Control of Plasma Nitric Oxide Bioactivity by Perfluorocarbons : Physiological

Mechanisms and Clinical Implications

Olga Rafikova, Elena Sokolova, Ruslan Rafikov and Evgeny Nudler
Circulation. 2004;110:3573-3580; originally published online November 22, 2004;
doi: 10.1161/01.CIR.0000148782.37563.F8
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2004 American Heart Association, Inc. All rights reserved.
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Control of Plasma Nitric Oxide Bioactivity

by Perfluorocarbons
Physiological Mechanisms and Clinical Implications
Olga Rafikova, MD; Elena Sokolova, MSc; Ruslan Rafikov, MSc; Evgeny Nudler, PhD
BackgroundPerfluorocarbons (PFCs) are promising blood substitutes because of their chemical inertness and
unparalleled ability to transport and upload O2 and CO2. Here, we report that PFC emulsions also efficiently absorb and
transport nitric oxide (NO).
Methods and ResultsAccumulation of NO and O2 in PFC micelles results in rapid NO oxidation and generation of
reactive NOx species. Such micellar catalysis of NO oxidation leads to formation of vasoactive S-nitrosothiols (RSNO)
in vitro and in vivo as detected electrochemically. The efficiency of PFC-mediated S-nitrosation depends on the amount
of PFC in aqueous solution. The optimal PFC concentration that produced the maximum level of RSNO was 1%
(vol/vol). Larger PFC amounts were progressively less efficient in generating RSNO and functioned simply as NO sink.
These results explain the characteristic hemodynamic effects of PFCs. Intravenous bolus application of PFC (0.14 g/kg,
1% vol/vol) to Wistar-Kyoto rats decreased mean arterial pressure significantly (10 mm Hg over 40 minutes).
PFC-induced hypotension could be further stimulated (17 mm Hg over 140 minutes) by exogenous thiols (cysteine
and glutathione). In contrast, a larger amount of PFC (1 g/kg, 7% vol/vol) exhibited a strong hypertensive effect
(11 mm Hg over 40 minutes).
ConclusionsThe present study reveals a physiologically significant pool of endogenous plasma NO and underscores the
crucial role of the circulating hydrophobic phase in modulating its bioactivity. The results also establish PFC as a
conceptually new pharmacological tool for various cardiovascular complications associated with NO imbalance.
(Circulation. 2004;110:3573-3580.)
Key Words: blood pressure hemodynamics nitric oxide oxygen fluorocarbons

itric oxide (NO) is enzymatically produced by various

types of cells and represents a central mediator within
the cardiovascular system.1 As a free radical, NO is highly
unstable in vivo. Its primary targets include heme proteins
2
such as guanylyl cyclase and free radical species, eg, O
2 .
Another physiologically significant aspect of NO biochemistry is the formation of thionitrite esters with cysteine (Cys)
and Cys residues (S-nitrosothiols; RSNO). Small RSNOs (eg,
S-nitrosoglutathione [GSNO]) and nitroso-derivatives of proteins such as albumin and hemoglobin are known to be
generated in vivo and exert NO-like activity. They induce
vasodilation, inhibit platelet aggregation, and participate in
various signal transduction pathways.37 Because RSNOs are
relatively stable and can release NO when required on
reaction with various reducing agents,8,9 they may serve as a
buffering system that magnifies the range of NO action along
the vascular tree.10,11
Although the pharmacological potential of RSNO is well
appreciated,1214 the role of plasma NO and the mechanism of
endogenous RSNO formation under physiological conditions

are a source of considerable debate.15 The apparent limitation

for plasma NO to exert its biological function is the constant
presence of abundant hemoglobin (10 mmol/L) from red
blood cells that converts NO into inactive metabolites at
near-diffusionlimited rates.16 Moreover, NO is unable to
react directly with thiols. It must be oxidized first to form
nitrosative agents, such as N2O3. Because of the slow rate of
the third-order reaction of NO with O2 (k 4106 mol/L2
per second),17,18 the formation of N2O3 and eventually RSNO
should depend primarily on the local concentration of NO and
the molecular environment. Indeed, strong acceleration of
NO oxidation occurs within lipid membranes19 and hydrophobic pockets of plasma proteins20 that effectively sequester
NO from the aqueous phase. Recently, we provided experimental evidence suggesting that such micellar catalysis of
NO oxidation and N2O3 formation by serum albumin accounts
for the presence of vasoactive RSNO in the circulation under
physiological conditions.21
In the present study, we explored a synthetic hydrophobic
phase (perfluorocarbon [PFC]) to manipulate plasma NO

