Nitric-oxide bioactivity depletion:

Jan. 1, 2009

There is more to a
blood transfusion than to increase the oxygen-carrying capacity of
red-blood cells (RBCs). Blood transfusions should be able to enhance
vasodilation, open blood vessels, and increase blood flow to hypoxic
tissue. Blood transfusions should also help improve RBC rheology to
facilitate flexible transport of RBCs through tiny capillaries. If,
however, blood vessels vasoconstrict or if the RBCs become rigid, then
the blood transfusion may be less effective.

An effective blood transfusion is one that not only
raises the hematocrit to the normal range but also enhances arterial
relaxation and vasodilation. In man, the enzyme that produces nitric oxide
(NO) from L-arginine is called nitric-oxide synthase The vasodilator gas,
nitric oxide, is carried by hemoglobin in the form of S-nitrosothiol (SNO)
(see Figure 1 online). Nitric oxide released from S-nitrosothiol helps relax
smooth muscle surrounding the tubules of arteries, arterioles, and
metarterioles (see Figure 2 online). When smooth muscle relaxes, the
arterial blood vessels expand, leading to vasodilation. Thus, wide-open
vessels improve the blood flow from arteries to capillaries, making the
transport of oxygen to tissue more efficient. Ultimately, in the
mitochondria, oxygen contributes to produce adenosine triphosphate (ATP)
from adenosine diphosphate (ADP) by electron-transport and oxidative
phosphorylation. ATP is used as an energy source by tissue. Blood
transfusions should be characterized by how well RBCs facilitate
nitric-oxide bioactivity to open blood vessels, rather than by how much
oxygen they can deliver to tissue.

Recently, Jonathan S. Stamler, MD, et al, and Timothy
J. McMahon, MD, PhD, et al, at Duke University Center (DUMC), have shown how
nitric-oxide bioactivity helps RBCs ferry oxygen to tissues by opening tiny
vessels during hypoxic vasodilation.1,2 They also report that
when RBCs leave the body during blood-banking donations, nitric-oxide
bioactivity and S-nitrosohemoglobin in RBCs begin breaking down almost
immediately. This type of storage lesion continues during the shelf life of
banked blood. The depletion of nitric-oxide bioactivity, along with the
decrease in concentration of 2,3-diphosphoglycerate, or 2,3-DPG, both affect
vasodilation, compromising oxygen delivery to tissues. Thus, it does not
matter how much oxygen hemoglobin carries; if the blood vessels do not relax
and open, oxygen cannot be delivered effectively to tissue. Therefore, the
unimpeded flow of blood throughout the blood-vessel bed is vital for the
efficient transport of oxygen.

The primary function of the cardiovascular system is
to supply oxygen to tissues and organs in the body RBC transfusions are
commonly used to treat anemia, including euvolemic patients with congestive
heart failure, to increase oxygen delivery to hypoxic tissue. The RBC
functions as an O2 sensor contributing to the regulation of blood
flow and oxygen delivery, by releasing nitric oxide, depending on the
oxygenation state of hemoglobin. RBCs depleted of nitric-oxide bioactivity
do not always improve oxygen delivery during blood transfusions. There are
recent concerns about the benefit of blood transfusions to critically ill
patients due to immunomodulation and storage lesions found in banked blood.3
Blood transfusions can lead to vasoconstriction, congesting the flow of
oxygen in narrow passageways in the cardiovascular system — damaging the
very heart tissue that the blood was transfused to help.

Blood transfusions that lack nitric-oxide bioactivity
may become associated with ischemia, a dangerous drop in blood flow.
Investigators are suggesting that re-nitrosylation — adding a purified
aqueous solution of nitric-oxide gas into banked blood before transfusions —
can raise the concentration of S-nitrosohemoglobin in vitro which, in
turn, can amplify vasodilation in vivo. In fact, Dr. Stamler and the
anesthesiology and biochemistry teams at DUMC have demonstrated that canine
coronary-blood flow was greater during the infusion of rejuvenated re-nitrosylated
RBCs than during infusion of S-nitrosothiol-depleted RBCs. This article
reviews the literature on depletion of nitric-oxide bioactivity in banked
blood affecting vasodilation, blood flow, and oxygen transport to tissue,
and elaborates on current research about the rejuvenation of donated banked
blood by adding nitric oxide prior to transfusion.

