NO Metabolomics Core Facility

NO Metabolomics Core Facility

NO Metabolomics @
Vascular Medicine Institute
E1240 BST
200 Lothrop St
Pittsburgh, PA 15261
412-624-0462

Nitric oxide (NO) is a gaseous signaling molecule that regulates a number of physiological processes. NO is enzymatically generated by Nitric Oxide Synthase (NOS), which catalyzes the conversion of L-arginine to L-citrulline. In addition to its immediate and local effects, recent data demonstrate that NO can be oxidized to the molecule nitrite (NO2-) to be transported in the vasculature, and this endocrine transport of NO allows for NO bioactivity in more distal microvasculature. NO can also be rapidly inactivated to the molecule nitrate (NO3-), through its reaction with oxygenated hemoglobin in the blood vessel or myoglobin in the heart as well as other enzymes (shown below). Dysregulation of the NO homeostasis can occur at several different points of the pathway and is a central component of a number of pathological processes.

The goal of the NO Metabolomics Facility is to assist investigators in determining changes in the NO pathway in any experimental model of interest.

The Facility is experienced in assays to measure each step in the NO pathway and currently offers the following assays:

Measurement of nitrite concentration
Measurement of nitrate concentration
Measurement of S-nitrosothiol/iron-nitrosyl concentration
Measurement of NOS expression levels
Measurement of NOS activity

Assays can be performed in a variety of experimental systems, including clinical samples as well as samples from animal or cell culture models.

For more information, please contact Sruti Shiva, PhD.

Services

Reductive Chemiluminescense

The facility houses two Sievers Nitric Oxide Analyzers for high sensitivity NO detection using ozone chemiluminescence. The principle of chemiluminescent NO detection is based on the rapid reaction of NO in the gas phase with ozone (O₃), which yields NO₂* in an excited state. As the excited electron returns to its ground state, a photon is emitted and is detected as chemiluminescence (hν):

NO + O₃ → NO₂* + O₂

NO₂* → NO₂ + hν

This emitted light is detected and amplified by a photomultiplier tube (PMT), to generate an electrical signal. The specificity of this method for NO is due to the unique properties of the NO molecule, including its ability to exist as a gas and its rapid reaction rate with ozone. Combined with reductive chemistry, the following NO-derived species can be measured in clinical specimens, biological samples, as well as cell culture models. For more information about chemiluminescence techniques, click here.

Nitrite & S-nitrosothiol – Tri-iodide reduction | Publications

Nitrate – Vanadium Chloride reduction | Publications

Iron-nitrosyl concentration – Potassium Ferricyanide reduction | Publications

Nitrite/nitrate reductase activity – No reduction required to detect NO production | Publications

Non-Chemiluminescense Based Assays

Measurement of NO synthase expression and activity –NOS activity is assessed by measuring the conversion of radiolabeled arginine to citrulline. This can be combined with Western blots for NOS expression and phosphorylation to obtain a full profile of NOS expression and activity. | Publications

Nitrotyrosine levels – Measured by ELISA and/or Western blot

S-nitrosation by biotin switch – Switch assay to label S-nitrosated proteins with biotin for Western blot detection | Publications

People

Sruti Shiva, PhD

Director, NO Metabolomics Core Facilty
Professor of Pharmacology and Chemical Biology

Office: 412-383-5854
Lab: 412-624-0462
Email: sss43@pitt.edu
View Profile

Yinna Wang, PhD

Senior Research Technician
NO Metabolomics Core Facility

Lab: 412-624-0462
Email: yiw24@pitt.edu

Publications

Chemiluminescense techniques

MacArthur PH, Shiva S, Gladwin MT. (2007). Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescense. J Chromatogr B Analyt Technol Biomed Life Sci. 851(1-2):93-105. PMID: 17208057.

Pelletier MM, Kleinbongard P, Ringwood L, Hito R, Hunter CJ, SChechter AN, Gladwin MT, Dejam A. (2006). The measurement of blood plasma nitrite by chemiluminescense: pitfalls and solutions. Free Radic Biol Med. 41(4):541-8. PMID: 16863986.

Nitrite & S-nitrosothiol

Cosyns SM, Shiva S, Lefebvre RA. (2015). Protective effect of exogenous nitrite in postoperative ileus. Br J Pharmacol. 172(20):4864-74. PMID: 26227770.

