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，我们的研究与镰状细胞性贫血的小鼠模型，我们已经分析了多年，我们的研究与美国国立卫生研究院临床中心输血医学部合作，对储存血液中亚硝酸盐水平的变化。 使用小鼠表达专门的镰状血红蛋白，我们介绍了一种新的实验模型镰状血管闭塞。镰状小鼠和对照小鼠在常压室中用10%O2中度缺氧2小时进行攻击，恢复至常氧，然后在18小时后采血测定血浆丙氨酸氨基转移酶（ALT）作为组织损伤的定量测量。镰状小鼠在缺氧-复氧（HR）激发后ALT升高4倍;野生型对照或非镰状集落对照小鼠在相同HR激发后ALT无变化。性二态性是明显的，雌性小鼠在基线时亚硝酸盐水平高15%，并且对HR激发的敏感性低于雄性小鼠。治疗性亚硝酸盐补充剂，无论是2.4纳摩尔/克的腹腔注射在HR或口服补充剂7天前HR的挑战，废除了镰状小鼠的ALT上升，增强再灌注后复杂II-IV线粒体呼吸。这些研究表明，NO生物利用度降低SCD的结果失调亚硝酸盐稳态。在该动物模型中，耗尽的亚硝酸盐储备与缺氧诱导的血管闭塞的增强损伤相关。通过腹膜内或口服递送的亚硝酸盐水平的治疗性恢复减少了这种损伤。 在以前对人类红细胞的研究中，我们发现从体内取出这些细胞后，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溶液，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通常具有约300纳摩尔的亚硝酸盐浓度，而全血水平约为此浓度的一半，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/或消耗的影响。 我们发现硝酸盐水平保持非常恒定，约为30微摩尔，但令我们惊讶的是，我们发现亚硝酸盐水平的最初快速下降逐渐减少，并且长达42天，储存的红细胞中仍存在显著的亚硝酸盐水平（约50纳摩尔）。 所有三种储存方法中的水平相当。 我们现在正在进行研究，以建立储存血液中部分亚硝酸盐保存的机制，并观察亚硝酸盐补充剂是否能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旨在确定亚硝酸盐临床用途的长期目标的研究。我们没有发现证据表明S-亚硝化血红蛋白在影响输血用红细胞的“储存损伤”中的作用。 在与NIH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动物大脑血流和功能的影响。我们已经制定了条件，使得在施用nNOS抑制剂的情况下全身或脑血流量没有变化，但发现它们是脑功能的显著变化，这是用某些一氧化氮供体（包括亚硝酸根离子）恢复的。 我们现在正在测试亚硝酸盐对大脑功能的药理作用，发现亚硝酸盐可以恢复神经血管偶联。 我们与NHLBI和临床中心的DTM一起开始了一个项目，研究NO在 在镰状细胞性贫血患者中引起痛苦的危机。 我们正在测量 溶血和NO被无细胞血红蛋白破坏的证据，以观察这些参数是否与疾病的表现相关。我们最近收到了国家卫生研究院的一笔实验室/床边资助， 为了这项工作，我们已经制定了两个临床方案，即将提交IRB批准，以便我们可以开始我们的临床研究。 在我们与美国红十字会合作的项目中， 亚硝酸根离子在人血小板的存活力和储存中的作用我们已经发现，在室温储存的5天期间，硝酸根水平几乎没有变化，亚硝酸根水平降低但仅约50%，然后保持稳定;其中一些似乎是从储存袋的塑料中浸出的。 我们正在研究这一发现的意义。

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 studies with a mouse model of sickle cell anemia that we have been analyzing 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. We find no evidence of a role of S-nitrosated hemoglobin in the proposed "storage lesion" affecting red blood cells used for transfusion. 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 with the administration of a nNOS inhibitor but find that their are significant changes in brain function, which is restored with certain nitric oxide donors, including nitrite ions. We are now testing the pharmacological effects of nitrite on brain function and find that nitrite can restore neurovascualar coupling. We have started a project with NHLBI and the DTM of the Clinical Center to study the role of NO depletion in causing painful crises in sickle cell anemia patients. We are measuring levels of hemolysis and evidence of NO destruction by cell-free hemoglobin to see if these parameters correlated with manifestations of the disease. We hve recently received an NIH Bench/Bedside grant for this work and have developed two clinical protocols which are about to be submitted for IRB approval so we can initiate our clinical studies. In our project in collaboration with the American Red Cross to study the role of nitrite ions in the viability and storage of human platelets we have found that there is little change in nitrate levels during five days of room temperature storage and that nitrite levels decrease but only about 50% and then remain stable; some of this appears to be leached from the plastic of the storage bags. We are studying the significance of this finding.