Surprises at the cellular level happen all the time.
Nitric oxide or nitrogen monoxide is a formidable toxin commonly found in
nature as a gas and as a component of air pollution. For instance, it is a
pollutant produced by automobile exhaust fumes and by power plants. Oxygen
from the air and nitrogen combine at combustion temperatures or in the
presence of electrical energy to form nitric oxide. Nitric oxide is produced
by many cells in the body; however, its production by the vascular
endothelium — the innermost cell layer of blood vessels — is particularly
important in the regulation of blood flow. Today, we know that nitric oxide
plays a function in the cardiovascular system, the immune system, and in the
central and peripheral nervous systems. Furthermore, this simple gas, nitric
oxide, affects a variety of complex biological processes, including
blood-pressure homeostasis, platelet aggregation, and transmission of
signals by the nervous system.4 NO also plays a key role in the
activation of macrophages and cellular defenses against microbial pathogens.
It is a major pathophysiological mediator of inflammation and host-defense

In the 1800s, Alfred Nobel invented dynamite, in
which one of the main components is the explosion-prone nitroglycerine.
Centuries later, nitroglycerine is being used as a vasodilator by patients
with chest pain, suffering with angina. Nitroglycerine is converted into
nitric oxide in the bloodstream, relaxing the muscle lining of vessels to
allow better blood flow.5 In the mid-1980s, scientists were
surprised to find out how nitric oxide was being produced meticulously in
human cells. Nitric oxide went from being an extraneous toxic and corrosive
gas to becoming an ubiquitous elixir of life. In 1987, Salvador E. Moncada,
MD, PhD, discovered the vital role of nitric oxide as a messenger in the
relaxation of muscle.6
In 1991, a team headed by K. E. Andersson of Lund University Hospital in
Sweden showed how nitric oxide was the principal neurotransmitter mediating
erectile function.7 In 1992, Science published a cover
story naming nitric oxide the molecule or the year. In 1998, Robert F.
Furchgott, MD, from the State University of New York; Louis J. Ignarro, MD,
from the University of California; and Ferid Murad, MD, PhD, from the
University of Texas, were given the Nobel Prize in Physiology for their
discoveries of nitric oxide as a signaling molecule in the cardiovascular

Non-biological functions of NO

oxide should not be confused with a) nitrous oxide (HN2O), a general
anesthetic; or b) nitrogen dioxide (NO2), which is another poisonous air
pollutant; or c) nitric acid (HNO3). Nitric oxide is a very unstable
free radical turning, within seconds, into univalent radicals of nitrate
(NO3) in vivo and into nitrite (NO2) in vitro. Nitric
oxide reacts with ozone in the air to form nitrogen dioxide (2NO + O2 ?

The synthesis of NO from molecular nitrogen and
oxygen ( N2 + O2 ? 2NO) requires elevated temperatures of greater than
1,000^0C. Internal-combustion engines have increased the concentration of NO
in the environment by automobile-exhaust fumes. The purpose of catalytic
converters is to minimize nitric-oxide emissions by catalytic conversion to
O2 and N2. Nitric oxide in the air can convert into nitric acid, which has
been implicated in acid rain.

Biological functions of nitric oxide

Nitric oxide is a lipophilic radical that readily moves across permeable
cell membranes via passive diffusion. Nitric oxide is one of the few
gaseous particles with biological-signaling capabilities. NO is known as
the endothelium-derived relaxing factor, or EDRF, and a liable free
radical with a half-life of about three to five seconds. It is
biosynthesized from L-arginine and oxygen to citrulline by several
nitric-oxide synthases, or NOS, enzymes and by the reduction of
inorganic nitrate. NO is known to be produced in bacteria but found to
act differently in mammals as a signaling molecule. Produced by many
types of cells including nerve cells and the endothelium, nitric oxide
is regulated by biofeedback and by the ability of superoxide anion and
superoxide dismutase to inactivate NO. Nitric oxide is also controlled
by the “on-and-off redox switch” — the reduction/oxidation potential
states of biochemical reactions.


Banked-blood packed RBCs have had
most of their leukocytes and plasma removed. Packed RBCs undergo
rigorous testing before their use. The blood-banking industry does an
extraordinary job of manufacturing a safe and effective product. In
order to prevent transfusion-transmitted diseases, the Food and Drug
Administration (FDA) mandates testing for viral markers including
hepatitis B; human immunodeficiency virus 1,2, or HIV 1,2; human T-lymphocytotrophic
virus 1,2, or HTLV-1,2; cytomegalovirus, or CMV; serologic test for
syphilis, nucleic-acid testing, or NAT, for West Nile virus, and
hepatitis C virus.9
In addition, in order to conserve red-cell survival and function, RBC
units are treated with additive solutions containing sodium chloride,
dextrose, adenine, monosodium phosphate, mannitol, sodium citrate, and
citric acid. RBCs also contain anticoagulants like citrate-phosphate
dextrose, or CPD; citrate-phosphate dextrose-dextrose, or CPD2D; or
citric-phosphate dextrose-adenine, or CPDA-1. The 42-day expiration date
of banked blood stored at 1^0C to 6^0C depends mainly on the type of
additive solutions used including AdsolR (Fernwall, Lake Zurich, IL),
NutricelR (Pall Life Sciences, Ann Arbor, MI), or OptisolR (Terumo,
Somerset, NJ).10 RBCs collected using the Trima Accel
Collection System (CaridianBCT, Lakewood, DO) also have a shelf life of
42 days.

