemoglobin stabilizing
protein bound to ferrous hemoglobin alpha-subunit. AHSP
on the right adopts an elongated three-helix bundle, whereas
a-subunit is composed of seven a–helices. AHSP binds aHb
on the side of the molecule opposite the heme pocket. In the
ferrous–aHb–AHSP complex, The F-helix (right to the heme)is
distorted and the heme surface is open to interactions with
solvent. On oxidation, the iron atom is reformed to generate
more oxidatively stable bis-histidyl configuration. The
structure was derived from PDB file 1Y01 (35). (To see this
illustration in color the reader is referred to the web version
of this article at www.liebertonline.com=ars).
BLOOD AGING AND TRANSFUSION 1721
initially in ferritin and later as the aggregated ferritin=protein
complex, hemosiderin.
The role of HO as a protective enzymatic system is estab-
lished extensively throughout the literature, and as a result
HO has been a target for upregulation in several disease
states. The end products of the HO-catalyzed reaction bili-
verdin and its metabolite bilirubin are believed to possess
antioxidant potential by removing reactive oxygen species
such as O
2
and hydroxyl radical (
OH) (104). Additionally,
ferritin has demonstrated cellular protective effects via anti-
oxidant activity on the vascular endothelium (10), while CO
can function as a vasodilatory gas similar to NO by increasing
cyclic guanosine monophosphate (cGMP), leading to de-
creased vascular resistance (78, 79). However, the majority of
scientific effort in the area of HO induction by heme focuses
on low and controlled levels which could differ significantly
from Hb exposures that can take place during both acquired
and genetic hemolytic anemias and when Hb is released from
stored RBCs. Figure 5 summarizes the interaction of Hb with
Hp, uptake by cell surface CD163 and Hb metabolic break-
down by HO.
Heme Storage and Transport
In circulation, free heme released from Hb upon hemolysis
of RBCs is instantly oxidized to its ferric state (often called
hemin) which may catalyze the formation of ROS that result in
the generation of oxidized forms of low density lipoproteins
(52). High and low density lipoproteins (HDL and LDL), he-
mopexin (HPX), and serum albumin (SA) bind most of the
free heme in plasma (Fig. 6). It is known that more than 80% of
heme immediately intercalates into LDL and HDL, whereas
the remaining *20% of heme are taken up by albumin and
HPX. However, the majority of LDL=HDL-bound heme will
be gradually transferred from lipoproteins to HPX and albu-
min. HPX has a higher binding affinity to heme than albumin
does, slowly binds most of heme, and transports it to specific
receptors on parenchymal liver cells where it undergoes
receptor-mediated endocytosis (51).
The dissociation of heme from Hb, including dissociation
from alpha and beta subunits of methemoglobin, and mea-
sured rate constants for dissociation and heme transfer to li-
poproteins, albumin, and hemopexin have been extensively
investigated (see Refs. 24, 45, 38, 46, and 82).
FIG. 5. Clearance of hemo-
globin by haptoglobin=
CD163. Aged red blood cells
represent a potential source of
extracellular hemoglobin (Hb)
(Tetramer, a-globin, white cir-
cles; b-globin, gray circles). (Top
left) Hb can then bind to
circulating haptoglobin (Hp)
(Center: gray-filled oval in
shape of a dumb-bell). Hb is
forced to dimerize and bind
to Hp, which is then bound
to monocyte=macrophage cell
surface CD163 (shown as a
nine domain cysteine rich
scavenger receptor tethered
to membrane of monocyte=
macrophage) and taken up
into the cell. The progression
of catabolism of heme (cross-
hatched circles) and generation
of critical by-products (carbon
monoxide (CO), biliverdin
(small gray circle)=bilirubin
( filled circle)) and sequestra-
tion of iron (Fe
2þ
) by binding
proteins are shown. Right top
shows the interaction of
chemically modified Hbs with
Hp (see (23) for detailed in-
teraction) demonstrating that
b crosslinked and polymer-
ized Hbs bind relatively well
to Hp, while a crosslinked
Hbs do not. The interaction
Hb with CD163 in the ab-
sence of Hp can proceed at a
low affinity with native and
crosslinked Hbs but not larger
multimeric Hbs (97).
