Shock 1998: Oxígeno, Oxido Nítrico y perspectivas
Simposio Internacional, Academia Nacional de Medicina
Buenos Aires, 30 abril 1998
Vasoactive properties of synthetic blood substitutes
F. Poli de Figueiredo
Cardiopneumonology, Faculty of Medicine, Universidade de São Paulo
and Department of Surgery, Universidade Federal de São Paulo, Brazil
Key words: blood substitutes, hemoglobin, shock, nitric oxide,
a great need for the development of a safe and efficient blood
substitute, to overcome the important limitations of homologous blood
transfusion. Currently available cell-free hemoglobin-based
oxygen-carrying solutions present oxygen transport and exchange
properties similar to blood and potential benefits over conventional
transfusion, including large supply, absence of transfusion reactions,
no need for cross-matching, no risk for transmission of disease and
long shelf life. Several experimental studies have suggested that
cell-free hemoglobin is a vasoactive agent. In animal models of
hemorrhagic shock, small doses of cell-free modified hemoglobin
restore arterial pressure, promote adequate tissue oxygenation, and
improve survival, when compared with fluids with no oxygen-carrying
capacity. On the other hand, it has been demonstrated that
hemoglobin-induced vasoconstriction may result in decreased cardiac
output, reduced blood flow to vital organs and severe pulmonary
hypertension. Cell-free hemoglobin solutions cause their pressor
effects by binding and scavenging nitric oxide. Although hemoglobin
within the red blood cells is the natural scavenger of NO, when the
hemoglobin is free in solution, NO is inactivated to a greater extend.
Cell-free hemoglobins are on advanced clinical trials, despite the
fact that several concerns raised by experimental studies have not
been adequately addressed in early clinical trials. The development of
a safe and efficient blood substitute depends on the availability of
these products for critical evaluation by the scientific community
before the widespread clinical use of these blood substitutes.
vasoactivas de sustitutos sintéticos de la sangre. Hay una urgente
necesidad de desarrollar un sustituto de la sangre con el fin de
resolver las importantes limitaciones de las transfusiones de sangre
homóloga. Las soluciones que aportan oxígeno a través de
hemoglobina libre de células ofrecen un transporte de oxígeno e
intercambio similares a la sangre con beneficios potenciales, frente a
la transfusión convencional, que incluyen un rápido suministro,
ausencia de reacciones transfusionales sin requerimiento de
cross-matching, ningún riesgo de transmisión de enfermedad y una
larga vida en el stock. Varios estudios experimentales sugieren que la
hemoglobina libre de células es un agente vasoactivo. En modelos
animales de shock hemorrágico, pequeñas dosis de hemoglobina
acelular restablecen la presión arterial, proveen adecuada
oxigenación tisular y aumentan la sobrevida, en comparación con
fluidos sin ninguna capacidad de proveer oxígeno. Por otro lado, se
ha demostrado que la vasoconstricción inducida por la hemoglobina
puede provocar una disminución en el volumen minuto, una disminución
en el flujo sanguíneo hacia órganos vitales y severa hipertensión
pulmonar. Soluciones de hemoglobina acelular inducen sus efectos
presores ya sea por unión o secuestro de óxido nítrico (NO). A
pesar de que la hemoglobina dentro de los eritrocitos es el natural
depurador de NO, la hemoglobina libre en solución inactiva NO aun
más. Hay ensayos clínicos en curso con hemoglobina acelular a pesar
de que todavía no se han resuelto algunos de los problemas que
surgieron en estudios previos. El desarrollo de un sustituto de la
sangre seguro y eficiente depende de la disponibilidad de estos
productos para una evaluación crítica por parte de la comunidad
científica antes de su distribución clínica.
Postal Address: Dr. Luiz F. Poli de Figueiredo, Instituto do
Coração, Fac. Medicina, Universidade de São Paulo, Av. Enéas de
Carvalho Aguiar, 44, São Paulo - SP 05403-000, Brasil. FAX:
55-11-853.7887; E-mail: firstname.lastname@example.org
Blood performs a great variety of important physiologic tasks, but
its most basic and critical function is to provide a continuous supply
of oxygen to the tissues. Since oxygen is poorly soluble in plasma,
the hemoglobin within the red blood cells (RBCs) is responsible for
the transport of more than 98% of this gas. Cell-free hemoglobin
solutions are the older and most studied “blood substitutes” or,
more accurately defined, “oxygen-carrying volume expanders”, since
these solutions do not have coagulant, immunological and other
functions that are performed by blood.
