Clinical Applications of Nitrite and Nitrate

在正常志愿者中注入亚硝酸盐和在实验动物中吸入NO的结果表明，NO可以作为激素转运，因此具有成为药理学试剂的潜力（即，药物）。然而，在我们的镰状细胞患者中，NO对血管的影响相对较小，这可能是由于循环血红蛋白的存在，这有助于这种和其他慢性和急性溶血综合征的病理生理学，特别是我们发现在老年患者中严重且频率高的肺动脉高压并发症。我们还发现，NO-生物活性的破坏似乎发生在组织中，在更大程度上比在血管床。我们也很幸运，能够使用重症监护医学部犬设施，并在缺氧犬中测试吸入的亚硝酸盐和硝酸盐离子，看看以这种方式施用NO生物活性是否具有治疗意义。我们测试了硝酸盐，因为有证据表明肺组织具有一定水平的酶，如黄嘌呤氧化还原酶，可以将硝酸盐离子还原为亚硝酸盐和NO。我们发现亚硝酸盐吸入对肺部和全身参数都有强烈的影响，正如其他动物模型所报道的那样，但硝酸盐的影响只是间歇性的。这些结果刚刚发表。然而，我们也发现，饮食限制硝酸盐和亚硝酸盐大大降低了这些水平（超过血液或肝脏中的水平，超过许多遗传操作），这些离子返回到饮食中导致快速积累，实际上，在某些情况下，水平“过冲”。 在动物和人中的硝酸盐-亚硝酸盐-NO代谢的详细代谢研究需要示踪剂研究，这可以使用无毒的重同位素。 我们在纽卡斯尔的合作者正在开发在动物身上并最终在人类身上进行这项研究的方案。他们从各种动物摄入研究中向我们发送了样本，我们正在使用我们高度灵敏和准确的化学发光方法来定量这些血液样本中的硝酸盐和亚硝酸盐水平。(One他们的工作人员访问了我们的实验室几个星期，学习如何进行这些测定。早期的人类研究已经检查了抗坏血酸对膳食硝酸盐和亚硝酸盐吸收的影响。我们在曼谷的合作者向地中海贫血患者给予低水平的亚硝酸盐，我们与他们合作，证明VASP磷酸化受到影响，作为测量血小板抑制的参数（如DK 025104所述）。 最令人鼓舞的是，我们已经与英国埃克塞特的肌肉生理学小组建立了强有力的合作关系，我们一直在测量人体组织中休息和运动时的硝酸盐水平，并证实了我们的动物结果适用于人类受试者，包括肌肉中的高硝酸盐水平及其在剧烈运动中的下降。 我们认为，在饮食干预中必须考虑肌肉硝酸盐水平，以产生药理作用或影响性能。这些结果最近也发表了。 最近启动了几个新项目。首先，我们正在与NIH研究人类微生物组的小组合作，看看这些药物中是否有任何一种对NO代谢途径有特别强烈的影响。我们还使用NIDDK质谱仪在各种动物模型中跟踪15 N标记的硝酸盐。我们已经开始测量整个啮齿动物眼睛中的硝酸盐和亚硝酸盐水平，作为在较大动物模型和人类样本中测量眼睛各个部分中这些水平的开始。我们在MedStar非常重要的猪研究中的新结果表明，泪腺将硝酸根离子从血液泵入泪液，在那里有几个过程可以将其转化为亚硝酸盐和/或NO吸附到眼睛中。我们计划与MedStar的兽医人员进行广泛的研究，以跟踪这些眼睛中的NO代谢途径，以及通过各种途径施用硝酸盐如何影响这些水平。 我们相信，这一过程有助于视网膜和眼睛的正常功能，并可能有助于治疗青光眼和黄斑变性等疾病。最近，我们一直在与NIDDK质谱单元合作，用15 N标记的硝酸盐研究这些过程。 这些动物模型研究导致了德克萨斯大学西南医学中心眼科系的临床研究，以获得具有正常眼功能的个体的泪液，然后与“干眼综合征”和其他条件一起观察我们的动物模型结果如何与正常和异常的人眼过程相一致。

The results of nitrite infusions in normal volunteers and NO inhalation in experimental animals suggest that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). Howeve, the relatively small vascular effects of NO in our sickle cell patients is likely due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes, especially the pulmonary hypertension complications which we have found to be severe and of high frequency in older patients. We also find that NO-bioactivity destruction appears to occur in tissues to a much greater extent than in the vascular bed. We have also been fortunate in being able to use the Critical Care Medicine Department canine facility and test inhaled nitrite and nitrate ions in hypoxic dogs to see if administering NO bioactivity in this way would be therapeutically possible. We tested nitrate as there is evidence that pulmonary tissue has some levels of enzymes, such as xanthine oxido-reductase that can reduce nitrate ions to nitrite and NO. We have found strong effects of nitrite inhalation on both pulmonary and systemic parameters, as has been reported for other animal models, but the effects of nitrate are only seen intermittently. These results have just been published. However, we also find that dietary limitations of nitrate and nitrite lower these levels greatly (more than in blood or liver and more than many genetic manipulations) and that return of these ions to the diet results in rapid accumulation and, indeed, in some cases an "overshoot" of the levels. Detailed metabolic studies of nitrate-nitrite-NO metabolism in animals and people require tracer studies which may be approached using non-toxic heavy isotopes. Our collaborators in Newcastle are developing protocols to do this in animals and eventually in human subjects. They have sent us samples from various animal ingestion studies and we are using our highly sensitive and accurate chemi-luminescence methods to quantity nitrate and nitrite levels in these blood samples. (One of their staff visited our laboratory for several weeks to learn how to do these assays.) Early human studies have examined the effects of ascorbic acid on dietary nitrate and nitrite absorption. Our collaborators in Bangkok have administered low levels of nitrite to thalassemia patients and we have worked with them to show that VASP phosphorylation is affected, as a parameter for measuring platelet inhibition (as described in DK 025104). Most encouraging, we have established a robust collaboration with a muscle physiology group in Exeter, UK and we have been measuring the levels of nitrate at rest and with exercise in the human tissues and have confirmed that our animal results obtain for human subjects, including the high levels of nitrate in muscle and its decrease with robust exercise. We believe that muscle levels of nitrate must be considered in dietary interventions, for pharmacological effects or for affecting performance. These results have also been recently published. Several new projects have very recently been initiated. First, we are working with groups at NIH who are studying the human microbiome to see if any of these agents have a particularly robust effect on NO metabolic pathways. We are also working with the NIDDK mass spectroscopy to follow 15N labelled nitrate in the various animal models. We have begun measuring nitrate and nitrite levels in whole rodent eyes as a beginning of measuring these levels in various compartments of the eye in larger animal models and perhaps in human samples. Our new results in porcine studies very importantly at MedStar suggest that the lacrimal glands pump nitrate ions from blood into the tears where several processes could convert it to nitrite and/or NO for adsorption into the eye. We have extensive studies planned with the Veterinary staff of MedStar to track NO metabolic pathways in these eyes and how administration of nitrate via various paths affect these levels. We believe that this process contributes to the normal function of the retina and eye and may be useful as therapy of conditions such as glaucoma and macular degeneration. Recently we have been working with the NIDDK mass spectrometry unit to study these processes with 15N-labeled nitrate. These animal model studies have led to clinical studies with the Department of Ophthalmology at the University of Texas Southwestern Medical Center to obtain tears from individuals with normal ocular function, and later with the "dry eye syndrome" and other conditions to see how our animal model results jibe with normal and abnormal human eye processes.