Harger, Jianfei Jiang, Bruce Pitt, Robert N. Taylor, Carl A. Hubel and Valerian E. Kagan
Robin E. Gandley, Vladimir A. Tyurin, Wan Huang, Antonio Arroyo, Ashi Daftary, Gail
Pregnancy and Preeclampsia Plasma
Mediated Relaxation Is Enhanced by Ascorbate and Copper : Effects in
Print ISSN: 0194-911X. Online ISSN: 1524-4563
Copyright © 2004 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
2005;45:21-27; originally published online November 29, 2004;
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S-Nitrosoalbumin–Mediated Relaxation Is Enhanced by
Ascorbate and Copper
Effects in Pregnancy and Preeclampsia Plasma
Robin E. Gandley, Vladimir A. Tyurin, Wan Huang, Antonio Arroyo, Ashi Daftary, Gail Harger,
Jianfei Jiang, Bruce Pitt, Robert N. Taylor, Carl A. Hubel, Valerian E. Kagan
Abstract—S-nitrosoalbumin (SNO-Alb) is a major reservoir of releasable nitric oxide (NO) in plasma. In preeclampsia, a
pregnancy-specific disorder associated with endothelial dysfunction, we previously found significant elevations in
plasma SNO-Alb concentrations and decreased plasma ascorbate (Asc) levels. This increased SNO-Alb may result from
low-plasma Asc if Asc, along with transition metals (eg, copper [Cu]) are necessary for release of NO from
S-nitrosothiols. We propose that vasodilator effects of SNO-Alb, mediated by release of NO, are fully realized only
when Asc/Cu availability is sufficient. Relaxation responses to SNO-Alb or the control reduced human serum albumin
(SH-Alb), and responses to pooled plasma from normal or preeclamptic pregnancies were examined in isolated mouse
arteries. Arteries preconstricted with phenylephrine were exposed to SNO-Alb or SH-Alb at physiologically relevant
concentrations. When free Cu was added in excess (10 ?mol/L), NO release was not dependent on Asc. However, when
Cu was added at lower (physiological) levels, NO release was dependent on Asc. The addition of Asc and Cu to
SNO-Alb stimulated vasodilatory responses in isolated arteries ?90%, whereas no change in the SH-Alb (5%) response
was observed. Preeclampsia plasma with higher levels of SNO-Alb caused arteries to relax 44.1?4.7%, whereas normal
pregnancy plasma caused 11.9?4.2% relaxation (P?0.007). These data indicate that SNO-Alb alone or in plasma can
act as a potent vasodilator, and that sufficient Asc/Cu promotes this action. We suggest that the higher circulating levels
of SNO-Alb, in women with preeclampsia, reflect a deficiency in Asc/Cu-mediated release of NO from SNO-Alb.
Key Words: preeclampsia ? pregnancy ? nitric oxide ? oxidative stress ? antioxidants
this pregnancy-specific disorder include hypertension and
proteinuria developing after 20 weeks of gestation. Placental
abnormalities and maternal endothelial dysfunction/activa-
tion are thought to contribute to vasospasm and the onset of
the maternal syndrome. It has been hypothesized that oxida-
tive stress plays a role in the pathophysiology of preeclamp-
sia. Evidence of oxidative stress in combination with pro-
found depletion of plasma ascorbate (Asc) has been reported
in women with preeclampsia.1–4Asc concentrations in
women with preeclampsia are nearly half those of matched
normal pregnant controls (?40 versus 25 ?mol/L).5The
implications of this reduction in plasma Asc are currently not
clear. Plasma Asc levels are inversely related to blood
pressures in men without a history of hypertension.6,7Asc and
vitamin E supplementation in women at high risk for pre-
eclampsia was associated with a reduction in markers of
reeclampsia is a major cause of maternal and neonatal
morbidity and mortality. The clinical manifestations of
endothelial dysfunction and in the incidence of preeclamp-
sia.5,8Although the decreased incidence of preeclampsia was
associated with decreased biochemical indices of oxidative
stress and poor placental function, the mechanism(s) driving
the apparent success of this treatment is currently unclear.
With the onset of several large clinical trials examining this
treatment regime during pregnancy, the role of Asc in the
pathogenesis of preeclampsia and other pregnancy complica-
tions has become an exceedingly pertinent question.
