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Pharmaceutica Analytica Acta
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Michael Klepser
Ferris State University
USA
HG Wang
Penn State College of
Medicine, USA
Srinath palakurthi
Jun Zhang
Development
Sciences of Abbott
Laboratories, USA
H Shintani
Chuo University
Japan
VF Samanidou
Texas A&M Health
Science Center, USA
Aristotle University of
Thessalonik, Europe
PS Hiremath
V Vemulapalli
Pharmaceutics
International Inc.,
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Xudong Yuan
Long Island University
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Bonnie A Avery
University of
Mississippi, USA
Mihaela Baniceru
University of Medicine
and Pharmacy
Romania
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The University of
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Josef Ozer
Ajay K Banga
Mercer University
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LEM Quintas
Federal University of
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Anand Iyer
Hampton University
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Lee Jia
Hideaki Hara
Gifu Pharmaceutical
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GS Erzinger
University of the
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Brazil
B Otieno Abonyo
FAI Habeeb
Zhe-Sheng Chen
Mansoura University
Egypt
Jacob Runyan
Virginia
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St. John’s University
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Abdel Halim
Acorda Therapeutics
Inc. USA
Daiichi Sankyo
Pharma
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Peisheng Xu
M F Desimone
Universidad de
Buenos Aires, USA
D Douroumis
Univeristy of
Greenwich, USA
Bodhi Chaudhuri
Αnastasios E
Diaconu Camelia C
National Cancer
Institute/NIH, USA
University of South
Carolina, USA
Syed A A Rizvi
Hosam Elbaz
Elena Garcia F
AR Mohammed
University of
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Alan Myers
Don E Farthing
COPHS, Drake
University, USA
Pfizer Biotech, PGRD
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West Virginia
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Alexander P
Swedish Herbal
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Joseph P Fuhr
Widener University
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Florida A&M University
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Pharmaceutics
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Nova Southeastern
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MRISoliman
Florida A&MUniversity
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Aston University
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Pasquale Maffia
University of Glasgow
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Pharmaceutica
Shintani, Pharm Anal Acta 2013, 4:6
http://dx.doi.org/10.4172/2153-2435.1000251
Analytica Acta
Short Communication
Open Access
Measurements of Nitric Oxide and Peroxynitrite
Hideharu Shintani*
Chuo University, School of Science, 1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan
Keywords: Nitric Oxide; Peroxynitrite; Measurement; Macrophage
The Measurement of NO Produced by Endothelial Cells
Introduction
Protocol
Although there is much interest in NO (nitric oxide) and
peroxynitrite (OONO), their measurement in biological media is
difficult because of their short lifetimes and low concentrations. Here,
the use of an NO-sensitive electrode is first described for direct realtime measurement of NO in endothelial cells. A method for detecting
peroxynitrite produced by the macrophage is also given. Brief
references to other methods are included in the comments sections.
NO measurement with NO-sensitive electrodes
Calibration [1]
Protocol
1. Prepare KH solution (HEPES-buffered modified KrebsHenseleit solution), containing NaCI (137 mM), KCI (1.1
mM), CaCI2・2H2O (0.18 mM), MgCI2・6H2O (0.1 mM),
NaHCO3 (4.2 mM), glucose (5.6 mM), and HEPES (5 mM).
After addition of optional reagents adjust pH to 7.4 with NaOH
(0.1 M).
1. Perfuse bovine aortic endothelial cells confluently cultured on
a cover slip (15 mmφ), with L-arginine solution (L-arg, MW =
174.2, 50 μM).
2. Place NO electrode as close as possible to the cover slip without
making contact. Start recording the basal level of electrode
current and wait until the current has stabilized (ca 30 min).
3. Switch the perfusate to 1 mM ATP/50 mM L-arg solution to
stimulate endothelial cells. ATP (Mr = 551) should be stored
at -20oC.
4. Re-switch the perfusate to 50 μM L-arf solution and confirm
that the current returns to the basal level.
Comments
1. Use a perfusion system as shown in Figure 1. It is important
to use a hydrostatic pressure of ca 40 cm H2O so that the flow
is constant, because fluctuations of the flow rate cause noise
on the electrode current. All perfusate should be prepared by
dissolving reagents in KH solution and adjusting the pH to 7.4.
2. Immerse NO electrode in KH solution (20 mL) and read basal
current after stabilization.
Control
3. Immerse NO electrode in freshly prepared SNAP solution (1
mM, 20 mL) and read NO current after stabilization (waiting
for ca 5 min).
+ATP
glass syringe
recorder
4. Repeat steps 2 and 3 three times and calculates mean NOdependent current.
Although calibration of the electrode can be performed with
authentic NO solution, this calibration is not practical because it is
difficult and troublesome to maintain the buffer solution anaerobic;
and the concentration of authentic NO solution should be determined
by other methods, e.g. the oxyhaemoglobin method [2].
Comments
1. NO measurements can be performed with an NO monitor
(NO-501, lnter Medical, Japan) with an NO-sensitive electrode
(NOE-47, 200 mmφ, Inter Medical, Japan), or with an
isolated nitric oxide meter (ISO-NO Mark II, World Precision
Instruments, USA) with an NO sensor (ISO-NOP 200,
World Precision Instruments, USA). All experiments must
be performed with electromagnetic shields, made of an iron
mesh screen, to avoid external noise. Plastics which are easily
charged with electricity can cause unexpected noise.
2. New electrodes (both working and counter electrodes from
Inter Medical) should be immersed for one day in buffer
solution before use. It takes more than 30 min to stabilize basal
current after first connecting the electrode and turning on the
NO monitor. The electrodes are stored with the tips immersed
in the buffer.
Pharm Anal Acta
ISSN: 2153-2435 PAA, an open access journal
40cmH2O
0.5mm i.d. Teflon tube
head
amp.
NO Monitor
electrode pair
gauge
strip
suction
endothelial cells
Figure 1: A perfusion system for measuring NO generated from endorhelial
vells.
*Corresponding author: Hideharu Shintani, Chuo University, School of
Science, 1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan, Tel: +81425922336;
E-mail: shintani@mail.hinocatv.ne.jp
Received May 20, 2013; Accepted June 17, 2013; Published June 20, 2013
Citation: Shintani H (2013) Measurements of Nitric Oxide and Peroxynitrite.
Pharm Anal Acta 4: 251. doi:10.4172/2153-2435.1000251
Copyright: © 2013 Shintani H. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Volume 4 • Issue 6 • 1000251
Citation: Shintani H (2013) Measurements of Nitric Oxide and Peroxynitrite. Pharm Anal Acta 4: 251. doi:10.4172/2153-2435.1000251
Page 2 of 2
Nitric oxide synthases (NOS) which produce NO in biological
systems consist of three isoforms. Among these NOS-l (neuronal type)
and NOS-3 (endothelial type) transiently produce a relatively small
amount of NO in response to external signalling molecules. To elucidate
these transient NO responses, highly sensitive and real-time methods
such as chemiluminescence methods [3,4] or an electrochemical
method [1] are required. On the other hand, spectrophotometric [5]
and fluorimetric [6] methods are convenient for determining total NO
production from NOS-2, which persistently produces a relatively large
amount of NO. These methods can be applied to assay NO2-, the end
products of NO, accumulated in incubation media for 6 to 48 h.
Quantitation of Peroxynitrite Produced by Macrophages
Protocol
1. Prepare Hank's balanced salt solution (HBSS) by dissolving
HBSS powder (Gibco, 450-1201) and NaHCO3 (0.35 g/L) in
doubly distilled water (pH 7.4).
2. Prepare a stock solution of phorbol・12・myristate-12 acetate
(PMA, Sigma; 1 mg/mL) in DMSO. Divide into 100-mL
aliquots, and store below -80oC (in the deep freeze).
3. Incubate macrophages (8×106 cells/mL) in HBSS containing
PMA (400 ng/mL), 4-hydroxyphenylacetic acid (4・HPA・
MW 152.15; 1 mM) and Cu, Zn-superoxide dismutase (SOD;
0.1 mg/mL) for 4 h at 37oC.
