USE OF A HEMOGLOBIN-TRAPPING APPROACH IN THE DETERMINATION OF NITRIC-OXIDE IN IN-VITRO AND IN-VIVO SYSTEMS

We describe methods for measuring the release of nitric oxide (NO) der ived from organic nitrates in vitro, using triple wavelength and diffe rence spectrophotometry in the presence and absence of concentric micr odialysis probes. These methods are based on the ability of NO to oxid ize oxyhemoglobin (OxyHb) to methemoglobin (MetHb) quantitatively in a queous solution. Isosorbide dinitrate (ISDN), a thiol-dependent organi c nitrate, increased MetHb concentration in 45 min from 2.47 +/- 0.47 to 4.15 +/- 0.12 mu M (p < 0.05) and decreased OxyHb concentration fro m 2.13 +/- 0.35 to 0.33 +/- 0.26 mu M (p < 0.05) at 37 degrees C. At 2 7 degrees C, the OxyHb concentration was not significantly altered (2. 04 +/- 0.23 to 1.60 +/- 0.04 mu M) by ISDN, nor was the MetHb concentr ation (from 2.68 +/- 0.50 to 2.59 +/- 0.25 mu M). Sodium nitroprusside (SNP), a thiol-independent organic nitrate, increased MetHb concentra tions in 30 min from 4.21 +/- 0.26 to 6.00 +/- 0.56 mu M (p < 0.05) at 37 degrees C, and from 4.23 +/- 0.39 to 5.90 +/- 0.43 mu M (p < 0.01) at 27 degrees C. SNP also decreased OxyHb concentrations in 30 min fr om 1.99 +/- 0.32 to 0.13 +/- 0.12 mu M (p < 0.01) at 37 degrees C, and from 2.25 +/- 0.31 to 0.13 +/- 0.09 mu M (p < 0.01) at 27 degrees C. Difference spectrophometry indicated that 0.25-5 mM SNP significantly increased NO production in a dose-dependent fashion. This hemoglobin-t rapping technique was also useful in quantifying the concentrations of NO released from SNP in aqueous solution in vitro, using concentric m icrodialysis probes. The NO concentration following exposure to SNP wa s 530 +/- 50 nM, as determined using the difference spectrophotometric technique. To demonstrate the applicability of this technique to in v ivo microdialysis, we implanted concentric microdialysis probes into h ippocampus and cerebellum of conscious and anesthetized rats. Baseline NO concentrations in hippocampus of conscious and anesthetized rats w ere 11 +/- 2 nM and 23 +/- 9 nM, respectively, while in the cerebellum NO concentrations were 28 +/- 9 nM and 41 +/- 20 nM, respectively. Th ese results demonstrate that microdialysis using a novel hemoglobin-tr apping technique possesses adequate sensitivity to measure the NO leve ls produced from organic nitrates in aqueous solutions, and further do cument the applicability of this approach to in vivo systems.