Rix PJ, Vick A, Attkins NJ, Barker GE, Bott AW, Alcorn H Jr, Gladwin MT, Shiva S, Bradley S, Hussaini A, Hoye WL, Parsley EL, Masamune H. (2015). Pharmacokinetics, pharmacodynamics, safety, and tolerability of nebulized sodium nitrite (AIR001) following repeat-dose inhalation in healthy subjects. Clin Pharmacokinet. 54(3):261-72. PMID: 25421879. PMCID: PMC4335132.

Neye N, Enigk F, Shiva S, Habazetti H, Plesnila N, Kuppe H, Gladwin MT, Kuebler WM. (2012). Inhalation of NO during myocardial ischemia reduces infarct size and improves cardiac function. Intensive Care Med. 38(8):1381-91. PMID: 22653370.

Zuckerbraun BS, Shiva S, Ifedigbo E, Mathier MA, Mollen KP, Rao J, Bauer PM, Choi JJ, Curtis E, Choi AM, Gladwin MT. (2010). Nitrite potently inhibits hypoxic and inflammatory pulmonary arterial hypertension and smooth musce proliferation via xanthine oxidoreductase-dependent nitric oxide generation. Circulation. 121(1):98-109. PMID: 20026772.

Zhao XJ, Wang L, Shiva S, Tejero J, Myerburg MM, Wang J, Frizzell S, Gladwin MT. (2013). Mechanisms for cellular NO oxidation and nitrite formation in lung epithelial cells. Free Radic Biol Med. 61:428-37. PMID: 23639566. PMCID: PMC3883890.

Nitrate

Curtis E, Hsu LL, Noguchi AC, Geary L, Shiva S. (2012). Oxygen regulates tissue nitrite metabolism. Antioxid Redox Signal. 17(7):951-61. PMID: 22098300. PMCID: PMC3411351.

Iron-nitrosyl concentration

Curtis E, Hsu LL, Noguchi AC, Geary L, Shiva S. (2012). Oxygen regulates tissue nitrite metabolism. Antioxid Redox Signal. 17(7):951-61. PMID: 22098300. PMCID: PMC3411351.

Nitrite/nitrate reductase activity

Curtis E, Hsu LL, Noguchi AC, Geary L, Shiva S. (2012). Oxygen regulates tissue nitrite metabolism. Antioxid Redox Signal. 17(7):951-61. PMID: 22098300. PMCID: PMC3411351.

Tiso M, Tejero J, Basu S, Azarov I Wang X, Simplaceanu V, Frizzell S, Jayaraman T, Geary L, Shapiro C, Ho C, Shiva S, Kim-Shapiro DB, Gladwin MT. (2011). Human neuroglobin functions as a redox-regulated nitrite reductase. J Biol Chem. 286(20):18277-89. PMID: 21296891. PMCID: PMC3093900.

Shiva S, Huang Z, Grubina R, Sun J, Ringwood LA, MacArthur PH, Xu X, Murphey E, Darley-Usmar VM, Gladwin MT. (2007). Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration. Circ Res. 100(5):654-61. PMID: 17293481.

Measurement of NO synthase expression and activity

Cosyns SM, Shiva S, Lefebvre RA. (2015). Protective effect of exogenous nitrite in postoperative ileus. Br J Pharmacol. 172(20):4864-74. PMID: 26227770.

Cruz JA, Bauer EM, Rodriguez AI, Gangopadhyay A, Zeineh NS, Wang Y, Shiva S, Champion HC, Bauer PM. (2012). Chronic hypoxia induces right failure in caveolin-1-/- mice. Am J Physiol Heart Circ Physiol. 302(12):H2518-27. PMID: 22505641. PMCID: PMC3378264.

S-nitrosation by biotin switch

Wang X, Kettenhofen NJ, Shiva S, Hogg N, Gladwin MT. (2008). Copper dependence of the biotin switch assay: modified assay for measuring cellular and blood nitrosated proteins. Free Radic Biol Med. 44(7):1362-72. PMID: 18211831. PMCID: PMC2396494.

Kamga Pride C, Mo L, Quesnelle K, Dagda RK, Murillo D, Geary L, Corey C, Portella R, Zharikov S, St Croix C, Maniar S, Chu CT, Khoo NK, Shiva S. (2014). Nitrite activates protein kinase A in normoxia to mediate mitochondrial fusion and tolerance to ischaemia/reperfusion. Cardiovasc Res. 101(1):57-68. PMID: 24081164. PMCID: PMC3868348.

Events

In March 2012, the Vascular Medicine Institute had several special visitors, including Lord David Trimble, House of Lords, UK, and Ryan Clark, former Pittsburgh Steelers Free Safety. They each volunteered to use the Nitric Oxide Analyzer (NOA) to measure the levels of nitric oxide in their sinuses. Here are the results!