Received May 21, 2004; revision received July 18, 2004; accepted August 2, 2004.
From the Department of Biochemistry, New York University Medical Center, New York, NY.
Correspondence to Dr Evgeny Nudler, New York University Medical Center, Department of Biochemistry, 550 First Ave, MSB #359, New York, NY
10016. E-mail evgeny.nudled@med.nyu.edu
2004 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org

DOI: 10.1161/01.CIR.0000148782.37563.F8

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December 7, 2004

bioactivity and establish its physiological role. PFCs are

highly hydrophobic, inert chemicals that are not miscible with
water and are capable of dissolving large quantities of gases,
including O2 and CO2. With sophisticated technology, it is
possible to generate stable PFC emulsions of small, uniform
particles that can be used as temporary intravascular oxygen
carriers.2224 Here, we show that by changing the volume and
properties of the circulating hydrophobic phase with PFCs,
one can rapidly and precisely modulate NO transport and
delivery, thus controlling blood pressure, platelet aggregation, and other NO-dependent processes. The results demonstrate the existence of a physiologically significant pool of
circulating plasma NO and the crucial role of nitrosative
chemistry in its vascular activity. We also suggest a conceptually new pharmacological approach for treating disorders
associated with NO disparity.

Methods
Chemicals and Reagent
One hundred milliliters of Perftoran emulsion (Perftoran Co) contains 13 g of perfluorodecalin (C10F18), 6.5 g of perfluoromethylcyclohexylpiperidin (C12F23N), 4.0 g of Proxanol 268, 0.6 g of NaCl, 39
mg of KCl, 19 mg of MgCl2, 65 mg of NaHCO3, 20 mg of NaH2PO4,
0.2 g of glucose, and H2O. Physical properties of Perftoran include
a particle size of 0.03 to 0.15 mol/L, osmolality of 280 to 340
mOsm, viscosity of 2.5 cP, pH 7.5, O2 solubility of 7.0 vol%
(pO2760 mm Hg, 20C), and CO2 solubility of 60 vol/%
(pCO2760 mm Hg, 20C). Saline in control experiments contained
all of the chemicals listed above except PFCs and Proxanol. Other
chemicals were from Sigma unless specifically indicated.

Determination of NO Partitioning (Q)

NO/H2O solution was prepared in an airtight device by bubbling NO
gas (Aldrich) that had been purified from higher oxides by passing it
through a 1-mol/L solution of KOH into water until the concentration
of dissolved NO reached 1 mmol/L. Water (Milli-Q grade) was
deaerated by boiling and then cooling under argon (Praxair). The NO
concentration was measured immediately before the reaction with an
ISO-NO Mark II nitric oxide electrode (WPI). To determine QNO for
PFC, the ice-cold Perftoran (10 mL) was degassed in the airtight
device by bubbling with argon for 6 hours. Next, 1.3 mL of Perftoran
was added to 12.2 mL of NO solution (11.2 mol/L) in the presence
of argon. The temperature was adjusted to 20C, and the ISO-NO
Mark II electrode was inserted into the solution under argon and
sealed. QNO is calculated as described in Figure 1.

Determination of Thiols and RSNOs

To prepare plasma samples (100 L), 0.5 mL of arterial blood was
collected into heparinized plastic tubes and centrifuged at 4500 rpm
at 4C for 3 minutes. Plasma was processed immediately after
preparation. Plasma GSH and Cys were detected by high-performance liquid chromatography (Waters 600E pump) at 230 nm with
a multiwavelength detector (Waters 490E) and C18 column with a
mobile phase of 99% 0.1 mol/L monochloroacetate buffer pH 3 and
1% methanol. In addition, thiols were detected with monobromo
(trimethylammonio)bimane bromide as described previously.25 The
total amount of RSNO in vitro or in plasma was determined
electrochemically with 1.5 mmol/L CuCl2 (Cu2/Cu) to rapidly
decompose all RSNO. The released NO was detected with the
ISO-NO Mark II electrode. GSNO and SNO-BSA were used as
standards. They were prepared by incubation of GSH or BSA with a
2-fold molar excess of NaNO2 in acidified water on ice. To estimate
the fraction of low-molecular-weight (LMW) RSNO in total RSNO,
the real-time kinetics of RSNO decomposition were traced as
described above except that 40 mol/L CuCl2 was used (Figure 5).