The potent vasodilator, nitric oxide (NO), is critical to
normal vascular function and endothelial integrity. NO is
important to the normal adaptive vasodilation of pregnancy in
animals and women, and decreased NO bioavailability is
thought to contribute to the development of preeclampsia.9A
functional loss of NO-mediated vasodilatation could result
from decreased production by the endothelium, through
improper storage, or oxidative degradation of NO and/or
impaired vascular smooth muscle responsiveness to NO.
Received March 31, 2004; first decision April 16, 2004; revision accepted September 15, 2004.
From the Magee-Womens Research Institute and Departments of Environmental and Occupational Health (R.E.G., V.A.T., W.H., A.A., J.J., B.P.,
C.A.H., V.E.K.) and Obstetrics, Gynecology, and Reproductive Sciences (A.D., G.H., C.A.H.), Center for Free Radical and Antioxidant Health (R.E.G.,
V.A.T., J.J., B.P.), University of Pittsburgh, Pittsburgh, Pa; and the Department of Obstetrics, Gynecology and Reproductive Sciences (R.N.T.),
University of California, San Francisco.
Correspondence to Robin E. Gandley, PhD, Magee-Womens Research Institute, 204 Craft Ave, Pittsburgh, PA 15213. E-mail firstname.lastname@example.org
© 2004 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.orgDOI: 10.1161/01.HYP.0000150158.42620.3e
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There is substantial evidence of oxidative degradation of NO
Nitrosolated thiols in the plasma are a circulating source of
stored NO, with the capacity to be released and have
biological activity.12Bioavailable reductants, such as Asc, in
the presence of transition metals (eg, copper [Cu]) are
required for release of biologically active NO from nitrosy-
lated thiols.13Cu in the presence of Asc is capable to
undergoing 1-electron oxidation-reduction conversion. Reac-
tion scheme typically includes the following reactions:
Formation of a Cu1?–S-nitrosoalbumin (SNO-Alb) interme-
diate weakens the S–N bond and strengthens the N–O bond,
thereby promoting NO release from SNO-Alb.14Serum
albumin is an important carrier protein and buffer for redox
active Cu in the circulation. Plasma Cu levels increase during
pregnancy.15,16We have previously reported that the Cu
binding capacity of Alb is impaired in women with pre-
eclampsia without an increase in the total circulating level.17
Total S-nitrosothiol and SNO-Alb concentrations were
significantly increased in the plasma of these patients.10
Although increased NO synthesis could explain these
increases, they alternatively might be caused by a de-
creased rate of decomposition of S-nitrosothiols in the
plasma of women with preeclampsia. High S-nitrosothiol
levels are associated with elevated blood pressure and
cardiac events in patients with end-stage renal disease.18It
is biologically plausible that the increased reserve of
SNO-Alb found in the plasma of women with preeclampsia
occurs in part because of an oxidative stress-induced
deficiency in plasma Asc. Previous in vitro data have
suggested that the release of NO from SNO-Alb is limited
in the absence of Asc or other suitable reductants.19
Therefore, we hypothesized that the vasodilatory effects of
SNO-Alb would be maximized only when Asc/Cu avail-
ability is sufficient. In the current work, we have deter-
mined that SNO-Alb, the major nitrosothiol in the plasma,
is a potent vasodilator when applied to isolated arteries.
This vasodilatory activity is modulated by the reductant
Asc and the presence of the transition metal Cu.
Materials and Methods
Preparation of SNO-Alb
Nitrosylated human serum albumin (SNO-Alb) was prepared using
the trans-nitrosylation reaction and S-nitrosoglutathione (GSNO) as
an NO donor.20GSNO was prepared by the reaction of acidified
NaNO2 with glutathione. Briefly, 100 mmol/L glutathione was
mixed with 100 mmol/L of NaNO2in 200 mmol/L HCl.21The
solution was used fresh for S-trans-nitrosylation of reduced human
serum albumin (SH-Alb). SH-Alb was mixed with a 50-fold excess
of GSNO and low-molecular-weight components were removed.20,22
SH-Alb and SNO-Alb was quantified using Ellman reagent and
DAF-2 assays, respectively, as previously described.23
Detection of NO Release From SNO-Alb
NO release was measured amperometrically using a Clark-type NO
electrode (Iso-NO with 2-mm shielded sensor; WPI, Sarasota, Fla).
