Teorías Relacionadas al Tema

Measurement of NO in biological specimens is difficult because of the small amounts present (nanomolar) and because it reacts readily to produce oxidation products, such as nitrate (NO3-), nitrite (NO2-) and NO2. Most methodologies chemically trap NO with an adduct and then use one of several detection strategies²⁹¹:

i) Measure the adduct; for example, by spectrophotometry with a diazotisation assay^292,293, or by electron paramagnetic resonance with nitroso or

haemoglobin traps.^294,295

ii) Spectrophotometric measurement of the conversion of reduced haemoglobin to met haemoglobin.^292,296

iii) An amperometric method involving oxidation of NO at the surface of an electrode.²⁹⁷

iv) Measure the light emission that occurs when ozone interacts with NO (chemiluminescence).^67,74,298

Chemiluminescence was the chosen method in this thesis since it is highly sensitive (detects < 1 part per billion of gas phase NO²⁹⁹) and allows large numbers of samples to be processed rapidly.²⁹¹ In the chemiluminescence assay detection of NO is based on the observation that ozone interacts with NO to form excited state •NO2, which then emits a photon during conversion to its ground state:

•NO + O3 → •NO2* (excited state) + O2

•NO2* → •NO2 (ground state) + photon

The luminecesence that is generated is directly proportional to NO levels and can be detected by a sensitive photomultiplier tube (PMT). If the specimen is gaseous then it is simply mixed with ozone gas in the ‘reaction chamber’. Alternatively, if the specimen is liquid the NO has to be driven (‘stripped’) into the gas phase first. This is achieved by bubbling the solution in a sample chamber (or purge chamber, figure 2.9) with an inert gas under vacuum conditions. In addition, acids or reducing agents are sometimes added to transform previously formed oxidation products back to NO and enhance

detection; e.g. vanadium and hydrochloric acid will convert nitrate and nitrite back to NO.

However, since these agents also release biologically inactive NO from nitroso compounds, inorganic nitrites and nitrosamines, there is a risk that NO levels will be overestimated.³⁰⁰ The gaseous NO that is formed by this process is then carried to a separate chemiluminescence reaction chamber. A ‘needle valve’ is used to regulate the flow and ensure that no foam or bubbles get through. Additional measures to avoid aspiration of bubbles into the reaction chamber include the use of antifoaming agents and de-proteinising samples prior to analysis. Ozone (generated by electrical discharge) is then mixed with the gaseous NO in front of a PMT. The PMT should be red sensitive, as the light emission occurs between 660 and 900 nm, and should be cooled, since this improves the signal / noise ratio.^291,301 The sensitivity threshold for detection of NO with chemiluminescence ranges between 20-50 pmol.^67,302

Method

The NO assay was based on a method which has previously been published in detail.⁴³⁶

Sample preparation

4.5ml citrated blood samples were taken from each patient at baseline and 60 minutes after treatment allocation. The samples were immediately centrifuged (Capricorn Laboratory Equipment, Hants, UK) at 3300 rpm for 12 minutes and the plasma was transferred to 5ml bijou bottles for storage at –80^oC. When required, they were allowed to thaw at room temperature and vortexed (Vortex Genie 2, Scientific Industries Inc, New York, USA) to mix the contents. The plasma was then deproteinised by mixing 100µl with 200µl of cold ethanol in microcentrifuge tubes. These were vortexed, allowed to stand at 0^oC for 30 minutes and then centrifuged at 13000 rpm (Biofuge Pico, Kendro Laboratory Products, Germany) for a further 15min. The deproteinised supernatant was then

removed for NOx analysis. The three-fold dilution of each sample was accounted for in the final NO calculation.

Preparation of the NO assay

Plasma NO was measured by chemiluminescence using a Seivers 280 NO Analyser (Analytix Ltd, County Durham, UK). First the analyser was switched on and allowed to warm up for 20 minutes. The purge chamber (figure 2.9) was filled with 5ml filtered vanadium (111) chloride reducing agent and 100µl of antifoaming agent. The reducing agent was prepared by adding 0.8g of vanadium chloride (VCl3, Fischer Scientific, Leicester, UK) to a volumetric flask containing 1M hydrochloric acid (Fischer Scientific, Leicester, UK). The flask was inverted several times until the solution turned blue. Then the contents were filtered and stored in a refrigerator. The antifoaming agent (Seivers Instruments, Boulder, Colorado, USA) was prepared by adding 1ml to a volumetric flask containing 29 ml of double deionised water (1:30 dilution). This was also mixed

thoroughly and stored in a refrigerator prior to use. For high conversion efficiency the purge chamber had to be heated to 90^oC. This was done using a hot water bath that was

connected to a heating jacket surrounding the purge chamber. Hose clamps were used to secure the rubber tubing to the heating jacket and the fittings on the water bath. To minimise damage to the NO analyser from hydrochloric acid vapour the purge vessel outlet was connected to a condenser and a gas bubbler filled with 20ml 1M sodium hydroxide (Fischer Scientific, Leicester, UK). Nitrogen (Air Products Plc, Crewe, UK) at 0.2 – 0.5 psi was then connected to the gas inlet of the purge vessel and the needle valve was adjusted until the ‘purging conditions’ were considered acceptable (when the

‘reaction cell pressure’ on the NO analyser was approximately 6 – 7 torr).

Calibration of the NO analyser

85mg of sodium nitrate (Fischer Scientific, Leicester, UK) and 10ml double deionised water were added to a volumetric flask to make 100mM of stock solution. 1:10

progressive serial dilutions were then performed to prepare a series of 1µM to 100µM standard solutions. A calibration curve was constructed by injecting 10µL of the standards into the purge vessel.

Sample analysis

A micro-syringe (Exmire Micro-syringe, Analtix Ltd, Co Durham, UK) was used to draw up 10µL of the sample and ‘pumped’ several times to remove any air bubbles. A paper towel was then used to wipe the outside of the needle before it was inserted into the purge vessel through an airtight septum. The plunger was then rapidly depressed to transfer the sample into the vessel. Before the next sample was inserted the syringe was removed, wiped and rinsed 2-3 times with de-ionised water.