| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701.
2
Department of Clinical Pathology, William Beaumont
Hospital, Royal Oak, MI 48073.
aAddress correspondence to this author at: Department of Chemistry and Biochemistry, Ohio University, Clippinger Laboratories, Athens, OH 45701. Fax 740-593-9641; e-mail malinski{at}ohio.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: We performed in vivo and ex vivo measurements of NO (electrochemical) and ex vivo in situ measurements of superoxide, peroxynitrite (chemiluminescence), and nitrite and nitrate (ultraviolet-visible spectroscopy). We determined the effect of lipopolysaccharide administration (20 mg/kg) on diffusible NO, total NO (diffusible plus consumed in chemical reactions), and superoxide and peroxynitrite release in the pulmonary arteries of rats.
Results: An increase in diffusible NO generated by constitutive NO synthase was observed immediately after administration of lipopolysaccharide, reaching a plateau (145 ± 18 nmol/L) after 540 ± 25 s. The plateau was followed by a decrease in NO concentration and its subsequent gradual increase after 45 min because of NO production by inducible NO synthase. The concentration of superoxide increased from 16 ± 2 nmol/L to 30 ± 3 nmol/L after 1 h and reached a plateau of 41 ± 4 nmol/L after 6 h. In contrast to the periodic changes in the concentration of diffusible NO, the total concentration of NO measured as a sum of nitrite and nitrate increased steadily during the entire period of endotoxemia, from 2.8 ± 0.2 µmol/L to 10 ± 1.8 µmol/L.
Conclusions: The direct measurement of NO concentrations in the rat pulmonary artery demonstrates dynamic changes throughout endotoxemia, which are related to the production of superoxide and the subsequent increase in peroxynitrite. Monitoring endotoxemia with total nitrate plus nitrite is not sensitive to these fluctuations in NO concentration.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NO is generated by two constitutive NOS (cNOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS), which control relaxation of smooth muscles and modify central and peripheral nervous system function. Dysfunction of cNOS is associated with reduced production of NO, which leads to severe ischemia/reperfusion injury, hypertension, and hypercholesterolemia (7). In contrast, excessive and continuous production of NO by iNOS over prolonged periods contributes significantly to circulatory failure and hypotension as observed during endotoxemia (8). Several endotoxins activate the transcription and translation of iNOS, the third NOS isoform (9). This induction of iNOS occurs mainly in leukocytes, macrophages, hepatocytes, cardiac myocytes, and, to a lesser degree, in vascular smooth muscle (8). Induction of iNOS at the site of infection is responsible for the massive and uncontrolled generation of NO, which occurs during endotoxemia (2)(6).
Cytotoxicity is often directly attributed to NO. However, NO is only
moderately reactive and shows low cytotoxicity at physiologic
concentrations (10). The major decay route for NO in
mammalian systems is by its extremely rapid reaction with superoxide
(O2-) to form peroxynitrite
(OONO-; Fig. 1
). It is one of the fastest reactions in mammalian physiology
(K = 6.7 x 109
L · mol-1 · s-1)
(11). The final product (peroxynitrite) is usually harmless.
However, after protonation (pKa =
6.8), peroxynitric acid is formed, which, at high concentrations, is
very cytotoxic. Peroxynitric acid undergoes either homolytic cleavage
to form the hydroxyl free radical (OH·)
and the nitrogen dioxide free radical
(NO2·) or heterolytic
cleavage to form the nitronium cation
(NO2+) and the hydroxide anion
(OH-) (12). These three extremely
reactive moieties (OH·,
NO2·, and
NO2+) are most likely the source
of the observed cytotoxicity erroneously attributed to NO
(13).
|
The total concentration of NO during endotoxemia has been estimated by the determination of the concentration of NO decay products (NO2- + NO3-). This indirect approach has demonstrated a significant increase in the concentration of NO2- + NO3- during endotoxemia as measured in a variety of body fluids (9)(14)(15). However, these measurements provide no information concerning diffusible active NO, which is capable of activating smooth muscle relaxation. The diffusible NO plays a crucial role in hypotension during endotoxemia. Therefore, the indirect measurement of NO degradation products (NO2- + NO3-) may provide very misleading information concerning the degree of vasorelaxation within the cardiovascular system.
