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 setup diagram: NO reaction, ozone generator, photomultiplier, cooler, vacuum inlet." class="xfigimg" src="/files/ftp_upload/63107/63107fig01.jpg" data-altupdatedat="1747911167000" 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="Purge and trap setup diagram for gas analysis; features NaOH trap, heated jacket, injection port." class="xfigimg" src="/files/ftp_upload/63107/63107fig02.jpg" data-altupdatedat="1747911167000" 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="Flowchart of chemical reaction process; PBS, HgCl₂, and sample incubation for compound measurement." class="xfigimg" src="/files/ftp_upload/63107/63107fig03.jpg" data-altupdatedat="1747911167000" 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

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...

Watch this Scientific Journal Video about Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds at JoVE.com

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

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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JoVE Journal

Medicine

3.8K Views.  Massachusetts General Hospital. Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity. 

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

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Cancer Research

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

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.

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.

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 ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Immunology and Infection

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

Capturing dynamic changes in the protein activation of enucleated red blood cells poses methodological challenges, like the preservation of dynamic changes to acute stimuli for later assessment. The presented protocol describes sample preparation and staining techniques that enable preservation and analysis of relevant protein changes and subsequent detection.

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute ...

Biochemistry

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Biology

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...

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

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Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

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En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements