Clinical Applications of Nitrite and Nitrate

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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). However, 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.