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Final haem in reaction chamber (M)

Fig. 2.3. Dose-dependent heme nitric oxide (NO) autocapture. Effect of heme NO scavenging in tri-iodide on signal height and area under curve analysis in the presence of 100 pmol (A) and 10 pmol NO (B). Squares represent area under curve analysis, triangles represent signal height. Values are presented as mean ± SEM (n = 4-7 for each concentration). *p < 0.05, **p < 0.01 in relation to 100% values; One-way ANOVA.

Fig. 2.3. Dose-dependent heme nitric oxide (NO) autocapture. Effect of heme NO scavenging in tri-iodide on signal height and area under curve analysis in the presence of 100 pmol (A) and 10 pmol NO (B). Squares represent area under curve analysis, triangles represent signal height. Values are presented as mean ± SEM (n = 4-7 for each concentration). *p < 0.05, **p < 0.01 in relation to 100% values; One-way ANOVA.

However, at high hemoglobin concentrations (and especially where NO-hemoglobin ratio is in the physiological range of >1:25,000) not only is signal height affected, but the AUC also is underestimated (Fig.2.3B). To overcome this problem, two techniques have been independently developed: (a) the use of high-pressure CO as the carrier gas in the NOA set up, and (b) the inclusion of KgFe^CN^ in the tri-iodide reagent (this latter methodology being specific for the tri-iodide reagent only). Both methods prevent the autocapture of the released NO by hemoglobin while not affecting the reductive capacity of the reagents (this means that the same species are measured in both the modified and unmodified assays). We will return to this in Section 3.3.

Improved accuracy of AUC measurement (especially for a weak signal) can be gained by exporting the recorded data from the NOA data collection program to a simulation package. We, and others, have found Origin to be particularly useful, where by adjacent signal averaging and peak analysis (enabling user placement of peak start and end) can be implemented to improve the signal-to-noise ratio, resulting in a more accurate determination of AUC and improved repeatability of measurement (Fig. 2.4). We have also found other software packages to be effective (e.g., Sigmaplot) but not as user-friendly.

2.6. Step-by-Step Setup 1. Switch on gas supplies to NOA and allow the machine to cool down to set temperature. Inside the machine, the PMT is cooled to less than -10°C (see Note 1).

2. A purge vessel loaded with the appropriate volume of reagent can then be loaded on to the system. The NOA can then be started; a vacuum pump should cut in to draw sample gas through (see Note 2).

Tri-iodide +KCN +K3FeIII(CN)6 +Both

Tri-iodide +KCN +K3FeIII(CN)6 +Both

0 200 400 600 800 1000 1200 1400 Time (seconds)

Fig. 2.4. Measurement of red blood cell lysate in different reaction mixtures. Reagents include tri-iodide alone; tri-iodide plus potassium cyanide (KCN); tri-iodide plus potassium ferricyanide (K3FeIII(CN)6) , and tri-iodide plus both cyanides. Example traces of chemiluminescence signals and the same data smoothed for analysis with Origin (version 7.0) are shown.

0 200 400 600 800 1000 1200 1400 Time (seconds)

Fig. 2.4. Measurement of red blood cell lysate in different reaction mixtures. Reagents include tri-iodide alone; tri-iodide plus potassium cyanide (KCN); tri-iodide plus potassium ferricyanide (K3FeIII(CN)6) , and tri-iodide plus both cyanides. Example traces of chemiluminescence signals and the same data smoothed for analysis with Origin (version 7.0) are shown.

3. Complete a standard curve once signal is at baseline.

4. After completion of a standard curve, change the purge vessel to begin sample testing (see Note 3).

It is vital that the vacuum has stopped; otherwise, an imbalance in the positive purge gas pressure, back pressure caused by filters, and the negative vacuum pressure can cause the reagent/sodium hydroxide trap to be drawn back through the system as the apparatus is dismantled.

