Analytical Techniques for Assaying Nitric Oxide Bioactivity

Summary

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Abstract

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)¹. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO₂^-) and nitrate (NO₃^-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

Protocol

1. Whole Blood Collection

1. Collect venous blood from humans or from experimental animals in NEM/EDTA containing tubes.
2. Immediately spin down blood in a benchtop centrifuge at 14,300 rcf (relative centrifugal force) for 7 minutes to prepare plasma and red blood cell pellet.
3. Prepare plasma samples for high performance liquid chromatography (HPLC) and chemiluminescence detection (CLD) analysis.
   HPLC: Add 1:1 volume of cold methanol to plasma, vortex and centrifuge at 13,200 rpm for 10 minutes to precipitate plasma proteins. Collect supernatant for HPLC analysis.
   CLD: aliquot a sample and preincubate with sulfanilamide and mercuric....

Discussion

The methods described here for the quantification of relevant NO metabolites in multiple biological compartments will allow for fingerprinting of NO biology in health and disease that can be correlated with functional measurements of NO by the endothelium. These methods require simple sample preparation with potential for adapting for high throughput. The relative quantity of these molecules can help understand the production of NO and its metabolic fate in a number of experimental models of disease and even serial sampl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
N-ethylmaleimide	Thermo Fisher Scientific, Inc.	23030
EDTA	Sigma-Aldrich	E7889
Potassium Ferricyanide	Fluka	60299
HPLC	Eicom Corp	ENO-20
Autosampler	Alcott
DMT Myograph	ADInstruments
PowerLab	ADInstruments
Chemiluminescent	EcoPhysics	CLD 88Y
Centrifuge	Eppendorf	5415D
Acetylcholine	Sigma-Aldrich	A6625
R-(-) Phenylephrine	Sigma-Aldrich	P6126