This report describes both direct electrochemical measurements of the diffusible concentration of NO (active NO concentration) and indirect measurements of the NO final decay products (total amount of NO produced is expressed as a sum of NO2- and NO3- concentration) during endotoxemia in an animal model. These results were correlated with chemiluminescence measurements of O2- and peroxynitrite, which are generated simultaneously with NO during endotoxemia.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
surgery
Rats were anesthetized with an intraperitoneal injection of a
mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. They were
intubated and ventilated with room air using a Harvard small animal
ventilator set at a tidal volume of 2.5 mL and a breathing rate of 100
breaths/min. The porphyrinic NO sensor was placed in a pulmonary artery
wall during the surgery, and its exact localization was determined
during postmortem examination. Polyurethane catheters were inserted
into the left jugular and left femoral veins for drug administration.
The endotoxin lipopolysaccharide [(LPS); Escherichia coli
serotype 0127:B8; Sigma] was dissolved in saline and subsequently
administered as an intravenous infusion (2
mg · kg-1 · min-1
for 10 min; total dose, 20 mg/kg).
NG-Monomethyl-L-arginine
(L-NMMA), a nonspecific cNOS and iNOS inhibitor,
was purchased from Sigma Chemical, and
L-N6-(1-iminoethyl)-lysine
(L-NIL), a selective iNOS inhibitor, was from LC
Laboratories Co. Each of the inhibitors was dissolved in 0.1 mol/L
phosphate buffer (pH 7.4) and then injected as an intravenous bolus (20
mg/kg) 45 min before the start of the LPS infusion. Another catheter
was placed in the right carotid artery for intermittent blood sampling.
preparation of pulmonary artery for in vitro measurements
The isolated pulmonary artery was placed in modified Hank’s
balanced salt solution (HBSS; Sigma) at 4 °C and pH 7.4. The
composition of HBSS is as follows: NaCl (137 mmol/L), Tris-HCl (10
mmol/L), MgCl2 (1 mmol/L), KCl (5 mmol/L),
CaCl2 (1.8 mmol/L), MgSO4
(0.8 mmol/L), KH2PO4 (0.44
mmol/L), Na2HPO4 (0.33
mmol/L), and L-arginine (0.1 mmol/L). Under the dissection
microscope (Wild M3G; Leica), the pulmonary artery was cleared of
adjacent tissue and cut into small segments (2–3 mm). When
polyethylene glycol–superoxide dismutase (PEG-SOD; Sigma) was used, it
was added to HBSS at a dose of 100 kU/L of medium 10 min before
injection of the calcium ionophore (A23187).
preparation of no sensors for in vitro and in vivo measurements
Two types of NO sensors were used for the in vivo and in vitro
experiments. Their design was based on previously developed and
well-characterized, chemically modified carbon-fiber technology
(16)(17)(18). The porphyrinic sensor is free of interference
from all reagents used in these experiments and from substances that
may have been found in mammalian blood to concentrations at least two
orders of magnitude greater than their expected concentrations. The NO
sensor is also insensitive to blood pressure changes (19)
and has a small thermal coefficient (1.8% of signal/°C).
Additionally, the sensor is free of piezoelectric noise and is not
affected by induced currents generated by contracting muscles of the
cardiac systems (16)(20). The catheter-protected
porphyrinic NO sensor for in vivo measurements was mounted on the tip
of a truncated needle. Briefly, the needle from an intravenous catheter
unit, 24 gauge and 25 mm long (Angiocath; Becton Dickinson), was
roughened along the shaft and then truncated and polished flat, so that
it was 3 mm shorter than its 24-gauge, protective catheter. A bare
copper wire with a diameter of 0.1 mm and coated with conducting epoxy
and a single carbon fiber (6 µm in diameter; protruding 3 mm) were
mounted inside the hollow needle on the protruding carbon-fiber tip. A
highly conductive polymeric porphyrin was deposited on the protruding
carbon-fiber tip by the use of 0.50 mmol/L nickel(II)
tetrakis(3-methoxy-4-hydroxyphenyl)porphyrin (TMHPPNi) in 0.1 mol/L
NaOH and the cyclic voltammetry deposition technique. After the
deposition process, the carbon fiber tip was immersed three times for
6–7 s in a 10 g/L alcohol solution of Nafion (Aldrich).