Samples were incubated at room temperature in a reaction chamber
under continuous stirring. Changes in current output (pA) were
recorded, and NO release was quantified using a standard curve
generated by the addition of NaNO2 in nitrite-free water under
reducing conditions (KI/H2SO4). Initial rate of NO release from
SNO-Alb was calculated as a difference in the amplitude of current
for the first 30 seconds after addition of Cu/Alb. To simulate the
isolated arterial bath conditions, NO release was measured in 3 mL
of HEPES-buffered physiological saline solution (HEPES-PSS, pH
7.4) in the presence of a mesenteric artery.
Isolated Arterial Preparation
Small resistance-sized mesenteric arteries (150 to 200 ?m) from
female mice were removed, flushed of residual blood, and mounted
in a dual-chamber pressurized arteriograph in HEPES-PSS.24Trans-
mural pressure and lumen diameters were monitored. Relaxation
responses to modified albumin (SNO-Alb or SH-Alb) or 1% hepa-
rinized pooled plasma from patients with normal or preeclamptic
pregnancies were assessed over a 15-minute period in phenylephrine
(PE) preconstricted arteries. The concentration of Alb was matched
to the level in 1% plasma, with a fraction of the Alb being SNO-Alb
(5.8 ?mol total Alb/L with 0.5 ?mol SNO-Alb/L). Reduced human
serum albumin (SH-Alb) was used to match the concentration of
albumin relative to the level of nitrosylation of albumin, keeping the
concentrations of both consistent. 1H-1,2,4 oxadiazolo (4,3-
a)quinoxalin-1-one (ODQ; 10 ?mol/L; Sigma), a guanylyl cyclase
inhibitor, was used to block the relaxation pathway of NO. L-Nitro-
arginine methyl ester (L-NAME; 0.25 mmol/L), an NO synthase
inhibitor, was used to block endogenous production of NO in the
isolated arteries. Asc and CuSO4solutions were used at 50 ?mol
Asc/L and 0.25 or 10 ?mol CuSO4/L buffer; 50 ?mol Asc/L was
chosen as a concentration comparable to that seen in the circulation5
(shown in Figure 1C) to maximize release of NO from SNO-Alb. Cu
was added at both a limiting dose and in excess compared with the
concentration of Alb present.
Study Subjects and Sample Collection
Heparinized plasma was collected from a total of 10 nulliparous
women with preeclampsia and 10 nulliparous women with uncom-
plicated pregnancies recruited at Magee-Womens Hospital in the
third trimester of pregnancy, before labor or therapeutic intravenous
administration of MgSO4. Preeclampsia was defined using the
criteria of gestational hypertension, proteinuria, and hyperuricemia,
and reversal of hypertension and proteinuria after delivery.25Preg-
nant controls were normotensive throughout gestation, did not have
proteinuria, and delivered at term. Pooled plasma samples from the
10 subjects in each group were prepared, aliquoted, and stored at
Determination of SNO-Alb in Plasma by Biotin
Switch Assay Using NitroGlo Kit
SNO-Alb in plasma was determined by Biotin Switch Assay using
NitroGlo Kit (PerkinElmer Life Sciences). Briefly, free SH groups in
plasma were blocked and proteins were precipitated by acetone.
SNO-Alb was then reduced by Asc in the presence of HPDP–biotin,
nonreducing buffer was added, and samples were electrophoresed
(8% SDS-PAGE) and immunoblotted. SNO-Alb was quantified
based on a calibration curve of biotinylated albumin.26
Two-way repeated measures ANOVA and post-hoc Bonferroni test
or Student t test were used where applicable. Data in graphs are
displayed as means?SE. Statistical significance was accepted if
More methods details are available in an online supplement at
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Role of Asc in NO Release from SNO-Alb
NO release from in vitro nitrosylated human serum albumin
(SNO-Alb; 0.5 ?mol/L) was measured amperometrically in 3
mL HEPES-PSS. As indicated in Figure 1A, no release of NO
was detected when SNO-Alb was diluted in HEPES-PSS.
Furthermore, the addition of Asc (50 ?mol/L) to this mixture
did not elicit release of NO during short-term incubation.
When excess (10 ?mol/L) free Cu was subsequently added, a
significant release of NO was detected. SH-Alb in the
presence of Asc and Cu did not release detectable levels of
NO (Figure 1A).