Figure 1. Micellar catalysis of NO oxidation by PFC. a, PFC

component of Perftoran emulsion. b, PFC as catalyst of NO oxidation and RSNO formation. Small and uniform PFC micelles of
Perftoran are stabilized by Proxanol surfactant. If partitioning (Q)
for NO and O2 is 1, PFC micelle would increase local concentration of both gases by sequestering them from aqueous phase
and accelerate NO oxidation and N2O3 formation. c, Determination of QNO for PFC. Equation used for QNO calculation: [NO]H2O
indicates NO concentration in aqueous phase (8.9 mol/L);
[NO]PFC, NO concentration in PFC micelle; VPFC, volume of PFC
(1.3 mL); and , change of [NO]H2O on addition of PFC. was
determined experimentally. d, Sequestration of NO by PFC.
Infusion of PFC into 12.2 mL of NO solution under O2-free argon
conditions results in immediate decrease in [NO]H2O from 11.2
(light gray bar) to 8.9 (dark gray bar) mol/L as determined electrochemically (see Methods). MeanSE (n10; P0.01).

Animals
Adult male Wistar rats (300 to 400 g) were used. Both femoral
arteries and the left femoral vein of anesthetized animals (urethane
1.2 g/kg IP) were cannulated with polyethylene tubing (PE-50, WPI).
An arterial catheter was used for continuous monitoring of aorta
blood pressure and heart rate and to withdraw blood samples. The
venous catheter was used for drug administration. The same infusion
rate of drugs or saline (0.2 mL/min) was applied for all animals. The
drugs were used as follows: L-NAME 20 mg/kg, GSH 2 mg/kg, Cys
0.8 mg/kg, NaNO2 1 mg/kg, and Perftoran 136 mg/kg (low dose) or
1 g/kg (high dose). Before drug administration, mean arterial
pressure (MAP) was recorded, and the initial level of RSNO was
determined in plasma as described above. MAP and heart rate were
detected with the pressure sensor connected to an amplifier. The
signal was digitally converted at a PowerLab 200 workstation
(ADInstruments) and transmitted to a desktop computer. All statistical calculations were performed with Origin 7.0 software (OriginLab Corp).

Results
Dramatic Acceleration of NO Oxidation by PFC
To study the effect of PFC on NO bioavailability, we chose
Perftoran, the Russian brand of clinically approved blood
substitute.26 Perftoran contains 13% (wt/vol) perfluorodecaline and 6.5% (wt/vol) perfluoromethylcyclopiperidine,
which forms 0.07-m micelles stabilized by a poloxamertype surfactant (Figure 1). O2 solubility by Perftoran is 12

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Rafikova et al
times greater than that in water or blood plasma.26 Unlike the
chemical binding of O2 to the porphyrin-iron sites of hemoglobin, O2 dissolution in PFC is a simple, passive process in
which hydrophobic gas molecules occupy PFC micelles. In
contrast to the characteristic sigmoid binding curve of O2 to
hemoglobin, O2 solubility in PFC emulsions increases linearly with partial pressure.2224
Because NO is hydrophobic, we reasoned that it should be
concentrated by PFC micelles along with O2 under aerobic
conditions, implying that PFC would accelerate NO oxidation
(Figure 1b). To estimate the maximum acceleration value (H)
of this reaction, the partition coefficient (QNO) for a PFC/H2O
system was determined. To calculate QNO, which is described
by the equation shown in Figure 1c, we used a Clark-type NO
electrode attached to an airtight O2-free gas/liquid mixing
device to monitor changes in NO concentration on addition of
PFC. We determined QNO(PFC/H2O) to be 200 (Figures 1c
and 1d). QO2(PFC/H2O) has been shown to be 12.26 For the
trimolecular reaction of N2O3 formation, the equation
HQ2NOQO22002125105 is used. This estimation implies that even a small quantity of PFC in mammalian blood
(eg, 1% vol/vol) would sequester a large amount of circulating NO. At the same time, it should boost production of
reactive N2O3 species hundreds of thousands of times, which
in turn would result in increased nitrosation of external
molecules such as thiols (Figure 1b).

