Name: Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds (Video) | JoVE Journal
Uploaded: 2022-02-16T14:00:00.000+00:00
Duration: 8 min 23 s
Description: Learn chemiluminescence-based assays for sensitive detection of nitric oxide metabolites in biological samples. This protocol enables precise measurement without dedicated personnel, supporting research in neuroscience, biochemistry, and therapeutic applications.

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The protocols reported in this manuscript were made possible by the accumulated contributions of previous fellows of Dr. Warren Zapol&#39;s laboratory of Anesthesia Research in Critical Care, Department of Anesthesia at Massachusetts General Hospital. We acknowledge the contribution of Drs. Akito Nakagawa, Francesco Zadek, Emanuele Vassena, Chong Lei, Yasuko Nagasaka, Ester Spagnolli and Emanuele Rezoagli.

L.B. receives salary support from K23 HL128882/NHLBI NIH as principal investigator for his work on hemolysis and nitric oxide. LB receives grants from "Fast Grants for COVID-19 research" at Mercatus Center of George Mason University and from iNO Therapeutics LLC. B.Y. is supported by grants from an NHLBI/#R21HL130956 and DOD/The Geneva Foundation (W81XWH-19-S-CCC1, Log DM190244). B.Y. received patents at MGH on the electric generation of nitric oxide.
L.B. and B.Y. have filed patent application for NO delivery in COVID-19 disease PCT application number: PCT/US2021/036269 filed on June 7, 2021. RWC receives salary support from Unitaid as the principal investigator for technology development aimed at decentralized diagnosis of tuberculosis in children located in low-resource settings.

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-</...