Homologous RBCs transfusion is currently safe, very efficient and,
most importantly, has been widely tested, usually in the most
critically ill patients, in very large doses, and in a great variety
of clinical settings. Moreover, the majority of the transfused RBCs
can survive for weeks or months and presents oxygen-carrying capacity
and elimination characteristics similar to native blood. For all these
reasons, RBCs transfusion is considered the “gold standard” to
which safety and efficacy of any other oxygen-carrying solution should
Despite the remarkable safety record for homologous RBCs transfusions,
they have very important limitations, including a major medical and
public concern with transmission of diseases like HIV, hepatitis, and
other bacterial, parasitic and viral blood-borne diseases2-4.
Additionally, there is a progressive increase in blood demand, while
blood supply may be critically decreased by the aging population and
inadequate rates of volunteer donations in the near future5. There are
other limitations, inherent to the current techniques employed for
RBCs transfusions, such as the requirement for compatibility testing,
the risks for transfusion reactions and human errors, the short shelf
life, limited to weeks, and the rigid storage requirements1, 6 .
These are the major reasons why there is a great need for the
development of a safe and efficient oxygen-carrying solution that
could replace RBCs transfusions. Cell-free hemoglobin-based
oxygen-carrying solutions that are currently available present oxygen
transport and exchange properties similar to blood and potential
benefits over conventional transfusion, including large supply,
absence of transfusion reactions, no need for cross-matching, no risk
for transmission of disease and long shelf life, up to one year if
frozen. They represent substantial improvement over the earlier
hemoglobin solutions, which caused marked toxicity and severe side
effects. Several highly purified, chemically modified hemoglobin-based
oxygen carriers are now undergoing clinical trials7, 8. However, there
is evidence that cell-free hemoglobin is a vasoactive agent. The aim
of this report is to discuss the vasoactive properties of the
hemoglobin-based oxygen carriers.
Effects of the earlier hemoglobin solutions
Earlier hemoglobin solutions were produced using RBCs that were
hemolyzed with distilled water, and made isotonic by adding salt9.
Oxygen transport and life were preserved in animal models of complete
exchange transfusion, in otherwise lethally low hematocrits1, 6, 10.
In small human trials, these solutions caused several reactions
including fever, nausea, vomiting, hypertension, bradycardia,
bleeding, intravascular coagulation, and marked oliguria. On the other
hand, some patients in shock showed restoration of arterial pressure
and improved mentation with small amount of hemoglobin solution11.
Improvement in the preparation resulted in hemoglobin solutions
without cell membrane residues, the stroma-free hemoglobin (SFH).
However, undesirable side effects were evident in a well conducted
clinical trial, which had shown that small doses of SFH, to healthy
normal volunteers, caused transient hypertension, bradycardia,
oliguria and gross hemoglobinuria12. This study proved that unmodified
human hemoglobin was toxic and highlighted the critical importance of
preventing the glomerular filtration of the dissociated hemoglobin
tetrameter that, precipitating in the proximal tubule, caused renal
damage1, 13. Moreover, SFH pre-sents a very short half life, because
hemoglobin dimers and monomers are quickly excreted by the kidneys,
and also a high oxygen affinity, caused by the loss of the effects of
2,3-diphosphoglycerate, limiting oxygen unloading at the tissues1, 13.
Modified cell-free hemoglobins
Chemical modifications of SFH solutions decreased oxygen affinity,
prolonged half-life and prevented renal damage, resulting in the
actual oxygen carrying hemoglobin-based blood substitutes that are
being tested clinically. All these modified hemoglobins are highly
purified, free of phospholipids, endotoxins, viral and bacterial
contaminants. Several different methods were employed, including
pyridoxylation, polymerization, conjugation, encapsulation,
intramolecullar crosslinking, the production of recombinant hemoglobin
and the use of bovine hemoglobin, which does not require 2,3-DPG and
has an oxygen affinity similar to human hemoglo- bin 1, 6, 9, 14.
These modifications resulted in hemoglobin solutions with P50 (oxygen
partial pressure resulting in a 50% hemoglobin saturation) values
similar to native blood, while half-life was prolonged to up to 36
hours and renal toxicity was decreased by the maintenance of the
tetrametric structure, reducing the rapid clearance of hemoglobin
diamers by the kidneys1, 6, 13.