Catalysis of RSNO Formation by PFC In Vitro

To investigate the stimulating effect of PFC on RSNO
formation, we compared the rate of GSNO accumulation as a
function of PFC volume in the probe. GSNO was detected
with Cu2/ followed by electrochemical detection of released
NO.21 A representative experiment (Figure 2a) shows that 1%
(vol/vol) PFC that has been aerobically saturated with water
solution of NO (0.5 mol/L) increased the rate of GSNO
formation 10-fold. A similar result was obtained with
L-cysteine (data not shown). Importantly, the efficiency of
PFC-mediated S-nitrosation depends on the amount of PFC in
the probe (Figure 2b). The resonance-like maximum was
observed at 1% (vol/vol) Perftoran. Larger or smaller
amounts of PFC were progressively less efficient. This fact is
readily explained in terms of micellar catalysis of NO
oxidation, in which a relatively small optimal volume of the
hydrophobic phase (in this case, PFC) generates maximum
acceleration of the reaction.21 These results suggest that the
use of various amounts of PFC can regulate the level of
endogenous vasoactive LMW RSNO.

PFC as a Powerful NO Sink In Vivo

To evaluate the ability of PFC to absorb NO in vivo, we
studied the effect of Perftoran on MAP in rats. Intravenous
bolus application of PFC (1 g/kg, 7% vol/vol) was characterized by extensive hypertension (11 mm Hg) over the first
40 minutes, followed by mild hypotension (Figure 3a).
Because the PFC solution was isoosmotic (see Methods) and
the rate of its infusion was slow (0.2 mL/min), PFC-induced
vasoconstriction could not be attributed to the change in the
osmotic status.

Control of Plasma NO Bioactivity by PFCs

3575

Figure 2. Catalysis of GSNO formation by PFC. a, Representative NO-electrode tracing of Cu2/-dependent NO production
after aerobic addition of 1 mmol/L GSH to 1 mol/L NO solution
(initial NO concentration was 100 mol/L) in presence or
absence of PFC (1% vol/vol). b, PFC-mediated GSNO formation
function of PFC concentration (experimental conditions as in
Figure 2a; see Methods; n6; P0.01).

To confirm that the PFC-mediated increase in blood

pressure was due to plasma NO sequestration, we administered a nonselective NO synthase inhibitor, NG-nitro-Larginine methyl ester (L-NAME; 50 mg/kg IP) to deplete the
vasculature of endogenous NO. This caused a characteristic
steep increase in MAP (Figure 3b). Because PFC failed to
increase MAP any further in this case, we concluded that
intravascular NO was essential for PFC-induced
vasoconstriction.
To further substantiate the link between PFC-mediated
hypertension and plasma NO, we used sodium nitrite, a
well-established NO source in vivo and a potent vasodilator.27
Reduction of nitrite to NO is catalyzed in vivo by xanthine
oxidoreductase28 and deoxyhemoglobin29 and also can occur
nonenzymatically.30 Moreover, a significant circulating arteriovenous plasma nitrite gradient has been reported, which
argues in favor of intravascular nitrite being a stable natural
source of NO.31,32 Infusion of NaNO2 (1 mg/kg IP) 90
minutes after L-NAME decreased MAP sharply (Figure 3c).
Subsequent PFC administration elevated MAP back to the
original L-NAME level (Figure 3c). Because nitrite is slowly
reduced to NO in vivo under physiological pH (hence its
vasorelaxation effect), an ability of PFC to specifically
counteract this effect confirms its mode of action as a
powerful NO sink.
Taken together, these results demonstrate the existence of
a sizable pool of circulating NO in mammalian blood that

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Figure 3. Relationship between PFC-mediated hemodynamic effects and NO. a, Effect of high dose of PFC on MAP of anesthetized
rats. Infusion of bolus 1 g/kg PFC (7% vol/vol of total blood) provokes steep increase in blood pressure followed by gradual asymptotic recovery and mild hypotension. Bars represent extremes of MAP during time course of continuous monitoring. MeanSE (n12,
P0.05). MAP indicates change in MAP. Arrow indicates time point of PFC or saline infusion. b, Inactivation of NOS abolishes hemodynamic effects of PFC. Infusion of L-NAME (50 mg/kg) causes prolonged and stable hypertension, which is not further affected by 1
g/kg PFC. MeanSE (n8, P0.05). c, Delivery of NO donor resumes hemodynamic effects of PFC under L-NAME conditions. Infusion
of NaNO2 1 mg/kg IP 110 minutes after L-NAME administration leads to partial recovery of MAP (24 mm Hg). In this case, subsequent infusion of PFC (1 g/kg) fully restored hypertensive effect of L-NAME (MAP28 mm Hg; n10; P0.05). d, Schematic interpretation of experimental results. Artery is shown with endothelial cells producing NO that diffuses in all directions. In a, PFC micelles (gray
circle) rapidly absorb plasma NO, thus causing vasoconstriction and hypertension. In b, NOS is inactivated, which limits the amount of
plasma NO and prevents further potential constriction by PFC. In c, reducing components of blood convert exogenous NO2 to NO,
thus causing partial relaxation from L-NAME. However, PFC micelles sequester exogenous NO and thus constrict vessel back to
L-NAME position.

