Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Summary

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

Abstract

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Introduction

Since Salvador Moncada and Nobel laureates Robert Furchgott, Louis Ignarro, and Ferid Murad identified nitric oxide (NO) as the previously known endothelial-derived relaxation factor (EDRF), the central role of NO has been established in several key mechanisms spanning throughout vascular biology, neurosciences, metabolism, and host response^{1,2,3,4,5,6,7}. Exogenous administration of NO gas has become an established treatment for respirato....

Protocol

The procedures indicated in this protocol are in accordance with the review board of Massachusetts General Hospital. The blood samples that were used had been collected during a previous study and were de-identified for the current purpose¹⁸.

NOTE: See manufacturer's instructions for specific guidance regarding optimal connections between tubing and glassware constituting the purge vessel, washing, and general maintenance. Connections need to be firm and carefully made not to damage the glassware. Identify the components of the glass purge vessel: gas inlet line, purge vessel with heating jacket and condenser, so....

Results

The NO-consumption by cell-free Hb assay was used in samples containing known concentrations of cell-free oxyHb (Figure 4). As one heme of oxyHb stoichiometrically releases one NO molecule in the assay, purified cell-free oxyHb is used to build the calibration curve for the assay (Supplemental Figure 3).

The dose-response relationship between cell-free Hb (measured with a colorimetric assay) and NO consumption in patients coming off cardiopulmonar.......

Discussion

Due to the high sensitivity, chemiluminescence-based assays for the determination of NO and related compounds have a high risk of NO₂^- contamination. Each reagent (especially the NO₂^- blocking solution) and dilutant (including ddH₂O) used in the experiment should be tested for its NO₂^- content to correct for background signal. Nitrite is extremely reactive with a half-life in whole blood around 10 min and rapidly generates NO₃^-

Materials

Name	Company	Catalog Number	Comments
Acetic Acid	Sigma	45754	500 mL - liquid
Antifoam B Emulsion	Sigma	A5757	250 mL - liquid
DETA NONOate	Cayman	82120	10 mg
Gibco DPBS (1x) no calcium, no magnesium	ThermoFisher	14190144	500 mL
Hydroochloric Acid 37% (1 M)	Sigma	258148	500 mL - liquid
Iodine	SAFC	207772	100 g - solid
Kimwipes	Kimtech	34155
Mercury (II) Chloride ACS reagent> 99.5%	Sigma	215465	100 g - solid (dissolve in water)
Mili-Q Water Purification System	Millipore
Model 705 RN 50 μL syringe	Hamilton	80530	Microliter syringe
Model 802 N 25 μL Syringe	Hamilton	84854	Microliter syringe
N-ethylmaleimide	Sigma	4260	25 g - crystalline
Nitric Oxide Analyzer + Bundle Software - Purge Vessel	Zysense	NOA 280i	Chemiluminescence Detector
Nonidet p-40  (NP-40)	ThermoFisher	85125	10% - 500 mL
Potassium hexacyanoferrate (III) ACS reagent≥ 99%	Sigma	244023	100 g - powder
Potassium Iodide ACS reagent> 99%	Sigma	221945	100 g - solid
Potassium Nitrite cryst. For analysis EMSURE ACS	Supelco	105067	250 g - crystalline
PowerGen 125	Fisher Scientific	14-359-251	Mechanic Homogenizer
RV3 Two Stage Rotary Vane Pump	Edwards	A65201906	Vacuum Pump - Bundled with analyzer
Sodium Heparin - BD Hemogard Clo	BD Biosciences	BD367871	75 USP Units
Sodium hydroxide anhydrous ACS reagent ≥ 97%	Sigma	795429	1 kg - pelletts
Sodium Nitrate ACS reagent ≥ 99%	Sigma	221341	500 g - powder
Sodium nitrite ≥ 99%	Sigma	S2252	500 g - crystalline
Sulfanilamide ≥ 98%	Sigma	S9251	100 g - solid
Vanadium (III) Chloride	Sigma	112393	25 g - solid - Caution (exothermic)
Whatman 1 Filter Paper	Sigma	WHA10010155