Clinical Applications of Nitrite

在最近完成的关于在亚硝酸盐生物活性相对不足的情况下亚硝酸盐作为一种可能的治疗选择的作用的研究中，我们使用我们已经研究了多年的镰状细胞性贫血的小鼠模型进行了研究，以及我们与美国国立卫生研究院临床中心输血医学部合作进行的关于储存血液中亚硝酸盐水平变化的研究。利用仅表达镰状血红蛋白的小鼠，我们介绍了一种新的镰状血管闭塞实验模型。镰刀鼠和对照组小鼠在常压舱中以10%O2中度缺氧2小时，恢复常氧，18小时后采血检测血浆丙氨酸氨基转移酶(ALT)，作为组织损伤的定量指标。在这种缺氧-复氧(HR)挑战中，镰刀鼠的ALT升高了4倍；野生型对照或相同HR挑战的非镰刀群对照小鼠的ALT没有变化。性别差异很明显，雌性小鼠的亚硝酸盐水平比雄性小鼠高15%，对HR挑战的敏感性比雄性小鼠低。治疗性补充亚硝酸盐，无论是在HR期间以2.4毫微克/克的剂量给药，还是在HR激发前连续7天口服，都能抑制镰刀鼠ALT的升高，并增强再灌流后复合体II-IV的线粒体呼吸。这些研究表明，SCD中NO生物利用度的降低导致了亚硝酸盐稳态的失调。在这个动物模型中，亚硝酸盐储备的耗尽与缺氧诱导的血管闭塞的损伤增加有关。通过治疗恢复亚硝酸盐水平，无论是通过腹膜内还是口服，可以减少这种损伤。在之前对人类红细胞的研究中，我们发现，在将这些细胞从体内移除后，细胞内亚硝酸盐的水平迅速下降，半衰期不到一小时；我们设计了一种使用铁氰化物、硫醇试剂和洗涤剂的保存液，并可以永久稳定这些水平。通过这些方法，我们发现人类红细胞的亚硝酸盐浓度通常约为300纳摩尔，而全血亚硝酸盐浓度约为其一半，这表明大多数血液亚硝酸盐存在于红细胞中。使用这些方法，我们系统地测量了储存的全血和红细胞中的亚硝酸盐和硝酸盐水平， 在没有白细胞减少的情况下，观察血液中其他成分对亚硝酸盐产生和/或消费的影响。我们发现硝酸盐水平在大约30微摩尔保持非常稳定，但令我们惊讶的是，我们发现亚硝酸盐水平最初的快速下降逐渐减少，并在长达42天的时间里，显著的亚硝酸盐水平(约50纳摩尔)保持在储存的红细胞中。在所有三种储存方法中，这些水平都是可比的。我们现在正在进行研究，以确定部分亚硝酸盐保存在储存的血液中的机制，并看看补充亚硝酸盐是否会改善这些红细胞的特性。此外，目前正在进行或计划进行其他几项长期目标为确定亚硝酸盐临床用途的研究。 在与美国国立卫生研究院成像中心的合作中，我们一直在研究一氧化氮水平的变化对啮齿类动物大脑血流和功能的影响。我们已经计算出了使全身或脑血流不变的条件，并发现它们是 大脑功能发生显著变化，在某些一氧化氮供体的帮助下，大脑功能得以恢复。我们是 现在在这些条件下测试亚硝酸盐，以确定它们的生理和药理作用。

Among the studies recently completed to characterize the role of nitrite as a possible therapeutic option in situations where there is a relative deficiency of nitrite bioactivity are our studied with a mouse model of sickle cell anemia that we have been studying for a number of years and our studies in collaboration with the Department of Transfusion Medicine of the NIH Clinical Center on changes in nitrite levels in stored blood. Using mice expressing exclusively sickle hemoglobin, we introduced a novel experimental model of sickle vaso-occlusion. Sickle mice and control mice were challenged with 2 hours of moderate hypoxia with 10% O2 in a normobaric chamber, restored to normoxia, and then 18 hours later had blood sampled for plasma alanine aminotransferase (ALT) as an quantitative measure of tissue injury. Sickle mice showed a 4- fold rise in ALT with this hypoxia-reoxygenation (HR) challenge; wild-type controls or non-sickling colony control mice with the same HR challenge had no change in ALT. Sexual dimorphism was evident such that female mice had 15% higher nitrite levels at baseline, and were less susceptible to HR challenge than males. Therapeutic nitrite supplementation, administered either as 2.4 nanomole/g intraperitoneal injection during HR or oral supplementation for 7 days before HR challenge, abrogated the ALT rise in sickle mice and augmented post-reperfusion complex II-IV mitochondrial respiration.These studies suggest that reduced NO bioavailability in SCD results in dysregulated nitrite homeostasis. Depleted nitrite reserves are associated with enhanced injury with hypoxia-induced vaso-occlusion in this animal model. Therapeutic restoration of nitrite levels, either by intraperitoneal or oral delivery, reduces this injury. In previous studies with human red cells, we found that upon removal of these cells from the body, levels of intracellular nitrite fell rapidly with a half life of less than an hour; we devised a preservation solution using ferricyanide, a thiol reagent and a detergent and could permanetly stabilize these levels. With these methods we found that human red cells normally have a nitrite concentration of about 300 nanomolar, while whole blood levels are about one-half of this, suggesting that most blood nitrite is in erythrocytes. Using these methods we have systematically measured nitrite and nitrate levels in stored whole blood, and red cells both with and without leukoreduction, to see the effects of other components of the blood on nitrite production and or consumption. We find that nitrate levels remain very constant at about 30 micromolar but, to our surprise, we find that the initial rapid fall in nitrite levels tapers and for as long as 42 days significant nitrite levels (about 50 nanomolar) remain in the stored red cells. The levels are comparable in all three methods of storage. We are now conducting studies to establish the mechanism of partial nitrite preservation in stored blood and to see if nitrite supplementation improves the properties of these red cells. In addition, several other studies with long term goals of defining clinical uses of nitrite are being done or being planned at present. In collaboration with the NIH Imaging Center we have been examining the effects of changes in nitric oxide levels on blood flow and function in the brains of rodents. We have worked out conditions so that there is no change in systemic or cerebral blood flow and find that their are significant changes in brain function, which is restored with certain nitric oxide donors. We are now testing nitrite salts under these conditions to ascertain their physiological and pharmacological effects.