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 response1,2,3,4,5,6,7. Exogenous administration of NO gas has become an established treatment for respiratory failure due to pulmonary hypertension in the newborn8. Nitric oxide gas has also been investigated for treatment of respiratory syncytial virus (RSV) infection, malaria and other infective diseases, ischemia-reperfusion injury, and for prevention of acute kidney injury in patients undergoing cardiac surgery9,10,11,12. The need for precise measurement techniques to assess the levels of NO, its metabolites, and those of its target proteins and compounds arises from both mechanistic and interventional studies.
Due to its high reactivity, NO may undergo different reactions depending on the biological matrix in which it is produced and/or released. In the absence of hemoglobin (Hb) or other oxy-hemoproteins, NO is oxidized almost completely to nitrite (NO2-).
2NO + O2 → 2NO2
NO2 + NO → N2O3
N2O3 + H2O → NO2- + H+
NO first undergoes autoxidation with molecular oxygen (O2) to yield nitrogen dioxide (NO2) and reacts with NO2 itself to generate dinitrogen trioxide (N2O3). One molecule of N2O3 reacts with water (H2O) to form two molecules of NO2- and a proton (H+)13. Within whole blood14,15, NO and NO2- are rapidly converted to nitrate (NO3-) as these molecules react avidly with the oxidized heme groups of Hb [Hb-Fe2+-O2 or oxyhemoglobin (oxyHb)] to yield NO3-. This reaction is coupled with the transition of the heme group to the ferric state [Hb-Fe3+ or methemoglobin (metHb)]:
Hb-Fe2+-O2 + NO → Hb-Fe3+ + NO3-
The red blood cell (RBC) barrier and the space immediately adjacent to the endothelium are the main factors limiting this reaction and allowing a small portion of the NO released by the endothelium to act as EDRF16,17. In fact, cell-free Hb in the circulation is known to disrupt vasodilation in experimental and clinical settings17,18. Within the RBC, depending on oxygenation and NO2- concentration, a portion of NO reacts with deoxyhemoglobin (Hb-Fe2+) to form iron-nitrosyl Hb (Hb-Fe2+-NO or HbNO):
Hb-Fe2+ + NO → Hb-Fe2+-NO
In the RBC15,17, NO2- can form Hb-Fe3+ by reducing Hb-Fe2+ leading to the release of NO, which in turn binds Hb-Fe2+-O2 (preferentially) or Hb-Fe2+.
The generation of NO-derivatives should not be considered strictly unidirectional as NO can be regenerated from NO2- and NO3- in various tissues and by different enzymes (e.g., by intestinal bacteria or within mitochondria, particularly under hypoxic conditions)19,20.
A variable amount of NO produced (or administered) leads to the downstream generation of S-nitrosothiols, mainly by thiol transnitrosation from N2O3 in the presence of a nucleophile creating a NO+ donor intermediate (Nuc-NO+-NO2-):
N2O3 + RS- → RS-NO + NO2-
Another possibility for S-nitrosothiols generation is nitrosylation of oxidized thiols (NO reacting with an oxidized thiol):
RS• + NO → RS-NO
This mechanism and direct thiol oxidation by NO2 might be possible only in very specific conditions which are described elsewhere21. S-nitrosothiols range from light molecules like S-nitrosoglutathione to large thiol-containing proteins. S-nitrosohemoglobin (S-NO-Hb) is formed by nitrosation of a thiol group of a conserved cysteine residue in the β-chain (β93C)22.
The generation and metabolism of S-nitrosothiols are part of important regulatory mechanisms. Examples include regulation of glutathione, caspases, N-methyl-D-Aspartate (NMDA), and ryanodine receptors23,24,25,26,27,28. Previously considered as a major mediator of NO biology in vivo, nitrosated albumin (S-nitroso-albumin) seems to be a NO/NO+ transporter without any specific additional biological activity29.
When measuring the concentration of NO and its derivatives from a specific biological sample within a biological matrix, it is important to consider characteristics such as acidity, oxygenation, temperature, and the presence of reagents. Examples include administered exogenous NO donors and, in the setting of acute inflammation, hydrogen peroxide (H2O2) reacting with NO2 leading to generation of supernormal concentration of free radicals like peroxynitrite (ONOO-)21. In addition to the analytical method that is employed, the preanalytical phase of sample preparation and storage is fundamental. Downstream reactions that do not represent the in vivo NO activity shall be predicted, considered, and blocked. A valid example is the instability of S-NO-Hb, requiring a dedicated treatment of blood samples when it is targeted for measurement22.
Chemiluminescence-based assays are the gold standard for detecting the levels of NO and its main metabolites [NO2-, NO3-, S-NO and iron-nitrosyl complexes (Fe-NO)] in any biological fluid, including tissue homogenates30,31. These methods rely upon the chemiluminescence detector (CLD), a device that houses the reaction of NO with ozone (O3), generating NO2 in an excited state (NO2•). Relaxation of NO2• causes emission of a photon of light that is detected by a photomultiplier tube, generating an electric signal that is directly proportional to the NO content of the sampled gas mixture32. A simplified schematic of the CLD is represented.
<img alt="Chemiluminescence detection diagram showing NO and O₃ reactions; photomultiplier tube setup." class="xfigimg" src="/files/ftp_upload/63107/63107fig01.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: Simplified schematic of a chemiluminescence detector for nitric oxide gas. Chemiluminescence-based detection of nitric oxide (NO) is the stoichiometric generation of one photon per NO gas molecule that is introduced in the chemiluminescence detector (CLD). The chemiluminescence reaction is obtained in a designated chamber supplied with ozone (O3) from an internal generator, which is kept at negative pressure by connection with an external pump, allowing continuous and constant inflow of sample gas. The generation of O3 requires diatomic oxygen (O2) that is supplied by a dedicated O2 tank connected with the CLD (other manufacturers provide CLDs operating with ambient air). Within the reaction chamber, each molecule of NO gas contained in the sampled gas reacts with oxygen to yield one molecule of nitrogen dioxide in the activated state (NO2*). By returning to its ground state, each NO2* molecule emits one photon that is detected by a photomultiplier tube (PMT) located adjacent to the reaction chamber. The PMT with the associated amplifier and central processing unit produces a signal proportional to the photon count and the number of NO molecules in the reaction chamber. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The sample inlet of the CLD can be connected to a glassware system containing a reaction chamber for liquid samples. The system is continuously purged with an inert gas such as nitrogen, helium, or argon, transferring NO from the reaction chamber to the CLD. Liquid-phase samples are injected through a dedicated membrane into the purge vessel.