To further examine the role of Asc and Cu in the release of
NO from SNO-Alb, the ratio of albumin to Cu was modu-
lated. We assessed the requirement of Asc to recycle Cu back
to its reduced form (Cu2?) for effective NO release to occur
(Figure 1B). Alb is an important transporter of redox-active
Cu in the circulation and binds Cu at a ratio of 1:1, avoiding
the redox-cycling activity of the metal.27Cu was added to
fixed amount of SNO-Alb either in excess (2 Cu/Alb) or
within the binding capacity (0.5 Cu/Alb) to establish condi-
tions for Cu-mediated NO release (Figure 1B). At a ratio of 2
Cu/Alb, the release of NO was detected independently of the
addition of Asc. The addition of Asc to this mixture resulted
in a small additional release of NO. When the ratio of Cu/Alb
was reduced to 0.5, the initial release of NO was very low,
and only after the addition of 50 ?mol/L Asc was a
significant release of NO detected.
Increasing concentrations of Asc added to a fixed amount
of S- NO-Alb (1 ?mol/L) and Cu (0.5 ?mol/L) increased the
initial rate of NO release from SNO-Alb. SNO-Alb decom-
position catalyzed by Cu/Alb (ratio 0.1:1) is strongly depen-
dent on the concentration of Asc (Figure 1C). This dose-
response established that at levels of Asc reported in plasma
(?mol/L), Asc could modulate release of NO from SNO-Alb.
Dose-Response Effects of Nitrosylated Human
Effects of the different Cu/Alb ratios on relaxation responses
in the arterial myograph were examined. First, preconstricted
arteries in the presence of a fixed concentration of SNO-Alb
(0.5 ?mol/L with a total of 5.8 ?mol Alb/L) were exposed to
2 concentrations of Cu (Figure 2A). The percent relaxation of
arteries exposed to 0.25 ?mol/L Cu (Cu/Alb ratio of 0.05)
was 50%, whereas arteries exposed to 10 ?mol/L Cu (Cu/Alb
ratio of 2) relaxed 70%, in line with our data on NO release
from SNO-Alb. Reduced albumin (SH-Alb) used as a control
verified that 10 ?mol/L Cu had no vasoactive properties
independent of SNO-Alb.
SNO-Alb is a major reservoir for NO in plasma12and is
elevated in plasma from women with preeclampsia.10To
establish that SNO-Alb is capable of mediating dose-related
vascular relaxation, the response of phenylephrine precon-
stricted arteries, in the presence of 50 ?mol/L Asc and
0.25 ?mol/L Cu, was assessed with increasing doses of
SNO-Alb (Figure 2B). The arteries began to respond to
SNO-Alb at submicromolar concentrations. For comparative
purposes, the relaxation response to the potent NO donor,
sodium nitroprusside, was also determined (Figure 2B). After
completion of the dose-response experiment, additional NO
release from albumin in buffer was undetectable by DAF2
assay, indicating that the release of NO from S- NO-Alb
during the arterial response was complete.
Figure 1. A, Cu induces release of NO from SNO-Alb (tracing a).
SNO-Alb in HEPES-PSS solution does not stimulate release NO.
The addition of ascorbate (50 ?mol/L) also does not cause
release of NO. Only after the addition of 10 ?mol/L of Cu was
NO released (trace a). Reduced Alb (SH-Alb, tracing b) was
used as a control and failed to release NO under all conditions.
B, When the ratio of Cu/Alb is low, Asc is required for the
release of NO from SNO-Alb. The tracing b indicates that Cu
induces the release of NO from SNO-Alb when the ratio of Cu
to Alb is high (2:1). Under this condition, the majority of NO is
released without a need for Asc. When the molar ratio of Cu/Alb
is reduced to 0.5:1 (tracing a), the addition of Cu alone caused
only a small release of NO, whereas the addition of Asc allowed
a dramatic increase in NO. C, Higher doses of Asc in the pres-
ence of a limited amount of Cu cause increased initial rates of
NO release from SNO-Alb catalyzed by Cu/Alb. SNO-Alb with a
total of 5 ?mol/L Alb and 0.5 ?mol/L Cu (Cu:Alb ratio?0.1:1)
were exposed to different concentrations of Asc in phosphate-
buffered saline at pH 7.4. When the ratio of Cu/Alb is very low,
the initial rate of NO release from SNO-Alb increases with
increasing concentrations of Asc.