1722 BUEHLER ET AL.
Table 2 summarizes the nature of heme linkages in Hb, in
the tetrameric and dimeric forms and in those proteins that
handle free heme derived from Hb. As can be seen among
plasma proteins, HPX has the tightest linkage (K
a
*1.910
13
M
1
) with heme via bis-His complex that is stabilized by
hydrophobic and electrostatic interactions within the heme
pocket. The affinity albumin towards heme is much lower (K
a
*510
7
to 2 10
9
M
1
); however, this has been compensated
by a remarkable abundance of albumin.
Hemopexin
HPX serves as the primary specific carrier of plasma heme
and participates in its clearance by transporting it to the liver,
and thus it functions as a major plasma protector against
oxidation because the heme–HPX complex is completely in-
active as an oxidant (44). HPX is an acute phase plasma pro-
tein, and its plasma levels vary, from 8 mM to 21 mM (102).
HPX competes with LDL for heme, and therefore it may also
regulate Hb oxidative activity (42). After transporting the
heme through a receptor on the parenchymal cell, the intra-
cellular HPX is recycled to its intact free form and released
into the blood stream (for review, see Ref. 7).
High and low density lipoproteins
Given the importance of LDL and HDL in cardiovascular
diseases, research efforts focused on the investigation of these
lipoproteins have provided a wealth of valuable information
during the last decade, including crystal structure and bio-
chemical characterization. Earlier studies pointed out that free
heme associates with proteins and lipids of the lipoprotein
particles at multiple sites (72). However, the transient heme
binding to HDL and LDL and the physiological mechanism
by which lipoproteins become oxidized remain unclear.
Published data suggest that heme released from Hb into the
circulation may compromise vascular endothelial cell integ-
rity through oxidative modification of LDL that activates cell
protective proteins, HO-1, and ferritin (52). Whereas plasma
lipoproteins, HDL and LDL, have a very high affinity to heme
(K
d
in between 10
10
M and 10
11
M) and rapidly accept most
of it, these plasma compartments are the most sensitive to
heme-induced oxidation. It is therefore important that the
heme release from HDL and LDL and its transition to HPX
proceed faster than the oxidation of lipoproteins triggered by
heme (73). Incorporation of heme into the LDL particle results
in peroxidation of the hydrophobic core lipids of the lipo-
protein, whereas free iron promotes oxidation of the surface
lipids only (106). Recently, it was shown that the heme-CO
monomer binds to the high affinity site of apolipoprotein B
(ApoB) on the LDL particle. Yet the precise mechanism by
which heme associates with LDL and HDL remain to be de-
termined [see recent reviews (85,100)].
Serum albumin
Due to its unique structure, albumin possesses remarkable
binding capacities for many endogenous and exogenous
molecules in the plasma (33). Therefore, it is involved in a
variety of metabolic processes, including the maintenance of
plasma antioxidant capacity by scavenging ROS and reactive
nitrogen species (RNS) and lipid peroxidation byproducts (94)
FIG. 6. Albumin and heme complex. The heme albumin
complex was derived from PDB crystal structure 109X for the
HA–myristate–heme complex (117). Heme (space-filling rep-
resentation) occupies a hydrophobic D-shaped cavity in sub-
domain 1B, also suitable for binding fatty acids. Heme
binding to albumin is stabilized by pp stacking interaction
provided by the two tyrosine residues, Y161 which is in
coordination with ferric heme iron, and Y138 whose phenolic
hydroxyl is directed to the exterior. Basic amino acid resi-
dues H146 and R114 provide additional stabilization via
electrostatic interactions and K190 via salt-bridges with
heme propionate groups. (To see this illustration in color the
reader is referred to the web version of this article at www
.liebertonline.com=ars).
Table 2. Heme Linkage with Proteins and Lipoprotein
Type of heme linkage
Proteins (Fe
II
=Fe
III
state) K
d
constants
High and low density
lipoproteins (HDL=LDL)
Transient binding to the lipoprotein (Fe
III
)K
d
* 10
11
M to 10
10
M
(73)
Hemopexin (Hpx) Hexacoordinate with low spin (Fe
III
)K
d
: < 10
13
M
(1)
Albumin (SA) Pentacoordinate with high spin (Fe
III
)K
d:
*10
8
M
(1)
Heme oxygenase-1 (HO-1) HO-1 binds and catabolizes heme into
biliverdin, free Fe
II
and CO
N=A
a
a
1
-Microglobulin Binding at hydrophobic pocket of lipocalin K
d
* 10
6
M
(63)
(a
1
M) b-barrel (Fe
II
to 0)
a
In cooperation with NADPH cytochrome P450 reductase.