<img alt="Chemical gas system diagram, showing purge vessel, NaOH trap, and gas flow setup." class="xfigimg" src="/files/ftp_upload/63107/63107fig02.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2: Structure of a purge vessel for chemiluminescence-based detection of nitric oxide gas The purge vessel (right) allows for the detection of nitric oxide (NO) gas or any other compound that can be readily converted to NO gas when released from a liquid phase reagent. The inert gas inlet is connected to a source (tank) of an inert gas such as Argon, Xeon, or diatomic nitrogen (N2). The needle valve (opens to the left) is used for pressure control within the purge vessel and can be completely removed to clean the vessel. The injection port is covered by a cap with a membrane septum for sample injection. The membrane should be replaced often. A heated jacket surrounds the reaction chamber and is connected to a hot water bath to perform the VCl3 in HCl assay. The purge vessel outlet is connected to the chemiluminescence detector (CLD) or to the NaOH trap (required for VCl3 in HCl assays). To drain the reaction chamber content, first close the stopcocks at the inert gas inlet and the purge vessel outlet, close the needle valve, remove the cap at the injection port and finally open the stopcock at the drain. The NaOH trap (left) is required to be placed inline between the purge vessel and the CLD if the VCl3 in HCl assay is performed because of the corrosivity of HCl. The connection to the CLD always requires an intense field dielectric (IFD) filter to be placed between the CLD and the output of the purge vessel (or the NaOH trap, if used). The IFD filter removes airborne particles and stops liquid from passing through the purge vessel. PTFE = polytetrafluoroethylene. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As a consequence, any compound that can be converted to NO through a specific and controlled chemical reaction can be detected with high sensitivity in any biological fluid and tissue homogenate24. Direct measurement of gas-phase NO through chemiluminescence is performed in both experimental and clinical settings. These techniques are extensively described elsewhere33,34,35. Measurement of NO2-, S-nitrosothiols, S-nitrosated proteins, and Fe-NOs can be performed by adding samples in a reaction mixture with triiodide (I3-), which stoichiometrically releases NO gas from all these compounds:
I3- → I2 + I-
2NO2− +2I− +4H+ → 2NO + I2 +2H2O
I3− + 2RS-NO → 3I− + RSSR + 2NO+
2NO+ + 2I− → 2NO + I2
while I3- does not react with NO3-15. Precise measurements of each compound are made possible by pre-treatment of sample aliquots with acidified sulfanilamide (AS) with or without mercuric chloride (HgCl2). Specifically, pre-treatment with AS removes all NO2- content. As a consequence, the NO content measured by the CLD only reflects the sum of S-NOs and Fe-NOs concentration. Injection of HgCl2 in a sample aliquot before AS injection causes NO2- to be released by S-NO. Treatment with AS (leading to NO2- removal) ensures that the measured NO content only reflects the concentration of Fe-NOs. A series of subtractions between the assessments allow to calculate the precise concentration of the three NO derivatives22.
<img alt="Static equilibrium diagram; chemical assay method; process flowchart; NO2−, S-NOs, Fe-NOs analysis." class="xfigimg" src="/files/ftp_upload/63107/63107fig03.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
Figure 3: Steps in sample preparation for the I3- in acetic acid chemiluminescence assay. The sequential steps for preparation of the I3- in acetic acid chemiluminescence assay are illustrated. Use of light-protected centrifuge tubes is required. Tubes 1, 2, and 3 are those used to prepare for the assay. Another sample aliquot (tube 4) is needed for the VCl3 in HCl assay if the measurement of nitrate (NO3-) is required. Steps are indicated by numbers in red. Prefill (Step 1) as indicated with phosphate buffer saline (PBS) or HgCl2 before adding the sample volume. Add the sample volume (2) as indicated, vortex, and incubate for 2 min at room temperature (RT). Add (3) PBS or acidified sulfanilamide (AS) as indicated,vortex, and incubate for 3 min at RT. Run the assay (4). The concentration measured by the assay is the sum of the concentration of the compounds reported under each tube. Tube number 1 will allow measurements of nitrite (NO2-), S-nitrosothiols (S-NO), and iron-nitrosyl complexes (Fe-NOs) as a single signal. For nitrate (NO3-) measurement, samples shall be run with both I3- in acetic acid and VCl3 in HCl assays, and the value obtained from tube 1 should be subtracted from the one obtained from tube 4. *suggested quantities to be used for Hb analysis for determination of residual NO2-, S-nitrosohemoglobin and iron-nitrosyl-hemoglobin. <a href="https://www.jove.com/files/ftp_upload/63107/63107fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For NO3- measurement, Vanadium (III) chloride (VCl3) in hydrochloric acid (HCl) is used for conversion of NO3- to NO in the purge vessel in order to measure NO3- stoichiometrically with the CLD:
2 NO3-+ 3V+3 + 2H2O → 2NO + 3VO2+ + 4H+
To achieve a sufficiently fast conversion, the reaction needs to be performed at 90-95 °C. Reduction from NO3- to NO2- is coupled with reduction of NO2- to NO by HCl. Vanadium metal also reduces S-NOs liberating their NO moiety22,36. The final concentration obtained by CLD with VCl3 in HCl reflects the aggregate concentration of NO3-, NO2, and other nitrosated compounds. Subtraction of the latter value from the concentration yielded with CLD with I3- allows for the calculation of NO3- concentration36,37 (Figure 3).
In the NO consumption assay, the continuous release of NO in the purge vessel by NO donors like (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) generates a stable signal allowing quantifying cell-free oxyHb in the injected samples. The amount of NO consumed in the purge vessel is in a stoichiometric relationship with the amount of oxyHb in the sample38.
Protocols for measurement of NO2-, NO3-, S-nitrosothiols, iron-nitrosyl complexes, and NO consumption by cell-free Hb in plasma samples are illustrated. Studies on NO in the RBC environment require specific sample treatment followed by exclusion chromatography to measure extremely fragile S-NO-Hb and Hb-NO coupled with the determination of total Hb concentration15,22. Sample preparation is instrumental in correcting measurement. Pre-existence of NO2- in H2O and release of NO2- during the assay can lead to measurement of artificially higher concentrations of NO derivatives such as S-NO-Hb14,39. Important aspects of sample preparation are also presented.

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

chemiluminescence-based assays for detection of nitric oxide and its derivatives from autoxidation and nitrosated compounds

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

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.

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...

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

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 ...

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Learn chemiluminescence-based assays for sensitive detection of nitric oxide metabolites in biological samples. This protocol enables precise measurement without dedicated personnel, supporting research in neuroscience, biochemistry, and therapeutic applications.

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

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