Experimental studies with modified hemoglobins
These solutions have been widely tested in animal models of
hemorrhagic shock and whole blood exchange, which demonstrated
maintenance of cardiovascular function and oxygen metabolism, and
long-term survival after partial and complete exchange
Vasoactivity of these modified hemoglobin solutions remained striking,
particularly in animal models of hemorrhagic shock, in which even
small doses of cell-free modified hemoglobin restored arterial
pressure, promoted adequate tissue oxygenation and improved survival,
when compared with fluids with no oxygen-carrying capacity21-26. It
has been suggested that these solutions, because of their
pharmacological actions and unique pressor-perfusion effects of
increased arterial pressure, cardiac output and organ blood
flows27-35, offer particular potential as a resuscitative fluid for
trauma and hemorrhagic shock. However, we and others have demonstrated
that hemoglobin-induced vasoconstriction resulted in decreased cardiac
output and reduced blood flow to the intestines, kidneys and heart,
using animal models of hemorrhagic shock, hemodilution, sepsis and
isolated organs36-44. The vasopressor effect is even more pronounced
in the pulmonary vasculature, causing severe pulmonary hypertension,
that can lead to hemodynamic instability and acute right ventricular
dysfunction38-40, 43, 44.
Figure 1 illustrates the vasoactive properties of one of the most
widely tested hemoglobin solutions, the alpha-alpha cross-linked
hemoglobin (aaHb), given to hemorrhaged pigs, which received a 2-min,
4 ml/kg bolus injection of either aaHb or an oncotically matched 7%
human albumin solution (ALBh) as the only treatment43. This amount of
fluid was equivalent to only one fourth of the shed blood to maintain
mean arterial pressure around 40 mmHg for 60 minutes. We can see the
immediate and sustained arterial pressure improvement after aaHb,
which was achieved only through vasoconstriction, with no improvement
in cardiac output. Pulmonary arterial pressure peaked immediately
after aaHb, and remained above baseline throughout the experiment.
Although some degree of systemic vasoconstriction may be desirable for
the initial resuscitation of hypovolemic shock, by restoring coronary
and cerebral perfusion pressures and brain blood flow43, pulmonary
hypertension and coronary vasoconstriction are highly undesirable side
effects, with potential for catastrophic hemodynamic events,
particularly in patients with the greatest need for blood substitutes,
such as the ones undergoing trauma and major cardiovascular and cancer
operations. Sustained vasoconstriction may also affect renal perfusion
and, although evidence of long term renal damage has not been reported
with these modified hemoglobins, most studies have been performed in
normal animals, with preserved organ functional reserve before
undergoing the acute insult. It is largely unknown the impact that
hemoglobin-induced vasoconstriction may produce in the kidneys and
other organs acutely or chronically compromised. Impairment of
functional capillary density, after hemodilution with modified
hemoglobin solution, has been demonstrated in a videomicroscopy study
of hamsters microcirculation, when compared with non-oxygen-carrying
colloids45. It has not been established whether this is mainly caused
by vasoconstriction or because modified hemoglobin carries too much
oxygen, eliciting a metabolic autoregulatory effect45. However, direct
measurement of tissue oxygen content has suggested that tissue
oxygenation with cell-free hemoglobin is reduced compared to RBCs46.
Mechanism for cell-free hemoglobin vasoactivity
There is large evidence that cell-free hemoglobin solutions produce
their pressor effects by binding and scavenging nitric oxide (NO), the
potent endothelium-derived vasodilator responsible for the normal
vasodilatory tone in the systemic and pulmonary circulation1, 6, 9,
35-38, 42, 47-51. Some authors have suggested that endothelin release
and other vasoconstrictors have also play a role34, 52. When the
hemoglobin is within the RBCs, NO is removed as it dissolves into the
plasma and ultimately interacts with hemoglobin. However, when the
hemoglobin is free in solution, NO is inactivated to a greater extend,
thereby causing vasoconstriction. Free-hemoglobin binds NO thousand
times more avidly than it binds oxygen and carbon monoxide6, 53.
The vasoactivity of the aaHb can be demonstrated by the tracings of
representative experiments performed on isolated blood vessels54,
demonstrating the endothelium-dependence of aaHb-induced contraction
(Figure 2), the endothelium-independence of sodium nitroprusside
(SNP)-induced relaxation in the presence of aaHb (Figure 3) and
time-dependent and endothelium-independent effects of aaHb on
SNP-induced relaxation (Figure 4)54.
