J Appl Physiol
90: 317–320, 2001.
A new method to measure nitrate/nitrite
with a NO-sensitive electrode
REINHARD BERKELS, SVENJA PUROL-SCHNABEL, AND RENATE ROESEN
Institut fuer Pharmakologie, Universitaet zu Koeln, 50931 Koeln, Germany
Received 22 December 1999; accepted in final form 11 August 2000
There are different methods of determining NO, which
have been described in detail elsewhere (1). Briefly, it
is possible to measure NO by using a bioassay (6), an
oxyhemoglobin assay (10), electron paramagnetic resonance (7), chemiluminescence (15), HPLC (11), the
Griess reaction (17), or different electrochemical electrodes (3, 13, 19). Moreover, it is possible to measure
NO indirectly via its second messenger, cGMP.
Up until now, the Griess reaction has been used to
measure nitrite/nitrate, although the detection limit of
this technique is in a micromolar range, which is usually only reached by induction of the inducible NOS.
This study presents a new, sensitive method to determine nitrite/NO with a commercially available electrochemical NO electrode (WPI, Sarasota, FL). We will
describe that it is possible to measure NO in aqueous
solutions from different biological models by a conversion of nitrite to NO. A big advantage of this method is
that the total (basal and agonist-induced) NO release
of cells in culture can be measured because the stable
degradation products of NO (nitrate/nitrite) accumulate in the incubation buffer.
nitric oxide; endothelium; nitrite
METHODS
(NO) is a small, diffusible molecule with a
short half-life (8) with various biological functions (4,
12, 14). It was first discovered by Furchgott (5) to be an
endothelium-derived relaxing factor, which was identified as being identical to NO by Palmer et al. (15) and
Ignarro et al. (9) in 1987. Up until now, three different
isoforms of NO synthases (NOS) have been identified
(4, 14). The neuronal NOS, which is calcium/calmodulin dependent, produces NO that acts as a neurotransmitter. After stimulation, macrophages express large
amounts of a calcium/calmodulin-independent NOSreleasing NO in high concentrations for bactericidal
purposes. The constitutive endothelial NOS causes
vascular relaxation via cGMP-mediated mechanisms,
and, moreover, albuminally released NO inhibits platelet aggregation and leukocyte adhesion (14). Because
NO has a short half-life (2–30 s) (8), it is difficult to
measure authentic NO. It rapidly decomposes to nitrate and nitrite, which may accumulate in the sample.
NITRIC OXIDE
Address for reprint requests and other correspondence: R. Berkels,
Institut fuer Pharmakologie, Gleueler Str. 24, 50931 Koeln, Germany (E-mail: [email protected]).
http://www.jap.org
Calibration of the NO electrode. The amperometric
ISO-NO electrode (WPI) measures NO by oxidizing it at the
working electrode. The resulting current is displayed in picoamperes and is proportional to NO concentrations (Fig. 1).
