Clinical Applications of Nitrite

在最近完成的研究中，亚硝酸盐作为一种可能的治疗选择在亚硝酸盐生物活性相对缺乏的情况下的作用是我们对镰状细胞性贫血小鼠模型的研究，我们已经研究了很多年，我们与NIH临床中心输血医学系合作研究了储存血液中亚硝酸盐水平的变化。我们利用单纯表达镰状血红蛋白的小鼠，建立了镰状血管闭塞的实验模型。将镰状小鼠和对照组小鼠置于常压室中，给予10%氧气2小时的中度缺氧刺激，恢复至常压状态，18小时后采血测定血浆丙氨酸转氨酶（ALT），作为组织损伤的定量指标。镰状小鼠在缺氧-再氧化（HR）刺激下ALT升高4倍；野生型对照或非镰状菌群对照小鼠在相同的HR攻击下，ALT没有变化。性别二态性很明显，雌性小鼠的亚硝酸盐水平在基线时高出15%，并且对HR攻击的敏感性低于雄性。治疗性亚硝酸盐补充，在HR期间以2.4纳摩尔/g腹腔注射或HR攻击前7天口服补充，消除了镰状小鼠ALT升高并增强了再灌注后复合体II-IV线粒体呼吸。这些研究表明，SCD中一氧化氮生物利用度降低导致亚硝酸盐稳态失调。在这个动物模型中，亚硝酸盐储备的耗尽与缺氧引起的血管闭塞损伤的增强有关。通过腹腔或口服给药治疗性地恢复亚硝酸盐水平，可减少这种损伤。在先前对人体红细胞的研究中，我们发现，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；我们用铁氰化物、硫醇试剂和洗涤剂设计了一种保存溶液，可以永久稳定这些水平。通过这些方法，我们发现人体红细胞的亚硝酸盐浓度通常约为300纳摩尔，而全血浓度约为这个浓度的一半，这表明大多数血液中的亚硝酸盐存在于红细胞中。利用这些方法，我们系统地测量了储存的全血和红细胞中亚硝酸盐和硝酸盐的水平

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 bioavailabity 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 permantly 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, but we find that keeping the blood in argon chambers significantly decreases these levels. 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.