Analysis of dietary nitrate incorporation in porcine ocular tissues after supplementing 15N-labeled sodium nitrate
Authors: Park Ji Won , Piknova Barbora , Tunau-Spencer Khalid , Cai Hongyi , Walter Peter , Jenkins Audrey , Hellinga David , Parver Leonard , Schechter Alan 
Published: May 2023

The mechanisms for the generation of nitric oxide (NO) include reduction of nitrate (NO3-) and nitrite (NO2-) as well as the classic nitric oxide synthase (NOS) pathways. In the reductive pathways, ingested nitrate is reduced to nitrite, mainly by oral bacteria and to some extent by tissue enzymes, then these nitrite ions can be reduced to NO by several endogenous proteins or, in stomach, by acid disproportionation. Dietary nitrate, rich in green leafy vegetables and beets, has beneficial effects in lowering blood pressure and improving vascular health by enhancing NO bioavailability. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite ions that are similar to those in blood. More importantly, cornea and sclera exhibited a nitrate reduction capability. To extend our knowledge on nitrate metabolism in the eye, in the present study, we fed pigs (Yorkshire domestic cross swine weighing 35-40kg, housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-accredited facility with environmental enrichment) with either NaCl or Na15NO3 and assessed the amount of 15NO3- and 15NO2- in various parts of the eye by LC-MS/MS at 3 or 24h after supplementation. We also measured the total concentrations of nitrate and nitrite ions using a standard chemiluminescence nitric oxide analyzer. At 3h, there was a marked increase in both 15NO3- and 15NO2- in all parts of the eye; in particular, aqueous and vitreous humor showed very high 15NO3- contents (77.5 and 74.5% respectively), similar to plasma (77.1%) and even higher 15NO2- contents (39.9% and 35.3% respectively) than plasma (19.8%). Cornea (64.4%) and sclera (63.4%) also showed notable increases in 15NO3- content at 3h, then retina (48.8%) and lens (44.8%) followed. In 15NO2- percent analysis at 3h, except aqueous and vitreous humor, other eye parts showed similar levels to plasma. At 24h, 15NO3- contents decreased significantly compared to the 3h point, but it was still higher than the baseline in all ocular components. The 15NO2- contents at 24h were almost back to the baseline. Absolute values of nitrate and nitrite ions in these tissues show similar patterns as shown in 15N analysis. These results indicate that dietary nitrate supplementation has a vast impact on the levels of nitrate and nitrite and likely NO as well in the eye. NO generation via the nitrate-nitrite reductive pathway may contribute to various physiological and pathophysiological processes in the eye and may be useful therapeutically. This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.