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，有我们多年来一直在分析的镰状细胞性贫血小鼠模型的研究，以及我们与NIH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。动物模型的结果已经发表，这里不再做进一步的总结。 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。我们没有发现S亚硝化血红蛋白在拟议的影响用于输血的红细胞的“储存损伤”中所起作用的证据。 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。我们已经计算出了这样的条件，即给予nNOS抑制剂后，全身或脑血流不会发生变化，但发现它们是大脑功能的显著变化，而某些一氧化氮供体，包括亚硝酸根离子，可以恢复大脑功能。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。此外，似乎大脑中高水平的细胞内和细胞外抗坏血酸可能有助于减少亚硝酸盐生成NO，我们现在正在用啮齿动物检验这一假说，这些啮齿动物本身不能合成抗坏血酸，因此我们可以通过外源给药来控制抗坏血酸水平。 我们已经与NHLBI、NINR和临床中心的DTM启动了一个项目，研究NO耗竭在导致镰状细胞性贫血患者痛苦危象中的作用。我们正在测量溶血水平和无细胞血红蛋白破坏的证据，看看这些参数是否与疾病的表现相关。我们最近收到了美国国立卫生研究院的长椅/床边补助金 为了这项工作，我们已经制定了两个临床方案，这两个方案已经得到了适当的IRB的批准，可以给镰状细胞患者管理疼痛日记，测量这些患者的亚硝酸盐、硝酸盐和呼出的NO水平，以及他们白细胞中的基因表达谱，看看我们是否可以确定疼痛严重程度的标记。 在我们与美国红十字会合作的项目中研究 亚硝酸盐离子在人体血小板存活和储存中的作用我们发现，在室温储存的五天中，硝酸盐水平有微小的变化，亚硝酸盐水平下降，但只有大约50%的水平，然后保持稳定；其中一些似乎是从储存袋的塑料中滤出的。我们正在研究这一发现的意义以及亚硝酸盐离子在保存过程中保持血小板活性的必要性。

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. The results with the animal model have been published and will not be summarized further here. 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 neurovascular coupling. Further it appears that the high levels of intracellular and extracellular ascorbate in the brain may contribute to the reduction of nitrite to generate NO and we are now testing this hypothesis with rodents that can not synthesize ascorbate themselves and thus we can control ascorbate levels by exogenous administration. We have started a project with NHLBI, NINR, 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 have recently received an NIH Bench/Bedside grant for this work and have developed two clinical protocols which have been approved by the appropriate IRB to administer pain diaries to sickle cell patients, to measure nitrite, nitrate and exhaled NO levels in these patients as well as gene expression profiles in their leukocytes to see if we can identify markers of pain severity. 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 a small change in nitrate levels during five days of room temperature storage and that nitrite levels decrease but only about to 50% levels 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 and the need for nitrite ions in the retention of platelet viability during storage.