serves as a systemic dilator. Limitation of its bioavailability

by PFC leads to systemic vasoconstriction.

PFC-Mediated Catalysis of RSNOs In Vivo and

Control of Blood Pressure
As discussed above (Figure 2), PFCs not only limit the
concentration of free NO but also accelerate its oxidation and
formation of RSNOs. Generation of vasoactive RSNOs can
explain the delayed dilation effects of PFC in the experiments
described in Figure 3. A modest but reproducible hypotension
was observed 60 minutes after PFC infusion (Figure 3a).
Furthermore, in the nitrite-treated rats, MAP dropped below
the baseline level 30 minutes after PFC infusion (Figure 3c),
whereas in the control group (without PFC), MAP remained
25 mm Hg above baseline (Figure 3c). We interpret these
results to mean that during the first phase of action, PFCs
rapidly absorb plasma NO, which causes the initial increase
in MAP; however, because local concentration of NO and O2
in PFC micelles becomes high, N2O3 forms at a much greater
rate, which leads to accumulation of vasoactive RSNOs.
To test whether PFCs can indeed decrease vascular tone
via generation of endogenous RSNOs, a series of in vivo
studies that used 10 times less PFC than that used in the
experiments reported in Figure 3 were performed. The reason
for using a smaller amount of PFC derives from the graph in
Figure 2b, which shows that 1% PFC in aqueous solution is
the optimal amount that generates the maximum acceleration

of NO oxidation and RSNO formation. Therefore, one would

expect that at this concentration in vivo, PFC would convert
plasma NO into vasoactive RSNO most efficiently. Indeed,
the bolus infusion of the low dose of PFC (136 mg/kg IV, 1%
vol/vol total blood) did not cause any increase in MAP (Figure
4a). Instead, it produced a more rapid and pronounced decrease
in MAP (10 mm Hg over 40 minutes) than that observed with
the high dose of PFC (compare Figures 4a and 3a).
To confirm that the observed PFC-mediated vasodilation
was due to endogenous RSNO formation, we measured LMW
RSNO in plasma before and after PFC administration (Figure
4b). Plasma LMW RSNOs were determined as described in
Figure 5, using a concentration of CuCl2 (40 mol/L) selected
to decompose predominantly LMW RSNO. More stable
high-molecular-weight (HMW) RSNOs (eg, S-NO-albumin)
were largely resistant to such a low concentration of Cu2/
during the experiment (Figure 5). Using this approach, we
found that the basal level of LMW RSNO in rat plasma is
106 nmol/L (n6) and total RSNO is 200 nmol/L (Figure
4). These numbers are in good agreement with the level of
RSNO previously detected in human plasma by a similar
electrochemical method.33,34 Because of RSNO rapid decomposition during sample preparation, the detected values of
RSNO reflect only a fraction of their actual amount in blood.
This explains at least in part a wide range of RSNO values in
the circulation determined by different methods.34 As Figure
4b shows, the level of plasma LMW RSNO increased

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Rafikova et al

Figure 4. Effect of low dose of PFC on MAP and RSNO formation in anesthetized rats. a, Systemic vasodilation response to
intravenous bolus of 136 mg/kg PFC (1% vol/vol of total blood).
Bars represent extremes of MAP during time course of continuous monitoring. MeanSE (n12, P0.05). Time of PFC or
saline infusion is indicated by arrow. MAP indicates change in
MAP. b, Stimulation of plasma LMW RSNO formation by PFC.
RSNO indicates change in plasma LMW RSNO. LMW RSNO in
plasma was detected as described in Figure 5. Total RSNO
concentration in control (PFC) group was 21025 (18020),
20520 (16015), and 19525 (19520) nmol/L at 0, 10, and
25 minutes, respectively (n10, P0.05). c, Schematic explaining vasodilating effect of PFC. At 1% vol/vol concentration, PFC
micelles (gray circle) absorb NO and O2 to generate reactive
N2O3 species via micellar catalysis of NO oxidation. N2O3 then
nitrosates plasma small thiols to form vasoactive RSNOs that
cause vasorelaxation.