Approximately 13.9 million units of blood are
transfused to 4.8 million patients each year in the United States, and the
basis for approved use is determined by meeting regulations during
collection, processing, and storage.11 Banked blood is a
biological product that is under the scrutiny of many regulatory agencies
and the scientific community. The AABB (American Association of Blood Banks)
also publishes guidelines for a safe transfusion. Blood has both benefits
and risks, and, therefore, should be evaluated in the same manner as
medications. To make a better product, however, the industry has pursued the
idea of introducing synthetic banked blood, but its success remains to be
proven. Until then, investigators are proposing ways to improve the product
that is already at hand today. The current interest in the literature is
about nitric-oxide bioactivity found in the form of S-nitrosothiol, which is
crucial for the delivery of oxygen to tissues. Nitric oxide is not only
needed for RBCs to transport oxygen but also may be responsible for the
flexibility of the RBCs. When nitric-oxide levels decrease, the RBCs become
stiffer, making it more difficult for them to adapt their shape in order to
travel through the tiny capillary spaces during the delivery of oxygen (see
Figure 3 online).

Storage lesions include RBC rheology, the loss of
shape, and flexibility,12 the decrease in the concentration of
molecular modulators of oxygen binding (e.g., 2,3-DPG), the decrease in
nitric-oxide bioactivity, and the increase in RBC adhesiveness during
prolonged storage. Storage lesions in banked blood have been found to be
responsible for adverse outcomes, like those leading to increased mortality
rates after blood transfusion. Alterations in RBC rheology and adhesion may
exacerbate rather than correct ongoing ischemia and — at least, partly —
account, for the adverse effects of blood transfusions.

At the Cleveland Clinic Foundation, Colleen G. Koch,
MD, et al, examined data from 1998 to 2006 for patients who received RBC
transfusions. A total of 2,872 patients received 8,802 units of blood that
had been stored for 14 days or less (“fresh blood”); 3,130 patients received
10,782 units of blood that had been stored for over 14 days (“aged blood”).
After cardiac-surgery patients who were transfused, “aged blood” had an
increased risk of postoperative complications and reduced chance for

Nobel laureate Dr. Ignarro, in his book No More
Heart Disease
, indicates that the endothelial cells can get sabotaged by
a variety of health conditions that compromise the production of nitric
oxide. Some of the health conditions that add more stress to the blood
vessels and that inflict endothelium-cell damage include high blood
pressure; atherosclerosis; high blood-cholesterol levels; elevated blood
glucose; high low-density lipoprotein, or LDL; and cigarette smoking. The
endothelial cells produce nitric oxide to protect us from many diseases by
regulating blood pressure and blood flow. The endothelial cells, however,
have a much harder job producing nitric oxide in patients with hypertension,
coronary heart disease, or stroke.14 Thus, patients with
underlying cardiovascular disease who are in need of blood transfusions have
a much bigger challenge to process nitric-oxide-depleted banked blood,
especially if it is more than 14 days old.

Furthermore, patients with sickle-cell anemia have
abnormal hemoglobin, which is needed to deliver oxygen and nitric oxide to
tissue. Hemoglobin-S has a lower affinity for oxygen and, once deoxygenated,
the RBCs become distorted or sickled. Hemoglobin-S does not transfer nitric
oxide from heme to thiol as well as normal hemoglobin during S-nitrosohemoglobin
conformation The symptoms of sickle-cell disease are attributed to the
physical obstruction of blood vessels by distorted or sickled and rigid
RBCs.15 Thus, sickle cells become fragile, demonstrate
vasooclusion, and lead to hemolytic anemia. Consequently, sickle cells have
added disadvantages pertaining to blood-vessel dilation when tissue
experiences oxygen deficiency during hypoxemia. Sickle-cell patients who
receive blood transfusions may have the transfused RBCs accumulate in their
vascular system, impeding the free flow of blood and transportation of
oxygen to tissue. Relieving the vasoconstriction and restoring nitric oxide
to RBC membranes may help prevent the painful symptoms of sickle-cell
disease. Thus, there is an opportunity for clinical trials on the
therapeutic use of nitric oxide with sickle-cell patients.