BLOOD AGING AND TRANSFUSION 1723
and the sequestering of free heme (8). Although the albumin
affinity for heme is significantly lower than that of lipopro-
teins and HPX, due to its abundance, albumin greatly con-
tributes to suppressing of free heme toxicity.
Earlier studies on albumin interactions with heme revealed
one primary binding site of high affinity that is specific to
heme and several weaker binding sites (15). A crystal struc-
ture resolved for albumin–heme–myristate complex (1:1:4)
identified a single heme molecule bound to the hydrophobic
D-shaped cavity in subdomain 1B, which is known to be a
fatty acid binding site, whereas myristate was bound to six
other fatty acid binding sites (117). Heme accommodation in
this highly hydrophobic binding site is stabilized by pp
stacking interaction provided by the tyrosine residues Y138
and Y161. The position of phenolic hydroxyl group of Y161
implicates coordination with ferric heme iron, whereas the
hydroxyl group of Y138 is directed to the exterior solvent.
Similar to Hb, but at the lower degree, albumin provides an
additional stabilization of heme by a coordination of the
propionate groups by basic residues of the binding site: H146
is involved in electrostatic interaction with one of heme car-
boxylates, the guanidinium group of R114 with the other (2.8
A
˚
), whereas K190 forms salt bridges with both propionate
groups (117). Heme binding to albumin is known to be allo-
sterically modulated; it can be inhibited and even prevented
by fatty acids binding at multiple sites, including the com-
petitive binding to the heme site.
a
1
-Microglobulin
a
1
-Microglobulin (a
1
-M) is a small glycoprotein (26 kDa)
that belongs to the lipocalin superfamily (2, 37). It participates
in heme scavenging due to its interactions with heme and Hb
(5). This small yellow-brown protein is charge and size het-
erogeneous. The exact function of a
1
-M is not fully under-
stood. a
1
-M binds covalently to many plasma proteins (17). In
the circulation, about 50% of a
1
-M exists in a 1:1 complex with
immunoglobulin A (IgA). Whereas free a
1
-M undergoes a
proteolytic cleavage, the a
1
-M=IgA complex serves as a res-
ervoir in which a
1
-M is preserved from proteolysis (due to
blocked C34 residue) and glomerular filtration (due to its
larger size). a
1
-M shares a typical sequence motits for lipoca-
lins eight-stranded b-barrel structure that forms a highly hy-
drophobic pocket suitable for various small ligands (37). The
free truncated a
1
-M (with C-terminal tetrapeptide cleaved)
binds the heme with K
d
* 10
6
M (63). The details of the
mechanism of heme degradation by a
1
-M are unknown. No-
teworthy, the truncated a
1
-M carries a heterogeneous mixture
of yellow-brown chromophores, presumably degradation
products of protoporphyrin, that are bound to cysteine and
lysine residues at the rim of the binding cavity, that is, C34,
K92, K118, and K130 (5, 17).
Whereas the heme scavenging by LDL=HDL, SA, and HPX
seems to be sufficient to minimize the levels free heme in
plasma, it is not excluded that other plasma proteins, less
abundant than SA and less specific to heme than HPX and SA,
may also contribute in the maintenance of heme in protein-
associated form, at least transiently. For example, heme as-
sociation with plasma a
1
-antitrypsin is of lower affinity
than that of SA (unpublished data), yet it illustrates the idea
that some other plasma proteins also may interact with free
heme.
Conclusion
Hemoglobin enzymatic activities such as NO removal (di-
oxygenase), NO production (nitrite reductase), and the con-
sumption of H
2
O
2
(peroxidase=pseudoperoxidase) can
trigger a number of side reactions that can be detrimental to
the protein and the surrounding tissues. However, nature has
provided an impressive array of proteins that are capable of
dealing effectively with released Hb and=or heme as RBC age
or during hemolysis due to disease states, therefore detoxi-
fying Hb and its oxidative byproducts. Indeed, we have re-
cently reported that when stroma-free Hb is complexed with
Hp, the Hb–Hp complex will lower blood pressure (due to
scavenging of NO by Hb) and prevent heme-mediated tissue
toxicity (due to oxidative chemistry of Hb) in dogs and in
guinea pigs in which their Hp levels were either induced by
drugs or co-infused with Hb, respectively (19). Moreover, and
surprisingly, the Hb–Hp complex retained its NO and O
2
binding characteristics. It may prove necessary to explore
some of theses naturally occurring antioxidative and clearing
mechanisms of acellular Hb which have survived selective
evolutionary pressures, as a basis of countering the side-
effects associated with free Hb in hemolytic anemias, oxygen
therapeutics, and possibly in controlling storage lesions rather
that relying on short-lived NO-based therapeutics.