5-fold 10 minutes after PFC injection (1% vol/vol) and then

gradually decreased over the next hour. The burst of plasma
RSNO induced by PFC explains the drop in MAP in Figure
4a. Significantly, despite the marked increase in the level of
LMW RSNO in response to PFC, the amount of total RSNO
undergoes only a slight change, which indicates that PFCmediated NO oxidation targets primarily the pool of LMW
RSH in the circulation. Indeed, only small thiols are capable
of penetrating PFC micelles efficiently. This result implies
that LMW RSNO has a much greater specific vasoactivity
than the more abundant HMW RSNO. The latter can be
considered as a relatively constant NO pool for storage and

Control of Plasma NO Bioactivity by PFCs

3577

Figure 5. Method for quantitative discrimination of LMW RSNO

from HMW RSNO. a, Electrochemical detection of GSNO in
mixture with SNO-albumin. A representative NO-electrode tracing of real-time Cu2/-dependent NO production from probe
containing 200 nmol/L GSNO and 200 nmol/L BSA-NO in
80 mmol/L Tris-HCl, pH 7.9. CuCl2 was added to 40 mol/L at
time 0. By 40 seconds, all GSNO but only 20% SNO-BSA had
been decomposed, which indicates marked difference between
2 types of RSNO in their sensitivity to low dose of Cu2/.
NO-Cys was more sensitive than GSNO and decomposed
within first 15 seconds (data not shown). b, Calibration curves of
GSNO and BSA-NO decomposition. Plot represents NO release
data from NO electrode at 40-second time point as function of
RSNO concentration. Under selected conditions (a), GSNO
(slope 0.9, r0.9998, P0.0001) is 90 times more sensitive to
CuCl2 (40 mol/L) than SNO-BSA (slope 0.01, r0.96, P0.02).
Such a difference allows estimation of amount of small RSNOs
in mixture of total RSNO. Sensitivity of method is 5 nm/L, linearity 10 to 5000 nmol/L, recovery 955%.

transport purposes.10 A slight drop of total plasma RSNO 10

minutes after PFC administration (the time of maximal LMW
RSNO increase) reflects the process of PFC-mediated redistribution of NO from its HMW depot to actively used LMW
RSNO.
We determined the concentration of the most common free
LMW thiols, GSH and L-CysH, in plasma of normally fed
adult rats to be 153 and 396 mol/L, respectively (see
Methods), which is consistent with previous measurements.35
Such a relatively low level of reduced small thiols in the
circulation could limit RSNO formation despite the high yield
of available N2O3 generated by PFC. To compensate for a
potential deficiency of intravascular small thiols, we administered exogenous LMW RSH to PFC-treated rats and examined their effects on LMW RSNO formation and vasodilation.
As Figure 6a demonstrates, coadministration of 2 mg/kg GSH
along with PFC resulted in a 68% increase in the amount of
plasma LMW RSNO (top panel) and a concomitant marked
decrease in MAP (bottom panel). Also, a strong effect on both

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Figure 6. Cumulative effect of exogenous thiols and PFC on vascular tone and RSNO formation. a, Systemic vasodilation (top) and
increase in plasma RSNO (bottom) in response to intravenous bolus infusion of 136 mg/kg PFC and 2 mg/kg GSH. Bars represent
extremes of MAP during time course of continuous monitoring. MeanSE from 11 independent experiments (n11, P0.05). Arrows
indicate when chemicals were infused. RSNO indicates change in plasma LMW RSNO. Total RSNOs in control (PFCGSH) group
was 21025 (18525), 20520 (19020), and 19525 (22025) nmol/L at 0, 10, and 25 minutes, respectively. b, Systemic vasodilation (top) and increase in plasma RSNOs (bottom) in response to intravenous bolus infusion of 136 mg/kg PFC and 0.8 mg/kg Cys.
MeanSE from 10 independent experiments (n10, P0.05). Total RSNOs in control (PFCCys) group was 21025 (20020),
20520 (18015), and 19525 (23020) nmol/L at 0, 10, and 25 minutes, respectively. c, Persistent hypotension in response to repetitive administration of 0.8 mg/kg Cys followed by singe dose of 136 mg/kg PFC (n6, P0.05). Arrows indicate when Cys was infused.
d, Schematics explaining vasodilating effect of exogenous thiols in combination with PFC. Increased concentration of LMW RSH in rat
vasculature leads to higher production of vasoactive RSNO via PFC-generated N2O3.

