Balcioglu A, Watkins C J, Maher T J
Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, Cambridge 02139, USA.
Neurochem Res. 1998 May;23(5):815-20. doi: 10.1023/a:1022475830200.
We describe methods for measuring the release of nitric oxide (NO) derived from organic nitrates in vitro, using triple wavelength and difference spectrophotometry in the presence and absence of concentric microdialysis probes. These methods are based on the ability of NO to oxidize oxyhemoglobin (OxyHb) to methemoglobin (MetHb) quantitatively in aqueous solution. Isosorbide dinitrate (ISDN), a thiol-dependent organic nitrate, increased MetHb concentration in 45 min from 2.47 +/- 0.47 to 4.15 +/- 0.12 microM (p < 0.05) and decreased OxyHb concentration from 2.13 +/- 0.35 to 0.33 +/- 0.26 microM (p < 0.05) at 37 degrees C. At 27 degrees C, the OxyHb concentration was not significantly altered (2.04 +/- 0.23 to 1.60 +/- 0.04 microM) by ISDN, nor was the MetHb concentration (from 2.68 +/- 0.50 to 2.59 +/- 0.25 microM). Sodium nitroprusside (SNP), a thiol-independent organic nitrate, increased MetHb concentrations in 30 min from 4.21 +/- 0.26 to 6.00 +/- 0.56 microM (p < 0.05) at 37 degrees C, and from 4.23 +/- 0.39 to 5.90 +/- 0.43 microM (p < 0.01) at 27 degrees C. SNP also decreased OxyHb concentrations in 30 min from 1.99 +/- 0.32 to 0.13 +/- 0.12 microM (p < 0.01) at 37 degrees C, and from 2.25 +/- 0.31 to 0.13 +/- 0.09 microM (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-trapping technique was also useful in quantifying the concentrations of NO released from SNP in aqueous solution in vitro, using concentric microdialysis probes. The NO concentration following exposure to SNP was 530 +/- 50 nM, as determined using the difference spectrophotometric technique. To demonstrate the applicability of this technique to in vivo microdialysis, we implanted concentric microdialysis probes into hippocampus and cerebellum of conscious and anesthetized rats. Baseline NO concentrations in hippocampus of conscious and anesthetized rats were 11 +/- 2 nM and 23 +/- 9 nM, respectively, while in the cerebellum NO concentrations were 28 +/- 9 nM and 41 +/- 20 nM, respectively. These results demonstrate that microdialysis using a novel hemoglobin-trapping technique possesses adequate sensitivity to measure the NO levels produced from organic nitrates in aqueous solutions, and further document the applicability of this approach to in vivo systems.
我们描述了在有和没有同心微透析探针的情况下，使用三波长和差示分光光度法在体外测量有机硝酸盐衍生的一氧化氮（NO）释放的方法。这些方法基于NO在水溶液中能够将氧合血红蛋白（OxyHb）定量氧化为高铁血红蛋白（MetHb）的能力。二硝酸异山梨酯（ISDN）是一种依赖硫醇的有机硝酸盐，在37℃下，45分钟内MetHb浓度从2.47±0.47微摩尔/升增加到4.15±0.12微摩尔/升（p<0.05），OxyHb浓度从2.13±0.35微摩尔/升降低到0.33±0.26微摩尔/升（p<0.05）。在27℃时，ISDN未显著改变OxyHb浓度（从2.04±0.23微摩尔/升变为1.60±0.04微摩尔/升），也未改变MetHb浓度（从2.68±0.50微摩尔/升变为2.59±0.25微摩尔/升）。硝普钠（SNP）是一种不依赖硫醇的有机硝酸盐，在37℃下30分钟内MetHb浓度从4.21±0.26微摩尔/升增加到6.00±0.56微摩尔/升（p<0.05），在27℃下从4.23±0.39微摩尔/升增加到5.90±0.43微摩尔/升（p<0.01）。SNP在30分钟内也使OxyHb浓度在37℃时从1.99±0.32微摩尔/升降低到0.13±0.12微摩尔/升（p<0.01），在27℃时从2.25±0.31微摩尔/升降低到0.13±0.09微摩尔/升（p<0.01）。差示分光光度法表明，0.25 - 5毫摩尔/升的SNP以剂量依赖方式显著增加NO生成。这种血红蛋白捕获技术在使用同心微透析探针体外定量SNP在水溶液中释放的NO浓度方面也很有用。使用差示分光光度技术测定，暴露于SNP后的NO浓度为530±50纳摩尔/升。为了证明该技术在体内微透析中的适用性，我们将同心微透析探针植入清醒和麻醉大鼠的海马体和小脑中。清醒和麻醉大鼠海马体中的基线NO浓度分别为11±2纳摩尔/升和23±9纳摩尔/升，而小脑中的NO浓度分别为28±9纳摩尔/升和41±20纳摩尔/升。这些结果表明，使用新型血红蛋白捕获技术的微透析具有足够的灵敏度来测量水溶液中有机硝酸盐产生的NO水平，并进一步证明了该方法在体内系统中的适用性。
