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Fresh 1 day 1 week 1 month 3 month 6 month Freezing time

Fresh 1 day 1 week 1 month 3 month 6 month Freezing time

Fig. 2.7. Effect of sample freezing on plasma nitrite and protein-bound nitric oxide levels (top) and plasma protein-bound nitric oxide (bottom) measured using tri-iodide in conjunction with chemiluminescence. Fresh represents snap frozen and immediately measured. Values presented as mean ± SEM (n = 5)

5.3. Changing Oxygen/pH

rosation) and K3FeIII(CN)6 (to stabilize the SNO in the sample). This cocktail is also touted to prevent the recapture of NO by free heme in the reaction vessel during the measurement, a critical practical consideration described above.

Alterations of pH can lead to in vitro chemistry between NO metabolite species. Even a minor decrease in pH below 7 is enough to reduce some nitrite back to NO. Changes in oxygen are also important in the case of RBC NO metabolite measurements. A switch in the conformational/oxygenation state of hemoglobin is believed to alter the location of NO within the hemoglobin structure (e.g., NO bound to the oxygen binding site [HbNO] or NO bound to a cysteine residue of the hemoglobin p chain [SNO-Hb]).

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