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To define the role of Asc in the relaxation response of
SNO-Alb, we looked at the response of preconstricted arteries
in the presence of 0.5 ?mol/L SNO-Alb to the addition of the
reductant Asc (50 ?mol/L) in the presence of only trace
amounts of transition metal (shown as Asc treatment; Figure
2C) relaxed 50%. After the addition of Asc, 0.25 ?mol/L
Cu (shown as Asc?Cu treatment) was added to the
preparation (Figure 2C). If Cu (0.25 ?mol/L) was also
added to the buffer, relaxation was increased to ?90%
(P?0.022). No relaxation responses were observed in
ODQ-treated arteries. To confirm that the observed vascu-
lar relaxation response was specific to SNO-Alb, precon-
stricted arteries were exposed to reduced albumin (SH-
Alb) at a matching dose of total albumin. No relaxation
response was observed on addition of Asc or Cu. Treat-
ment of arteries with ODQ followed by SH-Alb was not
significantly different from the response of SH-Alb alone
(data not shown). We have further found that treating
arteries with the NO synthase inhibitor, L-NAME, had no
effect on this relaxation response. These data indicate that
the nitrosylated albumin is acting as an NO donor, causing
relaxation in isolated arteries.
Response of Preconstricted Isolated Arteries to
Pooled Pregnancy Plasma
The amount of SNO-Alb in the plasma samples from the
normal pregnancies and preeclampsia pregnancies was deter-
mined using the biotin switch assay. Biotinylated albumin
standards were used to determine levels of SNO-Alb in the
normal pregnancy pool (2.9 ?mol/L) and the preeclampsia
plasma pool (7.2 ?mol/L) (Figure 3A inset). NO release from
plasma pools was also measured (Figure 3A). The assay was
validated using a pool of normal plasma with decomposed
SNO-Alb (trace 2) or spiked with 2 ?mol/L SNO-Alb (trace
3). The NO release detected in plasma from the normal preg-
nancy pool (trace 4) was approximately one-third that detected
from the preeclampsia pool (trace 5), which had a signal
comparable to the spiked normal pooled plasma (trace 3).
Pooled normal pregnancy plasma or preeclampsia plasma
(at a concentration of 1% plasma in buffer) was applied to
preconstricted arteries supplemented with 50 ?mol/L Asc
buffer. Asc supplementation was used to achieve levels of
Asc in both plasma samples sufficient to induce a maximal
relaxation response based on data in Figure 1C. Consistent
with their higher levels of SNO-Alb, preeclampsia plasma
caused arteries to relax 44.1?4.7%, whereas normal preg-
nancy plasma caused 11.9?4.2% relaxation (n?4/group;
P?0.007) (Figure 3B). Treatment with the guanylyl cyclase
inhibitor (ODQ) prevented plasma-mediated relaxation re-
sponses. Pretreatment of arteries with the NO synthase
inhibitor (L-NAME) had no effect on these relaxation re-
sponses (data not shown).
We found that SNO-Alb can act as a potent vasodilator
provided sufficient amounts of Asc and Cu are available to
promote this action. In vivo, Cu is normally bound to
ceruloplasmin and albumin and maintained in a redox-
inactive form. We previously found that modifications in Alb
can impair the redox control of Cu and ultimately contribute
to oxidative stress and depletion of Asc in the circulation of
women with preeclampsia. Mishandling of Cu is caused by
oxidative/nitrosative modifications in Alb, which are facili-
tated by excessive binding of free fatty acids.28–30This
modified Alb functions not as an antioxidant but as a
pro-oxidant.17,27We currently report that physiologically
Figure 2. A, Levels of Cu exceeding the binding capacity of Alb
cause a greater relaxation response to SNO-Alb compared with
a lower level of Cu. Phenylephrine preconstricted arteries were
exposed to a fixed concentration of SNO-Alb and total Alb or a
matching concentration of SH-Alb alone. The relaxation
response to the addition of Cu was determined. 10 ?mol/L Cu
(Cu/Alb ratio ?1:1) in the presence of 5.8 ?mol total Alb/L with
0.5 ?mol/L SNO-Alb caused greater relaxation than 0.25 ?mol/L
Cu (Cu/Alb ratio ?0.5:1). SH-Alb in combination with 10 ?mol/L
Cu was used as a control and did not induce significant relax-
ation. B, Preconstricted arteries relax in response to increasing
doses of SNO-Alb. Phenylephrine preconstricted arteries (n?6)
with 50 ?mol/L Asc and 0.25 ?mol/L Cu were exposed to
increasing doses of SNO-Alb. SNO-Alb (black symbols) caused
relaxation in a range of doses approaching the reference doses
of the potent NO donor, sodium nitroprusside (white symbols).