3. Cleavage Reagents and Specific Applications

3.1. Glacial Acetic Acid

Where measurement of nitrite is the primary aim, sample injection into acetic acid alone in an oxygen-free environment produces NO according to:

From nitrite standards, broad peaks (shorter and longer) are observed in acid alone compared with more-powerful reductive reagents. Acid alone is relatively specific for nitrite detection because it is not strong enough to reduce nitrate to NO. Also, S-nitrosothiols (RSNO; at least GSNO and albumin-SNO) is undetectable and considered stable to acid treatment, although in some cases (e.g., HbSNO), the activation energies of thiol groups is reported to be completely different, implying the same standards and techniques may not be completely appropriate. Examples in which direct injection into acid at 50°C detects nitrite efficiently include supernatant from cell culture experiments or assessment in saline or other medium.

3.2. Glacial Acetic Acid and Potassium Iodide

Originally, a reaction mix comprising potassium iodide in glacial acetic acidic was used to determine nitrite in biological fluids. However, this reagent was subsequently demonstrated to detect NO from other biological metabolite species (including S-nitrosothiols). The detection of RSNO was found to vary widely (1) because of the uncontrolled formation of free iodine from the following reaction:

The formation of free iodine (I2) allowed the production of tri-iodide (I3), a reagent capable of detecting RSNO and N-nitrosamine (RNNO) in addition to nitrite. Herein lay the major pitfall: The reductive power of the glacial acetic acid/KI reagent was not consistent in its yield return from RSNO.

The use of Kl/glacial acetic acid is only effective after the addition of KI to deoxygenated acid with the subsequent maintenance of an O2-free environment. If oxygen is introduced, even in small quantities, the formation of I3 results. Under such conditions, there is no difference between the reduction of nitrite to NO in KI/glacial acetic acid compared with tri-iodide.

3.3. Tri-iodide This reagent comprises the following ingredients:

1. 70 mL of glacial acetic acid.

3. 20 mL of HPLC-grade water.

The rationale behind the development of "tri-iodide" was to provide a reductive reagent that was capable of detecting other biological NO metabolite species (i.e., S-nitrosothiols) in addition to nitrite. This was achieved by the addition of free iodine to the glacial acetic acid/KI reagent to saturate the solution thus forcing the production of I3- in abundance (1).

I- + 2RSNO ® 31- + RS-SR + 2NO+ 2NO+ +2I- +2H+® 2NO + I2 + 2H2O

To make this reagent:

1. First, dissolve KI in 20 mL of HPLC water.

2. Add iodine to 70 mL of acetic acid.

3. Combine and stir the two solutions for 30 min until all the iodine has dissolved.

For biological samples (e.g., plasma) 5 mL of reductive reagent is loaded into the purge vessel with 20 pL of antifoam. Two to three samples (100-200 pL) can be tested before changing the reagent.

The measurement of red blood cell/hemoglobin-bound NO requires the use of a modified tri-iodide reagent (2). The modified tri-iodide reagent contains potassium ferricyanide to block hemoglobin from "auto-capturing" NO (see Sect. 2.5 and Fig. 2.4).

For the modified reagent:

1. Add 800 pL of a 250 mM solution of potassium ferricyanide (823 mg into 10 mL of HPLC-grade water) to 7.2 mL of tri-iodide (25 mM final) with 20 pL of antifoam. With regard to this modified reagent, two essential aspects should be considered. First, the timing of K3FeIII(CN)6 addition to tri-iodide should be kept the same for all samples because the reductive capacity of the reagent can change if left for a long period (>30 min). We add K3FeIII(CN)6 to the reagent 10 min before injection of the sample to allow time for the signal from contaminant nitrite in the K3Fem(CN)6 to decay and the temperature of the reagent to reach 50°C, thus providing a stable baseline OBC signal. Second, only one red blood cell (RBC) sample should be injected per reagent mix to limit the amount of hemoglobin in the reaction chamber (minimizing NO-hemoglobin interaction), which also ensures that the reductive potential of the reagent is the same for all samples (see Note 4).