For in vivo measurements, smooth muscle tissue was pierced with the catheter needle and positioned at the desired place in the pulmonary arterial wall. When the position of the catheter was secure, the needle was removed and replaced with the porphyrinic sensor. The auxiliary electrodes, platinum and silver/silver chloride (SSCE) electrodes, were placed in contact with the adjacent tissue. For in vitro measurements, an open arterial strip was placed in a thermostated organ chamber and two auxiliary electrodes of the three-electrode system (the platinum as a counter electrode and the silver/silver chloride as a reference electrode) were positioned in the organ chamber near the arterial strip. The porphyrinic sensor without catheter protection was lowered near the surface (5 ± 2 µm) of the previously positioned opened arterial ring with the aid of the dissecting microscope and micromanipulator. Chronoamperometry was used for fast (0.1-s response time) and continuous measurement of the change of NO concentration with time. The data were collected using a PAR model 273 voltammetric analyzer (EG&G Princeton Applied Research) interfaced with an IBM Pentium computer with data acquisition and control software (Research Electrochemistry Software, Ver. 4.30; EG&G Princeton Applied Research). Before and after in vivo and in vitro measurements, each sensor was calibrated using NO calibration solutions at NO concentrations of 2 x 10-9 to 2 x 10-5 mol/L prepared as described previously (21).
NO2- and
NO3- measurements
At the same time as the electrochemical measurements of active NO
in the tissue during endotoxemia, indirect measurements of
NO2- and
NO3- (final products of NO
decay) were performed using the Griess method (22). The
Griess reagent is specific for
NO2-; therefore,
NO3- was reduced to
NO2- by metallic cadmium.
NO2- and
NO3- content was determined as
NO2- from absorbance
measurements at 540 nm using a calibration curve.
superoxide measurements
The concentration of O2-
was determined by a chemiluminescence method (23).
O2- produced chemiluminescence
from lucigenin (bis-N-methylaridinium
NO3-; Sigma), which was
detected by a scintillation counter (Beckmann 6000 LS with a
single-photon monitor). Each tissue sample (0.8–1.5 mg) was immersed
in 2 mL of HBSS adjusted to pH 7.4 at 25 °C. Lucigenin was added to
the solution to a final concentration of 0.25 mmol/L. All measurements
of O2- were made in a similar
manner after a 2-min incubation in HBSS. For the nonbasal determination
of O2-, the 2-min incubation
period was followed by the injection of 20 µL of 1 mmol/L A23187
calcium ionophore. A23187 is a receptor-independent cNOS agonist and is
needed to measure maximal cNOS production of NO. Calibration curves
were constructed on the basis of the photons emitted by the
O2- generated after treatment
of xanthine with xanthine oxidase.
peroxynitrite measurements
A chemiluminescence method was also used to determine the
concentration of peroxynitrite (22). Photon counts were
recorded with a scintillation counter (Beckman 6000LS, with a
single-photon monitor). Each tissue sample (8–10 mg) was immersed in
0.5 mL of HBSS adjusted to pH 7.4 at 25 °C, and then calcium
ionophore A23187 solution was added to a final concentration of 20
µmol/L. Alkaline sodium bicarbonate luminol solution (1.0 mL) was
added after 3 s to achieve a final concentration of 400 µmol/L
luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and 50 mmol/L
bicarbonate at pH 10.0. The basal concentration of peroxynitrite was
obtained in the same manner in the absence of tissue.
statistical analysis
Values were expressed as means ± SE, with P
<0.05 considered statistically significant. Statistical evaluation was
done by ANOVA followed by unpaired Student t-test. All
analyses were performed with the statistical software Microcal Origin
(Microcal Software, Inc.).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). After 3 h of endotoxemia, a statistically significant
increase in the total concentration of
NO2- plus
NO3- was observed, which
reached a plateau of 10 ± 1.8 µmol/L after 5 h. The
plateau concentration was approximately fourfold higher than the basal
concentration.
|
However, the direct, in vivo electrochemical measurement of NO using
the porphyrinic sensor implanted into the rat pulmonary artery clearly
showed two phases of NO release during endotoxemia (Fig. 3a
). In the early acute phase, after administration of 20 mg/kg
LPS, a sharp increase in the production of NO above its basal
concentration of 52 ± 10 nmol/L was observed. The rate of
increase for NO was 0.19
nmol · L-1 · s-1.