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BLOOD AGING AND TRANSFUSION 1727
Address correspondence to:
Abdu I. Alayash, Ph.D., D.Sc.
CBER, FDA
NIH Bldg 29, Rm. 112
8800 Rockville Pike
Bethesda, MD 20892
E-mail: abdu.alayash@fda.hhs.gov
Date of first submission to ARS Central, July 2, 2010; date of
final revised submission, Oct 7, 2010; date of acceptance, Oct
14, 2010.
Abbreviations Used
2, 3-DPG ¼ 2, 3-diphosphoglycerate
a
1
-M ¼ a
1
-microglobulin
ACD ¼ acid citrate dextrose
AHSP ¼ alpha hemoglobin stabilizing protein
AS ¼ additive
CD163 ¼ macrophage scavenger receptor
CO ¼ carbon monoxide
CPDA ¼ citrate-phosphate dextrose adenine
EDRF ¼ endothelial derived relaxing factor
EPR ¼ electron paramagnetic resonance
GPX1 ¼ glutathione peroxidase 1
Hb
IV
¼ O
2
, ferryl hemoglobin
Hb
IV
¼ O
2
, ferryl hemoglobin radical
HbFe
II
-O
2
¼ ferrous (oxy)
HbFe
III
¼ ferric hemoglobin
HBOCs ¼ hemoglobin based oxygen carriers
HDL ¼ high density lipoproteins
HO ¼ heme oxygenase
H
2
O
2
¼ hydrogen peroxide
Hp ¼ haptoglobin
HPX ¼ hemopexin
IgA ¼ immunoglobulin A
LDL ¼ low density lipoproteins
MetHb ¼ methemoglobin
NO ¼ nitric oxide
NO
2
¼ nitrite
NO
3
¼ nitrate
Prx ¼ peroxiredoxin
RNS ¼ reactive nitrogen species
ROS ¼ reactive oxygen species
SA ¼ serum albumin
SNO ¼ s-nitrosolthiol,
TRALI ¼ transfusion-related acute lung injury
1728 BUEHLER ET AL.
    • "Exogenous sources of free heme include red blood cell (RBC) preparations during storage (Buehler et al., 2011), heme-based therapeutics (such as Panhematin, hemin for injection, and heme arginate) that are used for the treatment of porphyrias (Tenhunen and Mustajoki, 1998; Siegert and Holt, 2008), and Hb-based blood substitutes (Alayash, 2014). In the circulation, the free heme redox toxicity is mitigated by several innate defense mechanisms, including heme sequestration by plasmatic major heme scavengers hemopexin, albumin, and some other proteins (Ascenzi et al., 2005; Buehler et al., 2011). A1M, also known as protein HC, has emerged among anti-oxidative defense mechanisms as a tissue housekeeping protein which is capable of capturing heme and free radicals (Allhorn et al., 2002; Åkerström et al., 2007; Olsson et al., 2012; Åkerström and Gram, 2014 ), as well as reducing oxidative lesions (Allhorn et al., 2005; Olsson et al., 2008; Rutardottir et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme 1 and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions. Methods: To examine heme binding properties of A1M we utilized UV/Vis absorption spectroscopy, visible circular dichroism (CD), catalase-like activity, migration shift electrophoresis, and surface plasmon resonance (SPR), which was specifically developed for the assessment of His-tagged A1M. Results: The results of this study confirm that A1M is a heme binding protein that can accommodate heme at more than one binding site and/or in coordination with different amino acid residues depending upon heme concentration and ligand-to-protein molar ratio. UV/Vis titration of A1M with heme revealed an unusually large bathochromic shift, up to 38 nm, observed for heme binding to a primary binding site. UV/Vis spectroscopy, visible CD and catalase-like activity suggested that heme is accommodated inside His-tagged (tgA1M) and tagless A1M (ntA1M) in a rather similar fashion although the His-tag is very likely involved into coordination with iron of the heme molecule. SPR data indicated kinetic rate constants and equilibrium binding constants with K D values in a μM range. Conclusions: This study provided experimental evidence of the A1M heme binding properties by aid of different techniques and suggested an analytical methodology for a rapid evaluation of ligand-binding properties of recombinant A1M versions, also suitable for other His-tagged proteins.