C, Ascorbate combined with SNO-Alb causes relaxation of pre-
constricted arteries. Phenylephrine preconstricted arteries in
HEPES-PSS buffer (containing trace amounts of Cu) and
0.5 ?mol/L SNO-Alb buffer were exposed to Asc (50 ?mol/L)
and the percent relaxation was determined. Although SNO-Alb
with Asc caused modest relaxation, addition of 0.25 ?mol/L Cu
enhanced the vasorelaxation response. This effect was sensitive
to ODQ inhibition of guanylyl cyclase. No relaxation response
occurred in arteries exposed to the control, SH-Alb.
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relevant concentrations of Asc will facilitate release of NO
from SNO-Alb. SNO-Alb can act as a potent vasodilator in
small isolated arteries, and adequate levels of Asc can
facilitate both release and vascular relaxation. Cu at supra-
physiological concentrations can cause release of NO from
SNO-Alb independently of Asc. At much lower Cu concen-
trations (within the binding capacity of Alb), however, Asc
enhances Cu-mediated NO release. Both NO release and/or
trans-nitrosylation of low-molecular-weight thiols can be
involved as the physiological mechanism by which SNO-Alb
mediates its activity, as has been originally reported by
Stamler.31Under specific conditions of our experiments,
however, the lack of exogenously added low-molecular-
weight thiols make the latter mechanism unlikely. Because
the presence of both Asc and Cu was essential for achieving
maximal relaxation activity of SNO-Alb, we concluded that
Asc-driven redox-cycling of Cu was the major contributing
mechanism for its decomposition and NO? release. However,
small relaxation response was also detectable in the absence
of exogenously added Cu, indicating that some trans-nitrosa-
tion–dependent mechanism of Asc action32might be respon-
sible, at least in part, for its relaxation effects on mesenteric
Our data indicate that application of 1% plasma from
women with preeclampsia to preconstricted arteries causes a
greater relaxation response than plasma from women with
normal pregnancies. This relaxation response was mediated
through guanylyl cyclase and not by de novo generation of
NO by the isolated artery. The finding of greater relaxation
induced by preeclampsia plasma relative to normal pregnancy
plasma is consistent with higher S-nitrosothiol levels (repre-
senting a source of potentially releasable NO) in preeclamp-
sia.10Our previous work indicated that the majority of the
elevation in S-nitrosothiols in preeclampsia plasma was
caused by SNO-Alb.10The elevated levels of SNO-Alb in
preeclampsia plasma represent a pool of NO with the poten-
tial to mitigate the profound vasoconstriction typically ob-
served in this pathophysiological state. However, we cannot
attribute the entire relaxation response to SNO-Alb because it
is possible that the plasma samples have other contributing
vasodilators (ie, estrogen, angiotensin 1 to 7 peptide).33,34
SNO-Alb at levels in the submicromolar range can
induce relaxation in small isolated arteries. The sensitivity
of the isolated mouse mesenteric arteries to SNO-Alb is
within the dose range previously reported for SNO–bovine
serum albumin applied to rabbit aortic strips.12The levels
obtained in the plasma pools used in this study are
consistent with levels previously reported10,35and may be
an underestimate of the original levels in plasma, given the
instability of SNO-Alb in biological samples.36Method-
ological differences may be critical to the quantitative
aspects of SNO-Alb analysis of endogenous levels. Using
mild reducing conditions (provided by the addition of Asc)
to detect the amounts of SNO-Alb in plasma by either the
Figure 3. A, Assessment of SNO-Alb content by biotin switch assay (inset) and NO release. The first 5 lanes (1 to 5) represent biotinyl-
ated albumin standards obtained using known amounts of SNO-Alb (0.36, 0.72, 1.44, 2.16, and 2.88 pmol, respectively). Lanes 6 and 7
correspond to pooled normal pregnancy (1.10 pmol) and preeclampsia plasma (2.72 pmol) samples, respectively. Based on the calibra-
tion curve generated using the standards (lanes 1 to 5), concentrations of SNO-Alb were estimated as 2.9 and 7.2 ?mol/L for normal