After 540 ± 25 s, the NO concentration reached a plateau of
145 ± 18 nmol/L above the basal concentration that persisted for
4 ± 0.6 min. The plateau was followed by a decrease in the
concentration of NO at the rate of -0.07
nmol · L-1 · s-1.
After 45 min, a second increase in the concentration of NO was
observed, but at a much slower rate of <0.02
nmol · L-1 · s-1.
A second plateau of NO concentration was established 90 ± 10 min
after administration of LPS. The selective inhibitor of iNOS,
L-NIL, at the dose of 20 mg/kg decreased the NO
concentration only during the second phase of endotoxemia (
5 min
after LPS administration; Fig. 3B
). Administration of the
L-NMMA (20 mg/kg) nonselective inhibitor of eNOS, nNOS, and
iNOS decreased the concentration of NO during both the acute phase and
the second phase of endotoxemia (Fig. 3C
).
|
To estimate the potential maximum concentration of NO produced by eNOS
during endotoxemia, in vitro changes in endothelial NO concentration
were measured in freshly excised pulmonary artery after stimulation
with 10 µmol/L calcium ionophore A23187 (Fig. 4
). The single carbon-fiber porphyrinic sensor was placed close
(5 ± 2 µm) to the surface of the endothelial cells in an
excised segment of pulmonary artery, and NO concentrations were
recorded at different times after the administration of LPS. To
estimate the influence of O2-
on calcium-stimulated endothelial NO release, NO was measured after
endotoxemia in the presence or absence of 100 kU/L PEG-SOD
(membrane-permeable SOD). The basal concentration of NO was 45 ±
5 and 27 ± 8 nmol/L in control and endotoxic tissue,
respectively. In the presence of PEG-SOD, the basal concentration
significantly increased in endotoxic (40 ± 3 nmol/L), but not in
the control (48 ± 5 nmol/L). NO release stimulated by calcium
ionophore in the controls was increased by 15% from 437 ± 6
nmol/L to 504 ± 7 nmol/L in the presence of PEG-SOD. However, in
endotoxic artery, the concentration of NO increased by 50% in the
presence of PEG-SOD (increase from 262 ± 3 nmol/L to
547 ± 5 nmol/L; P <0.001; n = 6).
|
Endothelial NO production was substantially decreased during the first
3 h of endotoxemia (Figs. 4
and 5
) because of the increased generation of
O2-. The increase of
O2- was confirmed indirectly
with an experiment performed in the presence of PEG-SOD. In the
presence of PEG-SOD, the NO concentration was restored to the
concentration observed before endotoxemia. The maximal production of NO
by endothelium during different phases of endotoxemia was determined by
activating cNOS with the receptor-independent agent A23187 (Fig. 5
;
n = 6).
|
superoxide generation
The previous experiments using PEG-SOD suggest that the increase
in the production of O2- during
endotoxemia is responsible for the decrease in the concentration of NO
(Figs. 4
and 5
). During the time period (60 min) of endotoxemia, there
was no significant decrease in the eNOS protein as determined by
Western blot. Therefore, the decrease of the production of NO is
attributable solely to an increase in the generation of
O2-. To confirm this
hypothesis, a chemiluminescence method was used to directly measure the
concentration of O2- during
endotoxemia in freshly isolated tissue. The mean basal concentration
(before endotoxemia) of O2- in
the pulmonary artery was 16 ± 2 nmol/L (Fig. 6
). The change in the total concentration of
O2- with time after LPS
administration was measured after the maximal stimulation of NO release
by A23187 (Fig. 6
). One hour after LPS administration, the
concentration of O2- increased
to 30 ± 3 nmol/L, reaching a plateau of 41 ± 4 nmol/L after
6 h (Fig. 6
; n = 6). In the presence of L-NMMA,
the concentration of O2-
decreased 60%. The decrease in O2-
concentration is similar to the decrease in the NO concentration in the
presence of L-NMMA.