To prevent nitrate and nitrite from interfering with the
electrode, it is coated with a specialized membrane (WPI);
other diffusable gases such as CO are not measured because
of the potential at the electrode. The electrode was calibrated
by using a modification of the protocol (3, 19) given by the
manufacturer. A chemical titration calibration was performed by using an acidic iodide solution (0.1 mol/l H2SO4,
0.14 mol/l K2SO4, 0.1 mol/l KI), to which equal volumes of
different KNO2 solutions were added (Figs. 2 and 3). NO is
formed stoichiometrically and is measured directly. Briefly,
500 ␮l of KNO2 were added to 10 ml of the acidic solution at
room temperature while being stirred (100 rpm). The same
calibration was performed with increasing volumes of a
KNO2 solution of a definite concentration (5 ␮mol/l). Instantly the nitrite is reduced to NO, which is constantly
measured by the electrode that is placed in the solution. The
detection limit of the electrode is 2 nmol/l NO (2, 3) in an
aqueous solution, which means that a minimum of 50 ␮l of a
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
317
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
Berkels, Reinhard, Svenja Purol-Schnabel, and Renate Roesen. A new method to measure nitrate/nitrite with
a NO-sensitive electrode. J Appl Physiol 90: 317–320,
2001.—There are different methods to measure the unstable
molecule nitric oxide (NO). We will describe a new sensitive
method to measure NO by reconversion of nitrate/nitrite to
NO, which will be determined with an amperometric Clarktype electrode. Nitrate and nitrite are the degradation products of NO. First, nitrate is enzymatically converted to nitrite
with the use of the nitrate reductase. Second, nitrite is
reduced to equimolar NO concentrations by an acidic iodide
solution. The detection limit of the electrode in an aqueous
solution was 2 nmol/l NO (meaning the threshold was depending on the volume added: 500 ␮l of a 0.2 ␮mol/l nitrite
solution added to a 10-ml bath). This method provides the
ability to assess basal and agonist-stimulated NO releases of
different biological models. We measured basal and carbachol-stimulated NO release of native endothelial cells from
porcine coronary arteries and porcine aortic endothelial cell
cultures. Moreover, it was possible to measure the nitrate/
nitrite concentration in the coronary effluent of a guinea pig
heart. In conclusion, we present a valid, highly sensitive new
method of measuring nitrite/NO in biological systems with a
commercially available electrode.
318
NO MEASUREMENT BY CONVERSION OF NITRATE/NITRITE
Fig. 1. Electric current [picoampere (pA)] induced by the reduction of
nitric oxide (NO) at the electrode is directly proportional to the NO
concentration (nmol/l).
Fig. 2. Typical original recording of a calibration with KNO2 in
which increasing concentrations of KNO2 (500 ␮l) were added to a
10-ml solution (stirred, 100 rpm) in which the electrode was placed.
Cell culture of porcine aortic endothelial cells. Porcine
aortic endothelial cells (PAEC) were harvested and cultured
as described elsewhere (3). Briefly, porcine aorta were obtained fresh from a local abattoir, and the endothelial cells
were isolated enzymatically and cultured in medium 199
with 10% fetal calf serum. On confluence (2–3 days), the cells
were detached by treatment with trypsin, seeded onto sterile
glass coverslips, and grown to subconfluence (⬇70–80%).
Only cells from passage 2 were used for the experiments. The
purity of the PAEC culture was tested with different immunochemical markers.
Preparation of guinea pig hearts according to Langendorff.
Hearts of adult guinea pigs were prepared as previously
described (16). Briefly, the guinea pigs were heparinized
(1,000 IU heparin/kg), and the hearts were excised. They
were perfused at a constant perfusion pressure of 60 mmHg
with an oxygenated Tyrode solution at 37°C. The hearts were
beating spontaneously, and the left ventricular pressure was
recorded isovolumetrically.
Measurement of the nitrite/NO production. The vessel
pieces (30–50 mg wet wt) were fixed on a plastic rod with the
endothelium outside (3-mm diameter) and incubated for 30
min in 500 ␮l of HEPES buffer (in mmol/l: 5 HEPES, 140
NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 glucose, adjusted to a
pH of 7.4) at 25°C (without stirring). The incubate was then
treated with 50 ␮l of a NADPH-FAD mixture (1.5:0.03 mg/
ml) and 1 min later with 15 ␮l of nitrate reductase solution
(10 U/ml; Roche; final concentrations of 0.3 U/ml nitrate
reductase). The resulting mixture was incubated at room
temperature for another 30 min. The nitrite/NO concentration was estimated as described under Calibration of the NO
electrode.
