Quantitative determination of nitric oxide from tissue samples using liquid chromatography—Mass spectrometry

Figures

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  Scheme 1. Schematic representation of the reactions between NO and carboxy-PTIO. NO reacts with carboxy-PTIO to produce carboxy-PTI and NO2 in Reaction 1. NO2 then feeds into a cycle of Reactions 2 and 3, which consumes another molecule of NO. Both Reactions 2 and 3 (k = 1.5×107 M−1s−1 and 2.3×105 M−1s−1, respectively) are much faster than Reaction 1 (k = 6-10×103 M−1s−1) [19]. The cycle of Reactions 2 and 3 results in no loss of carboxy-PTIO or gain of carboxy-PTI. Together, these reactions result in an overall NO:carboxy-PTI stoichiometry between 1:1 and 2:1 [19].

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  Fig. 1. Titration curves of 100 nM, 400 nM, and 4000 nM carboxy-PTIO with DEANO. A, Sample chromatograph of carboxy-PTI (m/z=263.14, upper panel) and tryptophan-NH2 (m/z=188.06, lower panel). B, Normalized titration curves in water compared with readings from the Griess test. C-E, The regions of linearity for 100 nM (C), 400 nM (D) and 4000 nM (E) carboxy-PTIO are 5–90 nM, 100–900 nM and 40–1000 nM DEANO respectively. Griess test data are shown only when DEANO concentrations are within its range of linearity (i.e. D and E). Error bars represent ± 1 standard error of the mean.

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  Fig. 2. Testing the specificity of carboxy-PTIO for NO. A, At 4000 nM, carboxy-PTIO undergoes a slow reaction to form carboxy-PTI (●). Treating the solution with argon (▼), 200 pM DEANO (○) or a combination of both (△) significantly attenuated the reaction. B, Substitution of either 8 µM or 100 nM DEANO (black bars) with SulfoNO (gray bars) of equal molarity does not produce any carboxy-PTI. Dissociated DEANO (dotted), at 8000 nM, also does not produce carboxy-PTI. White bar shows the baseline carboxy-PTI relative abundance in absence of NO donors. All reactions here use 100 nM carboxy-PTIO. Error bars represent ± 1 standard error of the mean. P<0.001, one-way ANOVA with Tukey posthoc test.

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  Fig. 3. Quantitation of NO in frog aorta and heart. A, The calibration curve from titration of carboxy-PTIO with DEANO in frog Ringer solution. Error bars represent ± 1 standard error of the mean. B, NO was monitored using 100 nM carboxy-PTIO in the Ringer solution (see Materials and Methods) using the same method as the previous assays. Application of 0.5 mM carbachol (thick bar) at 60 min increases the NO production rate of the tissue sample to the equivalent of 44 nM DEANO per 30 min (solid circle). No increase in NO concentration is seen if the Ringer solution also contains 1 mM L-NAME, a nitric oxide synthase inhibitor (empty circle). The baseline NO is at the limit of detection. ***p<0.001, Student's t-test. Error bars represent ± 1 standard error of the mean. N = 3 animals per condition.