|
peroxynitrite production
Peroxynitrite release was measured in vitro during the maximal
stimulation of the release of NO by A23187 (Fig. 7
), and it paralleled the release of
O2- (Fig. 6
) throughout
endotoxemia. After 1 h, the concentration of peroxynitrite
increased approximately twofold compared with the control. A plateau
was established after 3 h of endotoxemia. The maximum
concentrations of NO, O2-, and
peroxynitrite before and 3 h after LPS administration are shown in
Fig. 7
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The first
increase in the concentration of NO occurred 10 min after the
administration of LPS and was attributable to the activation of eNOS. A
similar pattern of NO release is observed after stimulation of
endothelial cells with typical agonists of NO, such as bradykinin or
acetylcholine. The second increase in the diffusible concentration of
NO was observed 1 h after the onset of endotoxemia and was a result
of the activation of iNOS. These direct electrochemical measurements of
NO differ markedly from the indirect estimation of total NO by
spectroscopic measurements of
NO2- and
NO3- concentrations. This
indirect measurement of degradation products revealed a steady increase
in the concentration of NO with time during endotoxemia (Fig. 2
).
Therefore, the distinct biphasic temporal fluctuations detected in the
concentration of biologically active NO by the porphyrinic microsensor
were not detected with the less sensitive and more indirect method of
measuring NO degradation products. The in vivo measurement of NO may be
performed with the use of a modified porphyrinic microsensor placed in
a catheter or needle, which can be located in tissue or in circulating
blood for the direct determination of NO (17). Although
technically more demanding, this method provides a precise measurement
of fluctuations of NO concentrations with time. The measurement of NO and its metabolites in septic patients poses a challenge to the clinical laboratory. The indirect spectroscopic measurement of NO2- and NO3-, indicators of NOS activity and the total production of NO, is based on the detection of the degradation products of NO in plasma or urine (13)(22). However, experimental evidence supporting the validity of this assumption is limited. In a recent study, mice lacking the eNOS gene had 40% less plasma NO2- and NO3- compared with wild-type mice (24). Thus, the absence of eNOS leads to a reduction in plasma NO2- and NO3- not compensated by other NOS isoforms. However, this measurement still fails to account for temporal variations in concentrations of NO in plasma and specific organs (2).
The evaluation of NO2- and
NO3- in urine also has
important limitations. The rate of excretion of
NO2- and
NO3- in the urine is directly
related to renal function (25). NO is generated from the
following reaction: L-arginine + NADPH +
O2 
NG-hydroxyl-L-arginine
citrulline + NO + NADP+ (26). The cellular
elements in infected urine contain an increased amount of NO generated
by iNOS compared with uninfected control specimens. The iNOS represents
an endogenous source of NO2-
production in the urine specimen (27)(28). Under
appropriate physiological conditions, NO and, therefore, its
degradation products, NO2- and
NO3-, can be produced
nonenzymatically (29)(30)(31). Hydrogen peroxide generates NO
from D- or L-arginine by a
nonenzymatic pathway (30). The
O2- anion oxidizes the
intermediate,
NG-hydroxy-L-arginine,
to NO (30)(31). Therefore, inhibition of cNOS
and/or iNOS activity, increased in septic shock (32), will
not completely stop NO production because the nonenzymatic synthetic
pathways would not be altered. In acidic urine, some
NO2- is converted to NO
(33). A variety of disease states lead to increased or
decreased total production of NO and, therefore, alterations in
NO2- excretion in the urine
(22)(34)(35)(36). Consequently, the concentration of
NO2- and
NO3- in urine is not only an
indicator of a urinary tract infection, but also a monitor of the total
body production of NO
(13)(22)(33)(34)(35)(36).