RSNO formation (87% increase) and vasodilation

(17 mm Hg, 140 minutes) was observed when L-Cys (0.8
mg/kg IV) was used instead of GSH (Figure 6b). Without
PFC, neither glutathione nor cysteine had any effect on
plasma RSNO or blood pressure at these concentrations.
Obviously, the combined hemodynamic effect of PFC and
thiols was much greater than that of PFC alone (compare
Figures 4 and 6b), which provides direct evidence that
PFC-induced vasodilation was due to endogenous RSNO
formation.
Because the half-life of Perftoran in human and animal
blood is 24 hours,26 the eventual recovery of MAP after a
single dose of RSH was likely due to rapid RSH/RSNO
uptake by tissues rather than PFC inactivation. The data of
Figure 6b confirm this conclusion. On receiving a second
dose of L-Cys (0.8 mg/kg) 2 hours after the first thiol
infusion, PFC-treated animals responded by a second wave of
marked vasodilation that was virtually identical to the first
one. This result demonstrates that one can maintain the steady
level of circulating RSNO and control blood pressure for a
long period of time simply by supplying small amounts of
natural thiols to PFC-treated animals.

Discussion
The physiological and pharmacological significance of the
present study is several fold. First, it establishes the crucial

role of plasma NO in systemic hemodynamics. Second, it

directly implicates the intravascular hydrophobic phase in
regulation of plasma NO bioactivity via the micellar catalytic
mechanism of endogenous RSNO formation. Third, it establishes intrinsic plasma LMW RSNO as a potent relaxing
factor under noninflammatory conditions. Fourth, it explains
various hemodynamic effects associated with the use of PFC
in the clinic. Fifth, it provides a conceptionally new pharmacological approach to manipulate NO bioactivity.

Physiological Role of Plasma NO and the

Mechanism of Endogenous RSNO Formation
It has been widely assumed that a fraction of endotheliumderived NO that does not diffuse into the vessel wall gets
rapidly trapped and inactivated by hemoglobin from red
blood cells. Recently, we challenged this view by showing
that under physiological conditions, a significant amount of
NO accumulates in the hydrophobic compartments of serum
albumin.21 The high local concentration of NO within the
protein interior leads to accelerated NO oxidation and formation of nitrosative N2O3 species, which in turn are responsible
for maintaining the basal level of circulating vasoactive
RSNO.21 In support of this model, a number of studies,
including the present one, detected the basal level of RSNO in
mammalian plasma in the 20 to 200 nmol/L range.34 Moreover, intravenous application of NO was shown to exert a

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Rafikova et al
systemic hemodynamic effect via RSNO in humans.11 However, it has remained unclear whether intrinsic plasma NO or
RSNO played any significant role in cardiovascular processes. In the present study, we addressed this important issue
by devising an approach to specifically modulate the pool of
plasma NO and RSNO without boosting NO production or
delivering any exogenous NO into the system.
The approach is based on unique physical and chemical
properties of PFC emulsions.2224 By utilizing a commercial
PFC product, Perftoran (Figure 1a), we demonstrate that PFC
micelles adsorb NO from the aqueous phase, greatly accelerating NO oxidation and RSNO formation under aerobic
conditions (Figures 1 and 2). In accord with the theory of
micellar catalysis,21 the rate of NO oxidation and RSNO
formation depends on the volume of the hydrophobic phase,
with a resonance-like maximum at 1% vol/vol PFC (Figure
2b). Larger or smaller amounts of PFC were progressively
less effective. Next, we applied PFC as a tool for modulating
plasma NO bioavailability and RSNO formation in the
mammalian vasculature. Administration of a large dose of
PFC (7% vol/vol) caused extensive vasoconstriction due to
rapid sequestration of plasma NO (Figure 3). Alternatively, a
small dose of PFC (1% vol/vol) induced long-lasting systemic dilation (Figure 4). In this case, most of PFCsequestered NO was efficiently converted into N2O3 to elicit
endogenous vasoactive LMW RSNO (Figures 4 and 6).
Because the basal level of endogenous LMW RSNO can be
increased dozens of fold simply by changing the property of
the circulating hydrophobic phase, there must be a substantial
amount of available NO in mammalian plasma at any given
moment. Moreover, the level of plasma LMW RSNO can be
elevated further by supplementing PFC with exogenous
LMW RSH (Figure 6), which indicates that RSNO formation
is limited by circulating thiols rather than by plasma NO.
Importantly, the fate and physiological significance of plasma
NO are determined by the local hydrophobic environment.21
Taken together, the results of the present study directly
implicate intrinsic plasma NO in maintaining vascular tone
despite the presence of red blood cells or other potential NO
scavengers. Furthermore, our results underscore the physiological importance of nitrosative chemistry in plasma, establishing endogenous RSNO as a significant transport and
delivery system of bioactive NO in the mammalian
circulation.