pregnancy and preeclampsia plasma samples, respectively. Plasma samples (100 ?L) were mixed with 1 mL of acetone and then were
precipitated. After centrifugation of plasma samples, traces of acetone were removed by N2and pellets were resuspended in 400 ?L of
50 mmol/L Na–phosphate buffer pH 7.4. NO? was released by Cu (100 ?mol/L)/Asc (1 mmol/L) in normal pregnancy and preeclampsia
plasma samples in phosphate buffer pH 7.4. 1 indicates phosphate buffer alone; 2, normal pooled plasma in which endogenous S-NO-
thiols (SNO-Albumin) were decomposed by UV irradiation (?330 nm, 10 minutes) using an Oriel UV light source (model 66002) and cut-
off filter (Balzers, ?330 nm); 3, UV-irradiated normal pooled plasma plus 0.8 nmol of exogenous SNO-Alb; 4, normal pregnancy plasma;
5, preeclampsia plasma. Arrows indicate additions of Cu/Asc. B, Preeclampsia plasma, with a greater concentration of SNO-Alb,
causes a greater relaxation response in preconstricted mouse arteries than normal pregnancy plasma. Phenylephrine preconstricted
arteries were exposed to 1% pooled normal or preeclampsia plasma and 50 ?mol/L Asc. Percent relaxation was determined as the
change in diameter relative to the total amount of constriction of the artery. A group of arteries was pretreated with ODQ, an inhibitor
of guanylyl cyclase. ODQ blocked the relaxation response to plasma, supporting a role for NO in this response. Mean?SE (n?4 arter-
ies/group) is shown.
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amounts of SH-Alb formed (biotin switch assay) or the
amounts of NO released (using an NO?-sensitive electrode)
yielded results in reasonable agreement. It should also be
noted that the low-molecular-weight thiols present in our
plasma samples represent a small portion of the total
thiols.36Asc and Cu, at the concentrations used, were not
sufficient to induce vasorelaxation in the absence of
SNO-Alb. When the Cu/Alb ratio is high, the requirement
for Asc is less, likely because of the availability of free Cu.
At low Cu/Alb ratios, Asc appears to allow redox cycling
that enables the release of NO from SNO-Alb. We further
showed that the guanylyl cyclase inhibitor ODQ inhibited
the relaxation response, verifying that the NO signaling
pathway mediates this effect. If arteries were exposed to
SH-Alb, Asc, and/or Cu alone, no relaxation was observed.
The release of NO from SNO-Alb requires the presence
of transition metal and reductant. Moreover, a Cu1?che-
lator (neocuproine) produces concentration-dependent in-
hibition of the relaxations from S-nitrosothiols (GSNO,
SNAP) in the rat anococcygeus muscle, which indicates
that Cu1?participates in the relaxant action of RSNO.37
Likewise, depletion of Asc levels (as result of Cu/Alb-
catalyzed oxidation) has been shown to drastically de-
crease NO release from SNO-Alb and result in endothelial
dysfunction.38It is possible that profound conformational
changes of Alb on binding of fatty acids—particularly in
the presence of Cu tightly bound to its N-terminal tripep-
tide Asp-Ala-His site39(located in a close proximity to
S-nitrosylated Cys 34)—may lead to a decreased stability
of its S–NO bond dissociation energy.32In fact, we have
recently reported that albumin/Cu/FA complexes exert
dramatically increased rates of Asc-dependent SNO-Alb
decomposition.40We propose that the balance between Asc
and Cu necessary for normal decomposition of SNO-Alb is
lost in the setting of preeclampsia.
These data indicate that SNO-Alb can act as a potent
vasodilator, and that sufficient Asc and Cu promote this
action. It is likely that SNO-Alb, circulating at higher levels
in preeclampsia plasma, represents a potential pool of NO.
NO-mediated vasodilatory activity appears to be diminished
in women with preeclampsia. Interventions to promote re-
lease of NO from SNO-Alb, such as Asc supplementation,
might ameliorate the maternal and fetal vascular complica-
tions of this disease.
Financial support received from National Institutes of Health grant
numbers RO1 HL 64145, 2 PO1 HD 30367, 5M01 RR 00056, and
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