Clinical Applications of Nitrite and Nitrate

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

Abstract

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 possible. 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 and, as noted below, we are developing these methods in cell culture and animal experiments. Our collaborators in Perth, Australia 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. We are now initiating a project with them to study nirate and nitrite levels in samples from young and old individuals to see if muscle atrophy or even the sarcopenia syndrome is related to changes in these ions with aging and if administration of nitrate improves these conditions. 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 with the NIDDK mass spectroscopy group to follow 15N labelled nitrate in various animal models after ingestion of labelled nitrate and related compounds. We find that many tissues, including muscle and (perhaps surprisingly) the eye rapidly take up ingested nitrate suggesting the possible clinical use of these agents. In parallel studies we have been measuring steady state levels of nitrate in many organs and have found (considering its levels as well as the mass of muscle in the mammalian body)that skeletal muscle may be among the largest "reservoirs" for nitrate and then, assuming strong reductive reactions, NO bioactivity. Indeed we have been developing the idea of the general importance of such reservoirs, which may include bone and other tissues, as well as the metabolic pathways among organs, as determining the availability of NO to respond to various physiological needs. Such concepts may be useful to better understand the overall functions of NO in the body as well as pharmacological potentials. Second, we have begun measuring nitrate and nitrite levels in whole rodent eyes as a beginning of measung these levels in various compartments of the eye in larger animal models and perhaps in human samples. Our early studies show enhanced levels in the cornea and sclera, which may suggest surface reduction to nitrite or NO. Further, our results primarily in porcine studies 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 further NO metabolic pathways in these eyes and how administration of nitrate via various paths affect these levels. We believe that these processes contributes to the normal function of the retina and eye and may be useful as therapy of conditions such as glaucoma and macular degeneration. 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. We have been working with ophthalmologist Dr. David Parver, since the death of our collaborator Dr. Leonard Parver, on planing clinical studies of nitrate administration to the eyes of volunteers to further study these processes in human eyes. In studies with the diabetes and obesity groups of NIDDK, we have been measuring blood levels of nitrate and nitrite in patients with pathway- selective vs. non-selective insulin resistance and endothelial dysfunction to see if this pathway accounts for some of the processes associated with the loss of correct insulin action. In other studies we have shown that NO production by the reductive pathway increases to compensate with NOS inactivation, for example with the loss of the nNOS system for enzymatic synthesis from arginine. In parallel we have been working to validate a cyclicGMP assay in blood and tissues to determine more precisely how much NO-bioactivity occurs as a result of the nitrate-related pathways so as to better understand the basic biochemical mechanisms that are likely to have evolved in all mammals as a result of these complicated pathways. We believe these pathways have been optimized to supply NO-like activity to the body despite large variations in nitrogen oxides in the human diet.