Endothelial cell cultures were washed two times with
HEPES buffer and incubated for 120 min at 37°C. Defined
volumes (500 ␮l) of the supernatant (1.5 ml of HEPES buffer)
of PAEC cell cultures (10-cm dishes) were taken and treated
the same way as described above. The coronary effluent of the
guinea pig hearts was sampled for nitrite/NO analysis as
described above. If the nitrite concentration of the samples,
especially from the native coronary vessels, was out of our
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
solution that contains 2 ␮mol/l nitrite has to be added to the
10-ml bath to be detectable. Therefore, in an experiment, the
added nitrite sample is diluted according to its volume in the
10-ml bath, resulting in a much lower concentration. It is
important for the volume of the sample containing nitrite to
be greater than the detection limit in the 10-ml bath. It is
possible to add solutions up to a volume of 500 ␮l (single
addition) to the 10-ml solution without disturbing the measurement. When performing an experiment, we did not add
more than 1 ml (total sum of serial injections) into the 10-ml
bath; afterward we replaced the bath with a fresh solution to
perform further experiments. We always took the peak currents to quantify our values.
Preparation of porcine coronary arteries. Coronary arteries
of female pigs were prepared immediately after excision of
the hearts and stored in oxygenated Tyrode solution. Special
care was taken to preserve the endothelium during removal
of fat and connective tissue. The artery was cut into rings of
0.8 cm (30–50 mg wet wt, 2-mm diameter), opened longitudinally, fixed on customized needles, and placed in an organ
bath.
Fig. 3. Calibration curve with increasing KNO2 concentrations (500
␮l), which were added to a 10-ml bath. Linear regression yielded a
well-correlated straight line. Thin lines represent the 95% confidence
interval; arrows show the detection limit of a colorimetric assay and
a chemiluminescent Griess assay (data according to the manufacturers).
NO MEASUREMENT BY CONVERSION OF NITRATE/NITRITE
Fig. 4. Segments (10 mg) of porcine coronary vessels showed a
time-dependent basal release of nitrite/NO. Values are means ⫾ SE;
n ⫽ 4 segments.
RESULTS
Calibration. A calibration with a KNO2 solution (500
␮l) in increasing concentrations showed a concentration-dependent linear increase in NO with a detection
limit of 2 nmol/l in the 10-ml bath (Figs. 2 and 3).
Increasing volumes of a fixed concentration of KNO2
resulted in a linear signal too (2). A sharp increase in
NO was detected after application of the KNO2 solution, which slowly decreased down to basal levels,
depending on the height of the signal (Fig. 2). The
baseline stability did not change when several reductants were injected (up to 8 times). The detection limit
using our method was 0.2 ␮mol/l nitrite in 500 ␮l and,
therefore, was lower than the detection limit of a colorimetric assay, as well as a chemiluminescent Griess
assay (detection limits according to the manufacturers,
Roche and R&D) (Fig. 3).
Additionally, we could show in our system that the
spontaneous NO donor S-nitroso-N-acetyl-penicillamine (freshly prepared) released NO in a dose-dependent manner (data not shown). The buffer (HEPESbuffered saline solution) did not show any NO signals.
Addition of cysteine up to a concentration of 1 mmol/l
did not alter the recovery of nitrite. The same refers to
solutions of protein (BSA; up to 1 mg/ml). There was no
significant difference between the calibration curves of
solutions with or without proteins and CysSH (data not
shown).
Measurement of NO from native vessels. Isolated
porcine coronary arteries released nitrate/NO in a
time-dependent manner (Fig. 4) (n ⫽ 4). We standardized our experiments to a 30-min incubation of the
vessels. Basal nitrite/NO levels (per ring) released by
the constitutive NOS after 30 min of incubation were
350 ⫾ 17 pmol 䡠 10 mg⫺1 䡠 30 min⫺1 (n ⫽ 25) (Fig. 5).
Additionally, we induced an enhanced NO release of
the vessels by using carbachol as a conventional ago-
nist. After a period of 30 min, carbachol (1 ␮mol/l)
significantly elevated nitrite/NO levels (⫹50 ⫾ 9%; n ⫽
6; Fig. 5). The basal and the carbachol-induced NO
formation could be blocked by the NOS inhibitor NGnitro-L-arginine methyl ester (0.1 mmol/l), which
shows the specificity of our measurements (data not
shown).
Measurement of NO from endothelial cell cultures.
