100 mM stock (62.56 mg in

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

Blocks free thiols to prevent formation of additional RSNO compounds

10 mL of HPLC-grade water) Add 1 part to 9 parts sample

Blocks hemoglobin-NO formation and reported to stabilize hemoglobin-SNO

in the presence of sulphanilamide will be rendered undetectable to OBC (reactions C and D).

A very important point to consider is the fact that pretreat-ment is likely to either produce further NO-like species or at least perhaps create an imbalance in the NO metabolite profile of the original sample. Special care must therefore be taken when comparing treated versus untreated samples.

Sample separation or purification also needs careful consideration. Protein removal by the use of filters or by precipitation adds several potential stages for contamination. Column separation (such as with G25) is not effective in removing all NO2- or K3FeIII(CN)6/KCN from the sample; the latter can in turn give rise to an OBC signal when the sample is injected. In fact, the addition of diluent or washing the sample is a tricky business, not least because this introduces NO2- to the sample, which cannot be accounted for by simple removal of unreacted NO2 - at a later point (such as with sulfanila-mide/HCl) before measurement. Finally, even pure chemicals themselves (i.e., "pretreatments") are likely to contain trace amounts of NO~

4. Contamination

Nitrite represents the single most significant cause of unwanted OBC signal. Despite careful laboratory practice, nitrite can contaminate samples from a number of sources:

1. Buffers and water. Even the purer, high-quality reagents and water contain nitrite contamination. HPLC-grade water (Fisher) or water for injection (Braun) were found to contain the least contamination in our laboratory's experience. However, once open, these waters also accumulate further nitrite contamination over time. This contamination is unavoidable, although it can be minimized by good practices and accounted for by inclusion of a water injection in the standards, which are made up and tested on a daily basis.

2. Plasticware, glassware, and apparatus. All disposable and reusable apparatus will contain considerable nitrite contamination that can be minimized by repeat washing. Plasticware in particular can vary vastly depending on both source and batch. The use of filters and columns in this respect should be avoided. Glassware and syringes should be cleaned extensively (e.g., overnight in Decon®, followed by extensive rinsing with water).

3. Hamilton syringes. Syringes that are in regular use should be acid washed followed by repeat neutral washes on a regular basis, as residue builds up in both the needle and in the barrel of the syringe.

4. Chemicals. All reagents contain a degree of nitrite contamination.

Despite the challenge of contamination, with care and attention to detail this problem can be overcome.

5. Practical Considerations

5.1. Freezer Time The lengthy storage of sample in the freezer tends not to alter total NO metabolite levels when compared with freshly measured samples. However, the apportionment of NO between metabolite species does begin to alter at approximately the 7th day of storage (Figs. 2.6 and 2.7). As samples are taken, they should be immediately snap frozen in liquid nitrogen and subsequently stored at -80°C. Upon testing, samples should be rapidly thawed (within 3 min at 37°C) in the dark.

5.2. Sample Preparation

Generally, sample preparation should be kept to a minimum because of the aforementioned contamination issues. Experimental conditions may dictate that immediate measurement is not possible, for example, when a time-course of sampling is undertaken. Many of our studies that use human subjects dictate that the samples are snap frozen in liquid nitrogen and kept at -80°C for OBC at a later stage. Whether the sample (fresh or from the freezer) is then allowed to equilibrate at 37°C or pretreated for set incubation times can be critical, especially if comparing parallel samples incubated with versus without pretreatment. There is also the question of time for sample separation, e.g., via a G25 column. The key factor is the chemical stability of the species of interest and the potential for contamination (as discussed in Section 4). Some investigators have developed a "stabilizing" cocktail that is introduced to the sample as the sample is drawn. It contains NEM (to bind free thiol and prevent in vitro transnit-


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