Pharmacological Applications of PFC as a Potent

Modulator of NO Bioactivity
PFC emulsions have been clinically evaluated in many
countries as artificial O2 carriers to reduce allogeneic blood
transfusion and improve tissue oxygenation. They have been
successfully used to augment acute hemodilution after trauma
or surgery and to treat conditions such as cerebral or
myocardial ischemia.2224 Although PFCs are well tolerated
in animals and humans, their unexpected pulmonary and
systemic hemodynamic effects have limited their clinical
use.36,37 In particular, the higher doses of PFC have been
associated with progressive hypertension and decreased heart
rate.38 However, unexpected beneficial anti-inflammatory
and antithrombotic effects of PFCs have been noticed39,40 that

Control of Plasma NO Bioactivity by PFCs

3579

could not be explained merely by the ability of PFC to

transport and upload O2 or change blood viscosity.
The present study establishes PFC as a potent modulator of
NO bioactivity, thus explaining, at least in part, its dosedependent systemic effects. Most importantly, the present
findings raise an attractive possibility of using PFC as a
pharmacological tool to influence vascular tone and platelet
function by modulating plasma NO activity via endogenous
blood-borne NO carriers. In this regard, PFCs represent a
conceptually new drug for the treatment of cardiovascular
disorders associated with compromised and excessive NO
bioavailability. Indeed, depending on the dose, PFC functions
either as a potent NO stimulator or suppressor. The stimulating effect occurs at lower doses owing to the boosting of
endogenous RSNO production (Figures 4 and 6). At higher
doses, PFC suppresses NO activity by decreasing its available
concentration (Figure 3a).

Vasoregulation With Natural Thiols and PFC: A

Safe Alternative to Exogenous RSNO
RSNOs have a wide variety of therapeutic effects and may be
particularly useful alternatives to organic nitrates because
they do not elicit tolerance. As potent systemic vasodilators
and platelet inhibitors, RSNOs have been evaluated in the
treatment of various cardiovascular complications, including
thrombosis, vasospasm, preeclampsia, and ischemia/reperfusion injury.1214 Furthermore, because of their bronchodilating properties and the fact that the airway level of RSNOs is
reduced in asthma and cystic fibrosis patients, RSNOs are
considered as promising therapeutics for pulmonary conditions.41,42 Finally, RSNOs may be used as antiviral and
antimicrobial agents, because they inhibit viral protease
enzymes and possess a strong bacteriostatic effect.43,44 However, despite growing interest in the pharmacological potential of RSNOs, a number of general problems limit their
clinical use. The main shortcoming of RSNOs as a potential
drug is an unpredictable rate of decomposition in vivo and
instability in physiological vehicles. Excess RSNOs can lead
to hypotension and hemorrhage due to impaired platelet
function. Furthermore, synthetic drugs that have been
S-nitrosylated retain their own side effects.
In this regard, the use of PFC and PFC/RSH combinations
provides a unique pharmacological solution for safe delivery
of RSNOs. Because PFC-mediated generation of RSNOs
occurs in vivo and can be precisely controlled by PFC and
exogenous RSH concentrations, concerns about RSNO overdose and storage problems are eliminated. Furthermore,
chemically inert PFC emulsions (eg, clinically safe Perftoran)
and natural thiols, such as L-Cys or GSH, should have
minimal immunologic side effects. Finally, because PFC
uncouples RSNO formation from overall NO synthesis, it
creates a unique opportunity to relieve nitrosative stress, ie,
excessive nitration of macromolecules and tissue damage
during inflammation,45,46 while maintaining control over
vascular tone, blood clotting, and organ perfusion.

Acknowledgments
These studies were supported by the Irma T. Hirschl Career Award
and a grant from the National Institutes of Health.

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3580

Circulation

December 7, 2004

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