An ¹⁸oxygen inhalation method for determination of total body formation of nitric oxide in humans

Abstract

The formation of nitric oxide (NO) and the subsequent conversion of the NO formed into nitrate require molecular oxygen. Based on this fact, we have recently developed a method using inhalation of the stable oxygen isotope, i.e. 18O2, to determine total formation of NO in small laboratory animals. The method has now been further developed to be applicable also in humans. Five healthy awake male subjects inhaled a gas mixture of unlabelled and 18-labelled oxygen (approximate ratio 4:1) in nitrogen from a closed breathing system equipped with eliminators for carbon dioxide and water vapour. The ratio of unlabelled to 18-labelled oxygen, as well as the total oxygen concentration during the inhalation, were monitored. Venous blood samples were taken before and after the inhalation for analysis of unlabelled and 18O-labelled nitrate by gas chromatography/mass spectrometry. The procedure was repeated with the same protocol on a later occasion, during ongoing treatment with the NO synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA). The average nitrate level in plasma in the absence of L-NMMA was 26 micromol l-1. The rate of total synthesis of NO was estimated to be 0.38 +/- 0.06 mu mol kg-1 h-1, corresponding to a total body formation of 600-700 mu mol/24 h in an adult male. Infusion of L-NMMA caused an increase in mean arterial blood pressure from 86 +/- 4 to 99 +/- 5 mmHg (P<0.05). The average plasma level of nitrate during infusion of L-NMMA was 24 mu mol l-1. NO formation during infusion of L-NMMA was 0.17 +/- 0.03 mu mol kg-1 h-1, i.e. significantly (P<0.05) lower than in the absence of L-NMMA. We suggest that the described method allows direct determination of total NO formation in man. The method may be useful in the study of various experimental and pathophysiological conditions affecting NO formation.

Implication of steady state concentrations of nitrite and nitrate metabolites of nitric oxide in plasma and whole blood in healthy human subjects

“…When these kinetic parameters derived from the NO3load study were taken into account with NOx concentration at the steady state, it was possible to estimate the NOx formation rate under the steady state by an analogous process to determine the infusion rate of a drug to achieve a certain steady state (equation 4). The NOx formation rate derived of 0.5 mol/kg per h is consistent with values obtained as estimates of NO (or NOx) formation reported in anaesthetized and awake rats (0.33-0.85 moles/kg per h) using isotope-labelled oxygen, 23 as well as in humans (0.38-0.96 moles/kg per h) using either isotopelabelled oxygen, 24 isotope-labelled arginine 25 or theoretical analysis without an isotope-labelled substrate. 26 Because we evaluated kinetic parameters in whole blood and in erythrocytes for the first time, there are no data available for comparison.…”

New Methods to Evaluate Endothelial Function: A Search for a Marker of Nitric Oxide (NO) In Vivo: Re-evaluation of NOx in Plasma and Red Blood Cells and a Trial to Detect Nitrosothiols

“…According to the equation, we determined a NOx formation rate of about 0.5 m mol / kg × h, in concordance with values of NO formation estimated in anesthetized and awake rats (0.33 -0.85 m mol / kg × h) using isotopelabeled oxygen (41), in humans (0.38 -0.96 m mol / kg × h) using isotope-labeled oxygen (42) or arginine (43), or by theoretical analysis without isotope-labeled substrate (44). It should be noted that the NOx formation rate does not necessarily reflect NO formation directly in vivo; however, it would be acceptable as an alternative measure when the real NO formation rate is hard to evaluate.…”

Plasma Nitrate and Incidence of Cardiovascular Disease and All‐Cause Mortality in the Community: The Framingham Offspring Study

“…Urinary nitrate excretion usually exceeds dietary intake, which indicates a significant endogenous nitrate formation rate . NO synthesis has been identified as the major endogenous source of nitrite and nitrate . However, NO is highly reactive and has a short half‐life in vivo, which precludes its measurement outside of experimental settings.…”

“…Under physiological conditions, the major proportion of endogenous NO generation is generally attributed to endothelial NOS. Based on the highly sensitive 18 oxygen inhalation method, the rate of total endogenous NO synthesis in men is 0.6 to 0.7 mmol (corresponding to 37–43 mg nitrate)/24 h (≈0.4 μmol/kg per hour) . The endogenous NO synthesis may increase substantially under several conditions such as exercise or high‐altitude hypoxia .…”