    Full-text · Article · Dec 2014
    • "Exogenous sources of free heme include red blood cell (RBC) preparations during storage (Buehler et al., 2011), heme-based therapeutics (such as Panhematin, hemin for injection, and heme arginate) that are used for the treatment of porphyrias (Tenhunen and Mustajoki, 1998; Siegert and Holt, 2008), and Hb-based blood substitutes (Alayash, 2014). In the circulation, the free heme redox toxicity is mitigated by several innate defense mechanisms, including heme sequestration by plasmatic major heme scavengers hemopexin, albumin, and some other proteins (Ascenzi et al., 2005; Buehler et al., 2011). A1M, also known as protein HC, has emerged among anti-oxidative defense mechanisms as a tissue housekeeping protein which is capable of capturing heme and free radicals (Allhorn et al., 2002; Åkerström et al., 2007; Olsson et al., 2012; Åkerström and Gram, 2014 ), as well as reducing oxidative lesions (Allhorn et al., 2005; Olsson et al., 2008; Rutardottir et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions. To examine heme binding properties of A1M we utilized UV/Vis absorption spectroscopy, visible circular dichroism (CD), catalase-like activity, migration shift electrophoresis, and surface plasmon resonance (SPR), which was specifically developed for the assessment of His-tagged A1M. The results of this study confirm that A1M is a heme binding protein that can accommodate heme at more than one binding site and/or in coordination with different amino acid residues depending upon heme concentration and ligand-to-protein molar ratio. UV/Vis titration of A1M with heme revealed an unusually large bathochromic shift, up to 38 nm, observed for heme binding to a primary binding site. UV/Vis spectroscopy, visible CD and catalase-like activity suggested that heme is accommodated inside His-tagged (tgA1M) and tagless A1M (ntA1M) in a rather similar fashion although the His-tag is very likely involved into coordination with iron of the heme molecule. SPR data indicated kinetic rate constants and equilibrium binding constants with KD values in a μM range. This study provided experimental evidence of the A1M heme binding properties by aid of different techniques and suggested an analytical methodology for a rapid evaluation of ligand-binding properties of recombinant A1M versions, also suitable for other His-tagged proteins.
    Full-text · Article · Dec 2014
    • "The intracellular redox buffer comprises glutathione (GSH) and glutathione disulfide (GSSG), a reversible redox couple (GSSG,2H þ /2GSH). Maintaining an excess of GSH is integral in keeping cellular proteins reduced and functioning [9,13,1617181920. It has also been shown that increasing the intracellular level of GSH levels leads to healthier RBCs throughout the storage period [12]. "
    [Show abstract] [Hide abstract] ABSTRACT: Red blood cells (RBCs) collected for transfusion deteriorate during storage. This deterioration is termed the "RBC storage lesion". There is increasing concern over the safety, therapeutic efficacy, and toxicity of transfusing longer-stored units of blood. The severity of the RBC storage lesion is dependent on storage-time and varies markedly between individuals. Oxidative damage is considered a significant factor in development of the RBC storage lesion. In this study, the variability during storage and heritability of antioxidants and metabolites central to RBC integrity and function were investigated. In a classic twin study, we determined the heritability of glutathione (GSH), glutathione disulfide (GSSG), the status of the GSSG,2H(+)/2GSH couple (Ehc) and total glutathione (tGSH) in donated RBCs over 56 days of storage. Intracellular GSH and GSSG concentrations both decrease during storage (median net loss of 0.52±0.63mM (median±SD) and 0.032±0.107mM, respectively over 42 days). Taking into account the decline in pH, Ehc became more positive (oxidized) during storage (median net increase of 35±16mV). In our study population heritability estimates for GSH, GSSG, tGSH, and Ehc measured over 56 days of storage are 79%, 60%, 67%, and, 75% respectively. We conclude that susceptibility of stored RBCs to oxidative injury due to variations in the GSH-redox buffer are highly variable among individual donors and strongly heritable. Identifying the genes that regulate the storage-related changes in this redox buffer could lead to the development of new methods to minimize the RBC storage lesion.
    Full-text · Article · Aug 2014
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