Nitric Oxide

Overview:

Nitric oxide or nitrogen monoxide (NO) is a gas. It is highly reactive and is involved in many chemical reactions. NO is also a toxic air pollutant produced by automobile exhaust and power plants. However, it has many physiological functions and is an important signaling molecule in the body of mammals including humans.

NO is produced by many cells in the body from the amino acid L-arginine by the enzymatic action of nitric oxide synthase (NOS). It is produced in macrophages, endothelial cells, neutrophils and in brain cells. Its production by vascular endothelium is particularly important in the regulation of blood flow. Because of its importance in vascular function, the abnormal production of NO, in different disease states can adversely affect blood flow and other vascular functions. NO functions as an endogenous vasodilator, a modulator of inflammation and immunity, an inhibitor of platelet aggregation, and a neurotransmitter.

Method:

NO is formed in small amounts in vivo and is rapidly destroyed by interaction with oxygen, making measurement very difficult. Inhibitors of NO synthesis such as the arginine analogs or agents that inactivate NO, such as reduced haemoglobin, are commonly used as specific probes for NO, in vivo and in vitro. The most widely used assays include;

• Electron Paramagnetic Resonance (EPR)
  • NO is trapped by nitroso compounds, or reduced haemoglobin , forming a stable adduct that is detected by electron paramagnetic resonance (detection threshold ~ 1 nmol).
• Spectrophotometry (Absorbance)
  • NO rapidly oxidizes reduced haemoglobin (Fe²⁺) to methemoglobin (Fe³⁺) , which is detected by spectrophotometry (detection threshold ~ 1 nmol).
• Chemiluminescence
  • NO interacts with ozone to generate light which is measured by a sensitive photomultiplier tube in gas phase (detection threshold ~ 20 pmol). If the specimen is liquid, the NO is driven into the gas phase by bubbling the solution with an inert gas (e.g. helium) under vacuum (a process known as “stripping”).
• Electrochemical/Amperometric
  • Several amperometric microelectrode assays offer the potential to measure smaller amounts of NO (10 -20 M), permitting measurements in intact tissues and from single cells. Example: A metalloporphyrin-coated carbon fibre microsensor, where metalloporphyrin catalyzes the oxidation of NO and in doing so generates an electric current.

Note: NO detectors capable of providing a +/- 10V analog output, can be interfaced to a PowerLab system via a BNC connection for continuous real-time NO measurements.

Related applications may also include:
•    Oxygen Concentration
•    CO2 and NH3
•    Respiratory Gas Analysis
•    ECG
•    Blood Pressure - Intravascular

Software:

The LabChart Advantage:

(may require additional Modules and Extensions)

• Units Conversion for easy calibration into appropriate units such as µM, %NO, etc
• Multipoint Calibration LabChart Extension for Windows and Macintosh for multiple point (up to 12 points) calibration of non-linear electrodes.
• Comments can be added in real time, viewed or moved at a later stage
• Fast data extraction, analysis and export (e.g. csv. or txt.) to other applications using Timed Add to Data Pad and Multiple Add to Data Pad

LabChart

LabChart software (for Windows and Macintosh) together with a PowerLab data acquisition system offers up to 32 channels of real-time data acquisition, data integrity, easy selection of hardware settings, powerful online and offline analysis, procedure automation, seamless extraction of experimental data and flexible display options. Additional acquisition and analysis functionality is provided with the use of specialized LabChart Extensions and LabChart Modules. Modules are available as part of LabChart Pro while Extensions are free for download from the website for existing LabChart users.  

Multipoint Calibration

The Multipoint Calibration Extension  (Win and Mac) extends Units Conversion in LabChart to allow you to perform linear and non-linear corrections on any input using up to 12 standard points. The extension may be used online, while data is recording, or offline with previously recorded data. The calibration points should span the highest and lowest signals that you expect to encounter in your experiment. Various fitting functions are provided by the Multipoint Calibration Extension. The functions (except Point to Point) are fitted by least squares to your calibration points.

Note: Some calibration functions (especially polynomials) can work poorly if subsequently used with data outside the calibration region.

GLP and 21 CFR Part 11

For those researchers working within a laboratory requiring GLP and 21 CFR Part 11 compliance the GLP Client and GLP Server are available for use with LabChart (Windows only) and PowerLab data acquisition systems. For more information, visit the Good Laboratory Practice application page or contact your nearest ADInstruments representative.

Hardware:

PowerLab Data Acquisition Systems

The PowerLab is a high-performance data acquisition unit capable of recording at speeds of up to 400,000 samples per second continuously to disk (aggregate). PowerLab units are compatible with instruments, signal conditioners and transducers supplied by ADInstruments, as well as many other third-party companies. In addition to standard single-ended BNC inputs, 4 differential Pod ports are also available for direct connection of Pod signal conditioners and appropriate transducers. Research PowerLab units include:

• PL3504 PowerLab 4/35 - 4 Channels
• PL3508 PowerLab 8/35 – 8 Channels
• PL3516 PowerLab 16/35 – 16 Channels

Third-Party Equipment

ADInstruments do not supply any NO meters however, suitable NO meters with an analog output (+/- 10V) can interface with a PowerLab system via a BNC connection. Suggestions include:

• Chemiluminesence: Sievers Nitric Oxide Analyzer which can be used to detect nitric oxide in exhaled air.
• Electrochemical: Electrochemical meters and electrodes made by Diamond General, Innovative Instruments, Inter Medical, and WPI Inc.
• Spectrophotometry: Eicom ENO-10 NOx analysis system.

Age-related changes in carotid vascular responses to adenosine and nitric oxide in the rat: in vitro and in vivo studies.
Omar NM, Marshall JM, Journal of Applied Physiology, 305-313, 2010

The role of tetrahydrobiopterin and dihydrobiopterin in ischemia/reperfusion injury when given at reperfusion.
Chen Q, Kim EE, Elio K, Zambrano C, Krass S, Teng JC, Kay H, Perkins KA, Pershad S, McGraw S, Emrich J, Adams JS, Young LH., Advances in Pharmacological Sciences, ID 963914, 2010

Effects of a calcium-channel blocker (CV159) on hepatic ischemia/reperfusion injury in rats: evaluation with selective NO/pO2 electrodes and an electron paramagnetic resonance spin-trapping method.
Hataji K, Watanabe T, Oowada S, Nagaya M, Kamibayashi M, Murakami E, Kawakami H, Ishiuchi A, Kumai T, Nakano H, Kobayashi S, Otsubo T., Biological & Pharmaceutical Bulletin, 77-83, 2010