Cultured endothelial monolayers showed a basal NO
release of 186 ⫾ 17 pmol 䡠 10 cm⫺2 䡠 30 min⫺1. We used
shear stress to induce an increased NO formation,
which resulted in stimulation of the NO release (658 ⫾
23 pmol 䡠 10 cm⫺2 䡠 30 min⫺1; n ⫽ 3). To compare our
method with conventional Griess assays, we performed
measurements with a colorimetric assay (Roche) and a
chemiluminescent assay (R&D). We were not able to
detect nitrite in the supernatant of endothelial cell
cultures with these methods; thus it seemed that the
sensitivity in these systems was too low.
Measurement of NO in heart perfusate. The effluent
of a Langendorff-perused guinea pig heart was analyzed to measure nitrite/NO. The electrode was sensitive enough to monitor basal NO release after 30 min of
equilibration in the effluent. The basal NO concentration was 700 pmol/min at a flow of 11 ml/min.
Fig. 5. Original recording shows the nitrite/NO peak elicited by 100
␮l of the incubate solution in which the vessel pieces had been placed
(30 min). This aliquot was added to 10 ml of the acidic iodide
solution. After incubation with the agonist carbachol (1 ␮mol/l)
under the same conditions, the nitrite/NO content increased significantly.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
calibration curve (too high), we reduced the added volume
accordingly.
319
320
NO MEASUREMENT BY CONVERSION OF NITRATE/NITRITE
REFERENCES
We could show that it is possible to measure basal
and agonist-induced NO release of endothelial cells
with an electrochemical electrode by converting nitrite
back to NO. This method is very sensitive, with a
detection limit of 2 nmol/l, which means that, in our
system, a sample (500 ␮l) with a nitrite content of 0.2
␮mol/l would be detected. It even provides the ability to
measure NO in heart perfusate. The method is based
on the conversion of nitrite to NO in acidic iodide
solutions, which can be detected by the electrode. Nitrite is one of the two biological degradation products of
NO. The other product is nitrate. The enzymatic conversion of nitrate to nitrite is necessary to measure the
total amount of NO formed by the cells because the
iodide solution is not strong enough to reduce nitrate to
NO. Without a conversion, significantly lower levels of
NO are detected (data not shown).
Because the endothelial cells are incubated for a
fixed time period, we cannot monitor the actual NO
release online at a defined time, which is possible with
the electrode (3, 19). On the other hand, it is a big
advantage to be able to measure basal NO release,
which is hardly possible if the electrode is placed above
the endothelial cells and NO is measured directly,
because in this case the basal NO liberation is incorporated into the baseline and only a stimulated NO
release can be detected. Additionally, the total NO
amount formed would be obtained, even if the NO is
inactivated by oxygen radicals, because, at the end,
nitrite will be generated. Compared with the Griess
reaction, the typical assay used to measure nitrite
and nitrate, our detection limit is significantly lower
(Fig. 3) (18). Moreover, the Griess reaction might
interfere with thiols, proteins, and plasma constituents, whereas our methods seems not to be affected
up to very high unphysiological concentrations (18).
The chemiluminescent NO assay offers a detection
limit of the same range but is significantly more
expensive. Additionally, because nitrate and nitrite
are stable compounds, one does not need to worry
about the short half-life of NO, and it is possible to
freeze biological samples and measure them later.
Additionally, the volume that is needed to perform a
measurement is low (50–500 ␮l), and the time to
perform a measurement is short. To work with this
highly sensitive electrode, it is necessary to ground
the experimenter and the recording devices thoroughly, otherwise small changes in electric currents
will interfere with the recording. The same refers to
changes in pH, flow, and temperature.
In summary, we are presenting a highly sensitive
method to measure NO in biological samples by conversion of nitrite with a commercial NO electrode.
1. Archer S. Measurement of nitric oxide in biological models.
FASEB J 7: 349–360, 1993.