Because of this high affinity of free hemoglobin for NO, it has been
suggested for the treatment of septic shock, in which hypotension and
low peripheral vascular resistance are associated with excessive
production of NO35, 38. Given to septic rats, hemoglobin solutions
increased arterial pressure, improved regional perfusion to vital
organs and improved mortality35. Treatment with hemoglobin also
improved arterial pressure without a significant impairment in blood
flow to the kidneys and intestines in endotoxemic pigs; however,
hemoglobin caused a significant exacerbation of endotoxin-induced
pulmonary hypertension and arterial hypoxemia38. Hypoxemia,
ventilation-perfusion abnormalities and greater acidosis were also
observed with cell-free hemoglobin infusion in a model of canine
bacteremia55. We were able to selectively reverse aaHb-induced
pulmonary hypertension and decreased lung compliance with small doses
of inhaled nitric oxide (Figure 5)56.
Other potential problems with cell-free hemoglobin
Besides these undesirable hemodynamic and ventilatory effects,
there are other potential problems with the use of cell-free
hemoglobin for sepsis. Hemoglobin may bind the individual molecules of
lypopolysaccaride, breaking up the endotoxin mycelles and increasing
the biologic activity of bacterial endotoxin57, 58. Iron and heme may
cause bacteria to grow, worsening infections58. White cells and
platelets activation has been shown with cell-free hemoglobins, which
could promote the release of several proinflammatory cytokines and
Two important enzymes normally present within the RBCs, superoxide
dismutase and catalase, are able to remove oxygen radicals and
peroxides, and they are absent in cell-free hemoglobin solutions.
Hemoglobin breakdown products, the heme and iron, participate in redox
reactions and are capable of accelerating the generation of
oxyradicals by the Fenton and Haber-Weiss reactions, with potential
for increased lipid peroxidation and other forms of cell damage
related to reperfusion injury9, 62, 63. Iron clearance mechanisms may
became rapidly saturated with the rates of iron loss from the
cell-free hemoglobins64. Methemoglobin levels can be greatly increased
by cell-free hemoglobin, since a critical step for superoxide
production and for hemoglobin breakdown is the rate of methemoglobin
formation, and it does not carry oxygen40.
Other pharmacological disadvantage of cell-free hemoglobins include a
shorter intravascular half-live, from 8 to 36 hours1; they also have
colloid osmotic activity and changes in intravascular volume could be
expected as redistribution and clearance occurs. With cell-free
hemoglobin diffusion into the intersticial space, intravascular
concentration declines and extravascular concentration increases,
leading to intravascular fluid loss1, 9, 13, 65 . This fact, in
addition to hemoglobin-induced vasoconstriction and increased arterial
pressure, may mask a hypovolemic state after the use of large volumes
of hemoglobin. Modified hemoglobin solutions is scavenged primarily by
the reticuloendothelial system and long term effects have not been
Clinical experience with cell-free hemoglobins
Small doses of several cell-free hemoglobins were administrated to
healthy volunteers or in healthy anesthetized patients7, 8, with no
report of death, allergic reactions or major side effects. Although
safety has been claimed, these studies, unfortunately, have not been
published in the scientific literature, making a correlation between
the concerns above discussed and the clinical records.
We know that some early phase I safety trials were temporarily halted
by the FDA because of medical events including fever, flu-like
symptoms, headache, abdominal pain, gastrointestinal symptoms, muscle
aches, increased blood pressure, decreased heart rate, chest pain and
abnormal blood chemistries9,67. Human trials in Guatemala in 1990 and
in nine children with sickle cell anemia in Zaire showed no untoward
side effects, but surprisingly little information is available in
these full papers9,14. More recently, larger phase I and II clinical
trials, including hundreds of patients, are being performed but
limited data is available in abstract forms7, 8. Safety was evaluated
in 130 hemorrhagic shock patients at ten sites in the United States
and Europe with cell-free hemoglobin solution, but no other
information about these trials is available7.
In humans, the vasopressor effect with cell-free hemoglobin is evident
even in very low doses. It has been suggested that the vasopressor
effect could be beneficial for patients with hypotension during
hemodyalisis68 and for septic patients with low peripheral
resistance69. In one study, the vasopressor effect was also evident in
the pulmonary circulation, after a very low dose of cell-free
Marked elevations in amylase and lipase levels with synthetic
hemoglobin but with no clinical evidence of pancreatitis or other
major problems were reported in two studies71, 72. In addition to
increased levels of pancreatic enzymes, increased arterial pressure
was observed in awake patients during preoperative normovolemic
hemodilution, highlighting the marked vasopressor effect72.