The fact that the sample is plunged into hot acid and that most biological NO species can potentially be detected in I3 (as a result of its enhanced reductive potential) can cause trouble when interpreting results. A further issue is the fact that the mechanism of precisely how I3- works has yet to be elucidated. Nevertheless, I3- is probably the most widely used reagent in conjunction with chemical pretreatments that enable the putative measurement of individual metabolite species. Tri-iodide has been validated against standards of NO2-, GSNO, albumin-SNO, and Hb/RBC standards saturated with NO.

3.4. Vanadium Chloride This reagent comprises the following ingredients:

2. 20 mL of HPLC-grade water.

3. 80 mL of 1 M hydrochloric acid (HCl).

- Prepare a saturated solution of VCl3.

- After stirring filter the solution, it should appear turquoise blue in color.

- Load 8 mL of the reagent into the purge vessel with 20 pL of antifoam.

The VCl3 reagent can be used to measure nitrate (NO3-) in addition to those species observed with tri-iodide. Nitrate levels in biological samples tend to be orders of magnitude greater than nitrite levels.

3.5. CuCl/CSH This reagent comprises the following ingredients:

2. 0.1 mg of CuCl (made as 39.59 mg in 10 mL ofwater to create a 40 mM stock solution; then dilute 1/10 to give 4 mM, then add 10 mL below to create a 100 pM final concentration).

3. 390 mL of HPLC-grade water.

- Prepare the reagent fresh, daily.

- Mix well before pH correction to pH 7 with sodium hydroxide.

The CuCl/CSH reagent was developed specifically to quantify RSNO compounds (3). The reagent takes advantage of the trans-nitrosation reactions that occur between biological nitrosothiols and the cleavage of NO from RSNO compounds by copper ions. The neutrality of this reagent ensures that other metabolites such as nitrates and nitrites remain undetected, guaranteeing specificity.

Cu/CSH is useful because (like photolysis) it is reported to be specific for cleavage of the S-NO group, although the efficiency with which this occurs (30-60%) may depend on other factors (such as varying concentration of reactants, pH):

RS-NO + Cu+ + H+ ^ RSH + NO + Cu2+ 2RSH + 2Cu2+ ^ RS-SR + 2Cu+ + 2H+

Inclusion of cysteine improves NO yield to around 80%. There remains a modest lack of reproducibility, which has been attributed to the fact that decomposition of nitrosothiols under these conditions can occur by several pathways, some of which are described by the equations above. In addition, the presence of an oxidizer will decompose nitrosothiols to NO+ that in turn hydrolyses to nitrite. A more recent and important development for use with RBC samples is the simultaneous perfusion of the Cu/CSH reagent with CO at high reaction cell pressure to prevent autocapture of the released NO by free Hb in the reaction chamber (see ref. 4 and Fig. 2.5).

3.6. Photolysis This chapter has so far considered chemical cleavage reagents, but it is important to recognize that intense light at specific wavelengths can also be used to cleave NO from proteins and can be used in conjunction with OBC. Photolysis as a cleavage method has not been used in our laboratory but is used successfully by a select number of groups in the field and is worthy of consideration (5, 6). Samples are vaporized and irradiated at a wavelength of 340 nm, which is reported to specifically cleave RNNO, RONO, RSNO, and RMNO (where M is a metal). Nitrite is relatively resistant to photolysis, making this method ideal for the analysis of species that are perhaps not as accessible using chemical cleavage. In the instance of RSNO measurements, treatment with mercuric chloride can be used to validate the origin of the signal. A basic photolysis system is not generally available commercially and a bespoke setup can come at considerable cost.

Table 2.1

Summary of Different Reagents and the NO Metabolite Species Detectable

Cuprous chloride/

cysteine Tri-iodide Vanadium chloride

Table 2.1

Summary of Different Reagents and the NO Metabolite Species Detectable

Reagent temperature

5Ö (±1)°C

5Ö (±1)°C

85 (±1)0C

Species cleaved or reduced

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