Andrew G Pinder Stephen C Rogers Afshin Khalatbari Thomas E Ingram and Philip E James


A plethora of publications on techniques and methodologies for measuring nitric oxide (NO) or reaction products of NO (NO metabolites) has served in recent years to complicate and confuse the majority of researchers interested in this field. Here, we provide a practical approach and summarize the key issues and corresponding solutions regarding quantification with the use of ozone-based chemiluminescence, which is the most accurate, sensitive, and widely used NO detection method. We have drawn on the vast experience of leaders in the field to produce this consensus, but the views and implications presented herein represent our own, and we limit our advice to those techniques with which we have direct experience. Hopefully, this guide will allow authors to make more informed decisions regarding NO metabolite measurement methodology, without the need for each subsequent group to rediscover previously observed advantages and pitfalls.

Keywords: Nitric oxide, nitrite, nitrate, nitrosothiols, ozone-based chemiluminescence.

1. Introduction

The involvement of nitric oxide (NO) in a vast number of signaling pathways has created a need to accurately measure this free radical species in a variety of biological systems. However, because of the highly reactive nature of free NO, its measurement is practically difficult and extremely complex at best. Therefore, researchers have focused on the quantification of the reaction products of NO (NO metabolites). Particular emphasis has been made on the measurement of nitrate, nitrite, and protein-bound

John T. Hancock (ed.), Methods in Molecular Biology, Redox-Mediated Signal Transduction, vol. 476 © 2008 Humana Press, a part of Springer Science + Business Media, Totowa, NJ DOI: 10.1007/978-1-59745-129-1_2

NO species to obtain information regarding the in vivo production, consumption, and bioavailability of NO.

Ozone-based chemiluminescence (OBC) is generally recognized as the most accurate and sensitive technique available in which to measure NO. However, to directly measure metabolite NO, the NO must either be cleaved from the species of interest or the metabolite must be reduced back to NO. Several methods exist to achieve this state, primarily photolysis or the use of chemical cleavage reagents. However, researchers can be confused by the wealth of literature filled with speculation and counterargument regarding specificity. Over a number of years we have learned the hard way, undertaking the necessary experiments to identify key issues and confounding factors. With considerable success, we (and others) have gained an in-depth understanding and herein provide a useful and practical guide to NO metabolite measurement using chemical cleavage reagents linked to OBC.

2. General Methodology

2.1. Materials

2.2. Nitric Oxide Analysis by Ozone-Based Chemiluminescence (OBC)

1. High-performance liquid chromatography (HPLC)-grade water (Fisher Scientific, Leicestershire, UK).

2. Glacial acetic acid (Fisher Scientific UK).

3. Hydrochloric acid (Fisher Scientific UK).

4. Potassium iodide (KI: Sigma-Aldrich Co., Ltd., Dorset UK).

6. Cuprous chloride (CuCl: Sigma-Aldrich).

8. Vanadium chloride (VCl3: Sigma-Aldrich).

9. Antifoam 204 organic (Sigma-Aldrich).

10. Potassium hexacyanoferrate (K3FeHI(CN)6: Sigma-Aldrich).

11. Sulphanilamide (Sigma-Aldrich).

12. Mercury chloride (HgCl2: Sigma-Aldrich).

13. N-ethyl maleimide (NEM: Sigma Aldrich).

OBC is a measurement technique that exploits the luminescent nature of specific chemical reactions and can be used to quantify NO. As outlined previously in this chapter, before NO (or metabolite NO) can be measured, it must firstly be cleaved from its parent compound(s) or reduced back to its radical form. This can be achieved by the use of a number of chemical reagents, which will be discussed in more detail later. NO is subsequently carried in an inert gas stream (e.g., nitrogen [N2] or argon [Ar]

at a constant flow rate -100-150 cm3/min) to the nitric oxide analyser (NOA).

The NOA uses oxygen to generate ozone (O3) in its reaction cell. NO entering the NOA reacts with O3 to form nitrogen dioxide (NO2) and oxygen (O2). However, a proportion of the NO2 is formed in an electrically excited state (NO2*). Electrons in this state are unstable and, as they return to their original ground state, they release their excess energy as a photon. Released photons are focused via a low-pass filter lens (<900 nm wavelength) into a photomultiplier tube, which amplifies the signal to give an accurate, recordable millivolt signal.

In our laboratory, we currently use a Sievers NOA 280i (Ana-lytix, Durham, UK). It must be acknowledged that there is variation in sensitivity between machines, and considerable differences between manufacturers of NOA exist. It is therefore worthwhile investing some time in testing actual analysers if considering a purchase.

2.3. Experimental Setup

The inert carrier gas (N2) flows through a purge vessel that contains the cleavage reagent (Fig. 2.1 ) and then through a sodium hydroxide (25 mL of 1 N NaOH) trap and solvent filter (Whatman solvent IFD) placed in-line before the NOA. The NaOH and solvent filter serve to protect the NOA reaction cell from damage by hot acid vapor. The NaOH also ensures that N-oxide contaminants are not converted to NO and erroneously measured.

Fig. 2.1. Apparatus used for ozone-based chemiluminescence.

Commercially available purge vessels are provided with pressurized fittings and connectors. Depending on the specific application, they tend to have large reagent volumes that are ideal for multiple sample injections. However, they may not be ideal for certain measurements (for example, NO linked to hemoglobin), where it is essential that the reagent is changed between samples. This is the result of the ability of hemoglobin to scavenge NO within the purge vessel reagent (see Sect. 2.5.).

In our laboratory, we have opted for custom-designed and -built glassware. This has led to the creation of an interchangeable reagent vessel (~10 mL maximum volume) with side arm injection port and a T-shaped gas purging component, which forms a gas-tight fitting with the reagent vessel. Our setup allows for the quick replacement of the reagent and easy washing of the glassware (Fig. 2.1).

For different experimental setups, various temperatures are required. It is therefore essential to accurately maintain the temperature of the purge vessel. We achieve this by using a water bath on a thermostatically controlled hotplate (IKA® WERKE). Samples should be injected into the purge vessel with a glass Hamilton (Fisher) syringe through a rubber septum injection port on the side of the purge vessel.

2.4. Calibration Calibration should be performed on a daily basis to allow for day-

to-day variations in machine sensitivity. The standard used for the calibration curve depends on the sample to be measured and the cleavage reagent used.

1. I n general, nitrite or S-nitrosoglutathione (GSNO) is used as the standard.

2. Prepare standards of 62.5, 125, 250, 500, and 1000 nM in HPLC-grade ultrapure water of the volume to be used.

3. I nject a known volume for analysis (usually 100/200 pL) to generate a standard curve of NO (Fig. 2.2).

Alternatively, an NO donor (such as NONOates available from Axxora) can be injected into water. In this case, the half-life (t%) for NO release is critical. MAHMANOnoate has a of 2-3 min and is stable at pH 9. It can be conveniently stored on ice and quickly made up in water (pH 7) for injection and accurate production of NO in the purge vessel. It is extremely difficult, if not impossible, to synthesize biological standards that contain NO in physiological proportions, particularly in the case of blood-borne metabolite species.

4. Plot a standard curve of peak area under curve (AUC) against NO (either concentration or molar amount).

5. To generate a straight line relationship, the area under curve of the water injected is removed from the other NO standards to account for water contaminants.

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