It is surprising that human studies evaluating safety do not appear to
directly address the concerns raised in preclinical studies regarding
the vasoconstriction affecting the systemic and, particularly, the
pulmonary circulation, that could and should be easily evaluated with
echocardiography or with invasive techniques. Just a month ago,
through a Company Press Release on the Internet, we learned that
Baxter Healthcare halted its phase III trauma trial on the use of
Diaspirin aa-crosslinked hemoglobin, planned to include 850 patients,
because after 100 patients, mortality was greater with the use of
synthetic hemoglobin73. Surgical patients and critical care patients
are ideal candidates for a more extensive evaluation of these blood
substitutes, because if safety and efficacy are proven, they will
likely benefit those patients with multiple coexisting diseases and
limited organ reserves. In 1990, the journal Science addressing the
incredible relation between blood, money and research stated that “Solutions
of modified hemoglobin could replace whole blood in many transfusions
if researchers can learn how to avoid their potentially dangerous side
effects”74. Greater availability of these solutions for an
independent scientific community is crucial for a faster development
of a very much needed safe and efficacious blood substitute.
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Fig. 1.- Hemodynamic responses to a 4 ml/kg, 2-min bolus injection of
either 10% aaHb (n=7) or 7% human serum albumin (ALBh, n=7) to
hemorrhaged pigs (shed blood = 17±1.2 ml/kg).43
Fig. 2.- Endothelium-dependence of aaHb-induced contraction.
Representative tracings showing the contractile response of rat aortic
rings, precontracted with phenylephrine (PE, 10-3 M), exposed to
increasing concentrations of aaHb (1,8 x 10-8 to 1,8 x 10-6 M) with
and without endothelium. Endothelial removal was produced by rubbing
gently the internal surface of the rings with two wires. Tissue
viability was assured by the adequate contractile response to
phenylephrine (10-3 M). The presence of endothelium (+ENDO) was
established by the relaxation response to acetylcholine (ACh, 10-8,
10-7, and10-6 M), while the absence of endothelium (-ENDO) was
confirmed by the absence of relaxation in response to ACh. In the
presence of endothelium, aaHb presented a concentration-dependent
contractile response (B.+ENDO,+aaHb). In addition, there was a
decreased ACh-induced relaxation in the presence of aaHb. Without
endothelium, there was no contractile response to aaHb
Fig. 3.- Endothelium-independence of SNP-induced relaxation in the
presence of aaHb. Representative tracings showing the contractile
response of rat aortic rings, precontracted with phenylephrine (PE,
10-3 M), after ACh (10-6 M) and aaHb (1,8 x 10-8 to 1,8 x 10-4 M),
with and without endothelium. Experimental setup was described on
figure 2. ACh caused marked relaxation in the presence of endothelium
and the addition of aaHb produced concentration-dependent contractile
response, reversing completely ACh-induced relaxation. In the absence
of endothelium, there was no ACh-induced relaxation and no
aaHb-induced contraction. Sodium nitroprusside (SNP 10-7 and 10-6 M),
an NO donor, caused relaxation with and without endothelium.
Vasodilation induced by NO donors is independent of endothelium and
reverses aaHb-induced contraction.54
Fig. 4.- Time-dependent and endothelium-independent effects of aaHb on
SNP-induced relaxation. Representative tracings showing the
contractile response of rat aortic rings, precontracted with
phenylephrine (PE, 10-3 M), in which the relaxation response to sodium
nitroprusside (SNP, 10-9 e 10-8 M) was established with (+ENDO) and
without endothelium (-ENDO). Experimental setup was described on
Figure 2. SNP (10-7) induced marked and sustained relaxation with and
without endothelium (A.+ENDO, C.-ENDO). aaHb (1,8 x 10-5), produced
contraction only in the presence of endothelium (B.+ENDO, +aaHb). The
addition of SNP, 10-9 M, caused minimum relaxation while SNP, 10-8 M,
caused a partial and transient relaxation, with and without
endothelium. The repetition of SNP (SNP, 10-9 and 10-8 M) produced
similar partial and transient relaxation. These data suggest that, in
the presence of aaHb, NO donors will be required in higher doses and
for longer periods if they are to be used clinically to overcome
Fig. 5.- Effects of 2-ml/kg of 10% aaHb, infused over 20 minutes to
normovolemic, phentanyl-anesthetized pigs (n=5), on pulmonary arterial
pressure and lung compliance. aaHb produced marked increases in
pulmonary arterial pressure and decreases in lung compliance. Inhaled
nitric oxide (NO 5 ppm and 10 ppm), in 10-min cycles,, reversed
pulmonary hypertension and decreased lung compliance.56