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Samples also require extensive processing before testing which can lead to contamination issues.

3.7. Pretreatments Notwithstanding the other issues listed here, the various pretreat-

ment regimens have been well publicized and have been used in conjunction with both photolysis and I3- linked to OBC (Table 2.2). In brief, there are four to consider; mercuric chloride will selectively remove the S-NO component, sulfanilamide (in HCl) will remove NO2- over a short incubation time, NEM will prevent trans-nitrosation and in vitro chemistry involving free thiols, and K3FeIII(CN)6 is deemed to both convert heme-linked NO to NO2-and stabilize SNO. In general, these work well with standards.

The mechanisms by which these pretreatments act are not fully understood, but some mechanisms have been proposed. In the presence of acidified sulfanilamide (Ar-NH2 ), nitrite is reduced to NO2 (reaction A below) which, in turn is believed to react with sulfanilamide (reaction C). Reacted sulfanilamide ultimately forms a stable diazonium salt (reaction D). Mercuric chloride cleaves NO+ from RSNO compounds (reaction B) which

Table 2.2

Summary of Pretreatments Used to Differentiate Between NO Metabolite Species Treatment Concentration Use

Table 2.2

Summary of Pretreatments Used to Differentiate Between NO Metabolite Species Treatment Concentration Use

Acidified sulphanilamide

290 mM stock (500 mg in

10 mL of 1 M HCl) Add 1 part acidified sulfanilamide to 9 parts sample

Renders nitrite undetectable by OBC.

Requires minimum 15-min incubation in the dark

Mercuric chloride

5 mL of HPLC grade water). Add 1 part to 9 parts sample

Cleaves NO from RSNO compounds

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