The first phase of NO release during endotoxemia is catalyzed by
calcium-dependent eNOS, which is activated by an increase in unbound
cytosolic free calcium. Inhibition of calcium flux into the cell
inhibits NO release (1)(7). LPS activates
intracellular calcium gradients, which stimulate eNOS to produce NO,
water, and L-citrulline from
L-arginine and O2 (8)
(Fig. 1
). To estimate maximal cNOS activity at different times after
the induction of endotoxemia, we used the calcium ionophore A23187 to
increase intracellular calcium influx (
Figs. 4–7
). Concurrently,
during this first phase of NO release, the concentration of
O2- also increased (Fig. 6
).
This LPS-stimulated production of
O2- is inhibited by the
presence of the nonspecific NOS inhibitor L-NMMA. The
generation of O2- occurs during endotoxemia
after there is a local depletion of L-arginine of
<10-4 to 10-3 mol/L. At
this low concentration of L-arginine, there is an
insufficient concentration gradient for this substrate to bind to the
active site of cNOS (37). In the absence of
L-arginine or tetrahydrobiopterin, cNOS function
expands to the catalysis of two reactions: the one-electron reduction
of O2 to
O2- and the five-electron
oxidation of L-arginine to NO. When these two
reactions take place in close proximity, the extremely rapid reaction
of O2- with NO produces
peroxynitrite, which, after protonation, rearranges to form the stable
product NO3- (9).
Using isolated enzymes, Klatt et al. (38) found that both
eNOS and nNOS, but not iNOS, can produce
O2- in
L-arginine- or tetrahydrobiopterin-limiting
environments. Therefore, the decrease in the production of NO observed
throughout endotoxemia (Fig. 5
) can be attributed to the increased
production of O2-. eNOS appears
to be a major source of O2-.
However, in endothelial cells, there are several other sources of
O2- generation, including
prostaglandin metabolism, cytochrome P450, and processes stimulated by
protein kinase C or xanthine oxidase. Therefore, the net concentration
of NO measured by the porphyrinic sensor depends not only on NO
generated by cNOS, but also on the concentration of
O2-. The increasing
concentration of O2- reacts
with NO to produce peroxynitrite, which leads to a reduction in
bioactive NO (Fig. 5
), not a steady increase in NO as suggested by the
determination of NO2- and
NO3- (Fig. 2
).
In conclusion, the indirect measurements of NO using the concentration of NO2- and NO3- during endotoxemia fails to provide an accurate release of bioactive NO. Because of the fast consumption of NO by O2-, the actual concentration of active NO in the cardiovascular system is relatively low, and this concentration can be monitored only by using direct, real-time measurement.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 On sabbatical leave from: Department of Biochemistry, Medical
University of Gdansk, and Laboratory of Cellular and Molecular
Nephrology, Medical Research Center of the Polish Academy of Science,
80-210 Gdansk, Poland.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  G. Natarajan, M. Glibetic, V. Raykova, J. P Ofenstein, R. L Thomas, and J. V Aranda Nitric Oxide and Prostaglandin Response to Group B Streptococcal Infection in the Lung Ann. Clin. Lab. Sci., January 1, 2007; 37(2): 170 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Altunoluk, H. Soylemez, F. Oguz, E. Turkmen, and E. Fadillioglu An Angiotensin-Converting Enzyme Inhibitor, Zofenopril, Prevents Renal Ischemia/Reperfusion Injury in Rats Ann. Clin. Lab. Sci., January 1, 2006; 36(3): 326 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Cagli, C. Bagci, M. Gulec, B. Cengiz, O. Akyol, I. Sari, S. Cavdar, S. Pence, and H. Dinckan In Vivo Effects of Caffeic Acid Phenethyl Ester on Myocardial Ischemia-Reperfusion Injury and Apoptotic Changes in Rats Ann. Clin. Lab. Sci., October 1, 2005; 35(4): 440 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Bateman, C. G. Ellis, and D. J. Freeman Optimization of Nitric Oxide Chemiluminescence Operating Conditions for Measurement of Plasma Nitrite and Nitrate Clin. Chem., March 1, 2002; 48(3): 570 - 573. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and peroxynitrite (
) were measured after the
maximal stimulation of NO release with calcium ionophore (A23187; 10
µmol/L) after incubation with LPS (20 mg/kg; n = 6). Superoxide
was measured without (•) and with the addition of L-NMMA (
) and after 3 h of LPS (20 mg/kg) administration (
).