MSACL 2016 US Abstract

Quantification of 15N-nitrite and 15N-nitrate in Human Biological Matrices: An LC-MS/MS Application to Study the Development of Nitrate Tolerance

Elizabeth Axton (Presenter)
Oregon State University

Bio: I am a third-year PhD student pursuing a dual major in Pharmaceutical Sciences and Toxicology at Oregon State University. I am a graduate student in the lab of Dr. Fred Stevens, and my research is in the development of nitrate tolerance. I am primarily interested in the mechanism of bioactivation of nitroglycerin, as well as the role of vitamin C in nitric oxide bioavailability. I received a B.S. in Biological Sciences at the University of California at Davis, where I trained under Dr. Oliver Fiehn.

Authorship: Elizabeth Axton, Jan F. Stevens
The Linus Pauling Institute, Oregon State University

Short Abstract

Glycerol trinitrate (GTN) is a prodrug that is metabolized by xanthine oxidase (XO) to release nitric oxide (NO), inducing vasodilation in patients with cardiovascular diseases. Patients develop tolerance to GTN after a few weeks. We hypothesize that inactivation of XO by oxidative stress impairs GTN metabolism, which can be prevented with allopurinol (inhibitor of the molybdenum site of XO) and vitamin C (scavenger of reactive oxygen species). Our strategy is to quantify nitrite and nitrate, the stable metabolites of NO, as an endpoint of GTN metabolism. Our novel LC-MS/MS method quantifies 15N-nitrite and 15N-nitrate with a LLOQ of 1 nM. Isotope labeling removes interference from high background levels of nitrite and nitrate. We demonstrate that vitamin C and allopurinol can prevent nitrate tolerance in human endothelial cells, and the co-treatments enhance nitrite production from XO.

Long Abstract

Introduction: Glycerol trinitrate (GTN) is a prodrug that is metabolized to release nitric oxide (NO) to induce vasodilation. GTN is denitrified by xanthine oxidase (XO) to produce NO2-, and subsequently NO. GTN is prescribed to patients who are suffering from cardiac disease because it reduces blood pressure, increases blood flow, and relieves chest pain. However, after a few short weeks, patients develop tolerance to GTN. The current solution to this problem is having transdermal patches that are worn only during the day. Often, GTN is reserved as an emergency treatment during cases of severe chest pain and restriction. The mechanism of nitrate tolerance has been extensively studied, yet we do not have a complete understanding of the underlying mechanism. We hypothesize that irreversible inactivation of xanthine oxidase by oxidative stress causes reduced GTN metabolism. Inactivation of XO can be prevented by treating with allopurinol, and inhibitor of the molybdenum site of XO, and Vitamin C (ascorbic acid), a scavenger of reactive oxygen species (ROS). In order to measure GTN bioactivation, we measure nitrite and nitrate, the stable metabolites of NO, with a novel LC-MS/MS method developed in our lab. This method is a significant contribution to the field because it allows for the quantification of isotopologues of nitrite and nitrate, which is necessary to remove interference by high background levels of these analytes.

Methods: Nitrite reacts with 2,3-diaminonaphthalene (DAN) under acidic conditions to produce 2,3-naphthotriazole (NAT). NAT was chromatographically separated on a Shimadzu LC System with an Agilent Extend-C18 5 µm 2.1 x 150 mm column and detected using a multiple reaction monitoring (MRM) method on an ABSciex 3200 QTRAP mass spectrometer operated in positive mode. Mass spectrometry allows for the quantification of 14N-NAT (m/z 170.1, quantification transition 170.1→115.1) and 15N-NAT (m/z 171.1, quantification transition 171.1→115.1). Both nitrite and nitrate demonstrated a LLOQ of 1 nM and a linear detector response (1 nM – 10 µM, 1 nM – 100 nM, respectively), and had minimal matrix effects (>98% recovery in culture medium).

Human endothelial cells (EA.hy926) were maintained in culture in T75 flasks with DMEM media containing 4.5 g/L glucose, and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in a humidified incubator at 37°C with 5% CO2. For treatments, cells were plated in 6-well plates, and nitrate tolerance was induced with a 5-hour pre-treatment of GTN. The cells were washed, and then treated with 15N3-GTN (1-20 µM) and in the presence and absence of ascorbic acid (1 mM) and allopurinol (1 mM). 100 µL of culture medium was derivatized with DAN for nitrite analysis. Nitrate was enzymatically reduced to nitrite prior to derivatization.

Results: Background levels of 14N-nitrite are high in water (91 ± 3 nM), blank DMEM (339 ± 28 nM), and in EA.hy926 cells with no treatment (461 ± 61 nM). Quantification of 15N-nitrite effectively removed the interference of high background levels of nitrite. A dose-response of with 15N3-GTN demonstrated that pre-treatment with GTN induces nitrate tolerance in human endothelial cells, as evidenced by reduced 15N-nitrite concentration. 15N-nitrate concentration was not affected by GTN pre-treatment, indicating that nitrate is not a suitable biomarker for nitrate tolerance. Both 15N-nitrite and 15N-nitrate demonstrated a linear dose response from 15N3-GTN treatment (R2=0.996 and 0.976, respectively). Co-treatments with ascorbic acid and allopurinol effectively prevented nitrate tolerance in human endothelial cells. Pre-treatment with GTN caused a 66% decrease in 15N-nitrite concentration compared to the vehicle control. In contrast, co-treatment with ascorbic acid and allopurinol prevented nitrate tolerance, as evidenced by the fact that pre-treatment with GTN caused only a 7% decrease in 15N-nitrite concentration. We performed both an MTT Assay and an ATP CellTiter-Glo® Luminescent Cell Viability Assay on the EA.hy926 cells, which demonstrated that our treatments were not cytotoxic and did not affect cell proliferation.

We hypothesized that nitrate tolerance is caused by reduced bioactivation of GTN due to the progressive inactivation of xanthine oxidase (XO). To test this hypothesis we incubated microbial XO with 20 µM GTN in the presence and absence of ascorbic acid (1 mM) and allopurinol (1 mM) in aerobic conditions at 37°C. Co-treatments with ascorbic acid and allopurinol increased XO-mediated nitrite production by 240%. Our results suggest that ascorbic acid and allopurinol mediate XO activity to prevent the development of nitrate tolerance.

Conclusion: LC-MS/MS quantification of 15N-nitrite and 15N-nitrate is sensitive, accurate, and reproducible. This novel method demonstrated that 15N-nitrite can be quantified as an endpoint of GTN metabolism, and that ascorbic acid and allopurinol may be an effective co-treatment strategy to prevent the development of nitrate tolerance.

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