2. Berkels R, Bertsch A, Breitenbach T, Klaus W, and Roesen
R. The calcium antagonist nifedipine stimulates endothelial NO
release in therapeutical concentrations. Pharm Pharmacol Lett
6: 75–78, 1996.
3. Berkels R, Mueller A, Roesen R, and Klaus W. Nifedipine
and Bay K 8644 induce an increase of [Ca2⫹]i and NO in endothelial cells. J Cardiovasc Pharmacol Ther 4: 175–181, 1999.
4. Forstermann U, Closs EI, Pollock JS, Nakane M, Schwarz
P, Gath I, and Kleinert H. Nitric oxide synthase isozymes.
Characterization, purification, molecular cloning, and functions.
Hypertension 23: 1121–1131, 1994.
5. Furchgott RF and Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373–376, 1980.
6. Gryglewski RJ, Moncada S, and Palmer RM. Bioassay of
prostacyclin and endothelium-derived relaxing factor (EDRF)
from porcine aortic endothelial cells. Br J Pharmacol 87: 685–
694, 1986.
7. Henry Y, Ducrocq C, Drapier JC, Servent D, Pellat C, and
Guissani A. Nitric oxide, a biological effector. Electron paramagnetic resonance detection of nitrosyl-iron-protein complexes
in whole cells. Eur Biophys J 20: 1–15, 1991.
8. Ignarro LJ. Endothelium-derived nitric oxide: actions and properties. FASEB J 3: 31–36, 1989.
9. Ignarro LJ, Buga GM, Wood KS, Byrns RE, and Chaudhuri
G. Endothelium-derived relaxing factor produced and released
from artery and vein is nitric oxide. Proc Natl Acad Sci USA 84:
9265–9269, 1987.
10. Kelm M, Feelisch M, Spahr R, Piper HM, Noack E, and
Schrader J. Quantitative and kinetic characterization of nitric
oxide and EDRF released from cultured endothelial cells. Biochem Biophys Res Commun 154: 236–244, 1988.
11. Kelm M, Preik-Steinhoff H, Preik M, and Strauer BE.
Serum nitrite sensitively reflects endothelial NO formation in
human forearm vasculature: evidence for biochemical assessment of the endothelial L-arginine-NO pathway. Cardiovasc Res
41: 765–772, 1999.
12. Lane P and Gross SS. Cell signaling by nitric oxide. Semin
Nephrol 19: 215–229, 1999.
13. Malinski T, Mesaros S, Patton SR, and Mesarosova A.
Direct measurement of nitric oxide in the cardiovascular system.
Physiol Res 45: 279–284, 1996.
14. Moncada S, Palmer RM, and Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43:
109–142, 1991.
15. Palmer RM, Ferrige AG, and Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived
relaxing factor. Nature 327: 524–526, 1987.
16. Rosen R, Marsen A, and Klaus W. Local myocardial perfusion
and epicardial NADH-fluorescence after coronary artery ligation
in the isolated guinea pig heart. Basic Res Cardiol 79: 59–67,
1984.
17. Thomsen LL, Ching LM, and Baguley BC. Evidence for the
production of nitric oxide by activated macrophages treated with
the antitumor agents flavone-8-acetic acid and xanthenone-4acetic acid. Cancer Res 50: 6966–6970, 1990.
18. Tsikas D, Gutzki FM, Rossa S, Bauer H, Neumann C,
Dockendorff K, Sandmann J, and Frolich JC. Measurement
of nitrite and nitrate in biological fluids by gas chromatographymass spectrometry and by the Griess assay: problems with the
Griess assay–solutions by gas chromatography-mass spectrometry. Anal Biochem 244: 208–220, 1997.
19. Tsukahara H, Gordienko DV, and Goligorsky MS. Continuous monitoring of nitric oxide release from human umbilical
vein endothelial cells. Biochem Biophys Res Commun 193: 722–
729, 1993.
Part of this study was supported by a grant from the Köln-Fortune
program of the medical faculty of the University of Cologne (Cologne,
Germany).
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 17, 2017
DISCUSSION