There is growing interest to improve the safety of
our blood supply. Investigators at DUMC have shown that the level of S-nitrosohemoglobin
was reduced by 85% to 95% at storage days seven and 43 compared to day one.
They also noted a deficiency in vasodilotary activity in banked blood when
compared with “fresh blood.” Banked blood used for transfusion still has
some shortcomings, but researchers are now contemplating reducing some of
the storage lesions by replenishing nitrosylation in banked blood before
using it for transfusion.

Replenishing bioactivity modulators in banked blood
is nothing new. During the Vietnam era, United States Navy Physician C.
Robert Valeri and N. M. Hirsch showed that ''spiking'' stored RBCs with
diphosphate glycerate and ATP precursors led to significant improvements in
cardiovascular function.16 Some establishments use rejuvenating
solutions, like RejuvesolR (Cytosol Labs, Lenoir, NC) which contains
pyruvate, inosine, phosphate, and adenine, to restore oxygen transport and
improve post-transfusion survival of RBCs.

It is being suggested that adding nitric oxide to
banked blood before its use could, theoretically, improve hemoglobin
nitrosylation and the ability of nitric oxide in S-nitrosothiols to dilate
and open blood vessels and, thus, prevent heart attacks and even death.
Investigators have demonstrated that replenishing banked blood with nitric-
oxide gas raises S-nitrosohemoglobin, or SNO-Hb, concentrations and restores
the hypoxic vasodilatory activity of RBCs.

The RBC is more than a “passive bag” full of
hemoglobin that transports oxygen. In fact, the RBC is a regulator of its
own destination. The matching of oxygen supply with demand requires
nitric-oxide bioactivity to increase blood flow in response to decreased
levels of oxygen in tissue. Increasing the hematocrit into the normal range
after a blood transfusion should be supplemented by increasing vasodilation,
blood flow, and oxygen transport. This can be achieved by adding nitric
oxide to banked blood prior to blood transfusions. Therefore, we support the
new paradigm from Joseph Bonaventura, PhD, for testing nitric-oxide
bioactivity in banked blood, once re-nitrosylation of banked blood becomes a
There exist opportunities to further investigate nitric-oxide bioactivity as
suggested by Bonaventura which include testing of arterial and venous RBC S-nitrosohemoglobin
as a diagnostic indicator for transfusion; assaying hemoglobin re-nitrosylation
treatment of stored RBCs; verification of normalized RBC rheology before
transfusions; and verification of normalized RBC vasoactivity prior to

Allogenic, autologous, and directed blood
transfusions are not scrutinized with a risk/benefit analysis common for all
biologics. Furthermore, there are no regulations or clinical standards aimed
at examining the clinical outcome of an effective blood transfusion in
patients with respect to nitric-oxide bioactivity in vasodilation, blood
flow, and oxygen transport to tissue. Consequently, an opportunity exists
for clinical trials to evaluate the outcome and effects of transfusing
improved re-nitrosylated banked-blood products to patients. Thus, further
research is needed to measure the effectiveness of transfusions by testing
the concentration of nitric oxide in the form of S-nitrosothiols or S-nitrosohemoglobin
in the peripheral blood of patients who receive blood transfusions.

Adding soluble portions of nitric oxide to banked
blood is in its infancy, but this seems to be more promising than the
current results and developments seen with the manufacturing of a synthetic
blood product. The addition of nitric oxide to banked blood eventually may
need to undergo rigorous clinical trials, FDA approval, and re-evaluation of
the current 42-day expiration date. Nonetheless, the growing concern of
transfusing banked blood that is over 14 days old in patients undergoing
cardiac surgeries may help expedite more research and diligent acceptance by
the medical community and regulatory agencies. Re-nitrosylation of banked
blood with nitric oxide is the most promising project undertaken by DUMC
investigators to preserve more of our blood supply.

Faon Rodriguez, MS,
is section supervisor at Florida Hospital, Celebration Health, FL, and
Diana Ramirez, MS
, is transfusion service supervisor at Osceola Regional
Medical Center, Kissimmee, FL.

The authors want to thank these colleagues for their advice and comments
after reading this manuscript: Patrick J. O'Sullivan, laboratory
director, Florida Hospital, Orlando, FL; Pamela Hargrave-Thomas,
laboratory director, Osceola Regional Medical Center, Kissimmee, FL;
Theresa Palmer
, assistant director, Florida Hospital, Kissimmee, FL; and
Kathryn Pearson, assistant director;
Gail S. Borysko, laboratory supervisor; and
Sonaly Cosme, medical technologist — all from Florida Hospital,
Celebration Health, FL.


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