4. Centrifuge the solution (1000 rpm for 10 min) to remove
macrophages.
5. Acidify supernatant with H3PO4 (10%) and add acetonitrile
(final conｭ centration 20% v/v).
6. Pass through a 0. 45-μm membrane filter.
7. Apply the sample to a 4.6 mm×150 mm C-18 (ODS) reversedphase HPLC column equilibrated with 20:80 (v/v) acetonitrilephosphate buffer (pH 3.2, 10%).
8. Elute the column with a linear gradient of 20 to 60% acetonitrile
over 10 min at a flow rate of 1 mL/min.
9. Monitor UV absorption at 360 nm to assay 4-hydroxy-3nitrophenyl.acetic acid (NO2-HPA).
Comments
Peroxynitrite nitrates phenol derivatives in the presence of redox-
active metal complexes such as Fe3+-EDTA (1 mM) and Cu, Zn-SOD
(0.1g/mL). Because relatively large amounts of Fe3+-EDTA have toxic
side-effects and promote reactions of reactive oxygen species, Cu, ZnSOD is used as the copper catalyst although it scavenges superoxide and
might reduce peroxynitrite generation. In this method, peroxynitrite is
assayed as a nitrated phenol derivative (NO2-HPA) and separated by
HPLC. It has also been reported that a manganese porphyrin complex
has negligible SOD activity, and is thus an excellent catalyst for this
nitration reaction [7].
Peroxynitrite formation in vivo can also be estimated by measuring
nitrotyrosine by means of an HPLC [8,9] or immunohistochemical [10]
method. Because there are several routes for nitrotyrosine formation
(for example, nitrite + peroxidase + tyrosine), nitrotyrosine itself is
not such a specific indicator for peroxynitrite, although the efficiency
of peroxynitrite at producing nitrotyrosine is high. Thus, control
experiments with NOS inhibitors and scavengers of reactive oxygen
species should be performed to determine the origin of nitrotyrosine.
References
1. Ishimori K, lshida H, Fukahori M, Nakazawa H, Murakami1 E (1994) Practical
nitric oxide measurement employing a nitric oxide-sensitive electrode. Rev Sci
Instrum 65: 2714-2718.
2. Murphy ME, Noack E (1994) Nitric oxide assay using hemoglobin method.
Methods Enzymol 233: 240-250.
3. Brien JF, McLaughlin BE, Nakatsu K, Marks GS (1991) Quantitation of nitric
oxide formation from nitrovasodilator drugs by chemiluminescence analysis of
headspace gas. J Pharmacol Methods 25: 19-27.
4. Kikuchi K, Nagano T, Hayakawa H, Hirata Y, Hirobe M (1993) Real
time measurement of nitric oxide produced ex vivo by luminol-H2O2
chemiluminescence method. J Biol Chem 268: 23106-23110.
5. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, et al. (1982)
Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem
126: 131-138.
6. Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG (1993) A
fluorometric assay for the measurement of nitrite in biological samples. Anal
Biochem 214: 11-16.
7. Ischiropoulos H, Zhu L, Beckman JS (1992) Peroxynitrite formation from
macrophage-derived nitric oxide. Arch Biochem Biophys 298: 446-451.
8. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA (1990) Apparent
hydroxyl radical production by peroxynitrite: implications for endothelial injury
from nitric oxide and superoxide. Proc Natl Acad Sci U S A 87: 1620-1624.
9. Kaur H, Halliwell B (1994) Evidence for nitric oxide-mediated oxidative
damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from
rheumatoid patients. FEBS Lett 350: 9-12.
10.Ye YZ, Strong M, Huang ZQ, Beckman JS (1996) Antibodies that recognize
nitrotyrosine. Methods Enzymol 269: 201-209.
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Citation: Shintani H (2013) Measurements of Nitric Oxide and Peroxynitrite.
Pharm Anal Acta 4: 251. doi:10.4172/2153-2435.1000251
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