Name: chemiluminescence-based assays for detection of nitric oxide and its derivatives from autoxidation and nitrosated compounds
Uploaded: 2022-02-16T14:00:00.000+00:00
Duration: 8 min 23 s
Description: 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

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-. The time that elapses between blood collection and centrifugation, or NO2- blocking, may therefore cause preanalytical errors and should be minimized15. In an assay using plasma samples, collect blood preferably in heparin tubes and centrifuge at 750 x g for 5 min at 4 &#176;C after thiol group blockade with 40 &#956;L of 200 nM NEM produced by dissolving 250 mg of NEM into 10 mL of phosphate-buffered saline (PBS). The stock solution can be stored at 4 &#176;C. Compounds such as nitroarginine, nitroprusside, and nitroglycerin that inhibit NO synthase (NOS) undergo slow non-quantitative conversion to NO, resulting in a baseline elevation. If the use of NOS inhibitors is part of the investigation, it is advised that blockers that do not contain nitro groups be used43. The inlet pressure in the purge vessel should match the continuous negative pressure made by the CLD for sampling. If the inlet pressure is low, the vacuum distillation of the medium may lead to contaminate gas purging lines and chambers, resulting in signal distortion. Furthermore, small volumes of air may enter the purge vessel and react with the solution to generate excessive NO. On the contrary, excessive pressure caused by a flow rate that is higher than sample withdrawal flow might release NO into the atmosphere leading to an underestimation of the signal15. In addition, both the VCl3 in HCl and the I3- in acetic acid protocol require multiple dilutions during preparation and multiple calculations to yield sample concentration. Every step should be carefully recorded as dilutions and calculations are recognized by many groups as the main source of error44.
Addition of NO2- blocking solution (step 2.1.2) causes red blood cells to lyse, reducing all OxyHb to MetHb. It avoids the rapid reduction of iron-containing proteins (primarily OxyHb) by NO2- with subsequent generation of NO3-. Treatment of samples with this solution upon collection is required to correctly measure NO2- and/or NO3-. In contrast, the solution should not be used when planning the NO consumption by cell-free Hb assay. Aliquots of the NO2- blocking solution should be stored together with the samples and used to remove the portion of signal caused by the solution itself. Finally, the dilution factors should be taken into account for calculating the final concentration of the measured metabolite. In the I3- in acetic acid assay, the NO2- concentration is obtained by subtracting the concentration of the aliquot containing AS without HgCl2 (S-NO and Fe-NO) from that of aliquot containing only PBS (NO2-, S-NO, Fe-NO). To calculate the concentration of S-NO (mercury-labile proteins), the concentration of the aliquot treated with AS with HgCl2 (Fe-NOs or mercury-stable nitroso-proteins) should be subtracted from that of the aliquot treated with AS without HgCl2. Before subtracting, the user should recall multiplying each aliquot&#39;s concentration by 0.8181 (270/330) to take into account the dilution factor used during sample preparations (Figure 3). The same concept applies for S-NO-Hb and Hb-NO measurement, where absolute concentrations are a fraction of measured Hb22. Depending on the type of sample, a precise count of the NO3- concentration may require running both the VCl3 in HCl protocol and the I3- in acetic acid protocol to finally subtract the concentration of NO2- from those of NO3- and NO2-. This is not always necessary in whole blood and plasma samples, for example, where NO3- typically far exceeds NO2-.
It is very important to remind that the CLD will be damaged if the NaOH trap is not in proper place when performing a VCl3 in HCl assay due to the corrosivity of HCl. The 1 M NaOH solution used to fill the NaOH trap can be purchased or prepared either by dissolving 4 g of NaOH salt in 100 mL of ddH2O or by diluting 5 mL of 50% NaOH in 100 mL of ddH2O. When further diluted to 10 &#181;M with ddH2O, the NaOH is very useful to remove protein debris by injection in the purge vessel.
The baseline signal detected by the CLD should be stable and within 0-5 mV. A higher baseline signal could be caused by air pollution (by NO and derivatives), and this can be verified by leaving the inlet in the open air. If a higher voltage is observed only in the purge vessel, while contamination of the tank with inert gas should be considered, it is most often due to the persistence of organic remnants or the presence of NO2- in the tube. Washing the purge vessel multiple times with ddH2O can help minimize the baseline voltage. If unsuccessful, the incubation of the purge vessel with 100 mM NaOH for 24-48 h followed by multiple washes with ddH2O helps eliminate pollutants. In the NO consumption by cell-free Hb assay, the required CLD signal is 70-100 mV for at least 20-30 min. If the signal is not stable (i.e., voltage decreases), an extra dose of 10 &#956;L of DETA NONOate can be added to the purge vessel. This can be repeated as needed. It is essential to remember that both unused powder and already expanded DETA NONOate should be kept at -80 &#176;C. This reagent decays rapidly despite optimal storage and maybe underperforming when not freshly prepared, requiring multiple injections into the purge vessel and leading to a less stable isoelectric signal. More importantly, it should be expanded in PBS with pH 7.4 as this optimizes its half-life (56 h at pH 7.4 at room temperature vs. quasi-instant dissociation at pH of 5.0)45. The syringe used for injection of DETA NONOate should be exclusively dedicated to that action and not used for injection of samples.
If the injection of a sample generates a peak that is higher than the upper point of the calibration curve, especially if it generates a voltage higher than 700 mv, it is advised to dilute the sample (2x or 4x) to minimize error. This also applies to the NO consumption assay if the dip caused by sample injection is deeper than the one caused by the injection of the most concentrated sample used in the calibration curve. The evidence of a peak (or a through) in signal after injection that is less than expected or absent may indicate clogging of the precision syringe used for injection. Due to the small volumes used (10-20 &#956;L/sample), particularly if plasma or urine assays are performed, it may be difficult to distinguish between a filled syringe and the absence of the sample due to clogging of the syringe. It is advised to pay attention to the resistance made by the plunger. After confirmation by inspection of the syringe, removal of the obstruction may be attempted by repeatedly washing the syringe with ddH2O and by rubbing the syringe on a delicate task wipe if the obstruction is externally visible. When in doubt, it is advised to eject the sample volume on a task wipe and go back to wash the syringe with ddH2O and try to repeat sample administration. Foaming is a common complication and forces the experiment to be terminated once the height of the liquid column exceeds that of the reaction column, causing signal instability. Exceeding the antifoaming agent dose indicated in the protocol may lead to an increase in the baseline signal and should be avoided. The number of samples causing the formation of excessive foaming becomes predictable when repeated assays are performed, a phenomenon that should inform how the experiment can be optimally planned. An optional step to decrease foaming is to mix plasma or tissue supernatant with cold methanol (ratio 1:1) and centrifuge for 15 min, 13000 x g at 4 &#176;C. It should be considered that methanol causes proteins to precipitate. Only light S-NOs and Fe-NOs that are not proteic in nature are measured. Thereby, this option cannot be adopted in studies on whole blood or in any other case where there is interest in S-NO and Fe-NO proteins.
More reagent solutions other than VCl3 in HCl and the I3-&#160;in acetic acid have been described and successfully adopted. Ascorbic acid specifically reduces NO2-, does not react with NO3- nor S-NOs, and can be used to reduce the bulk of injected samples and calculations in situations where NO2- is the only molecule of interest. The main pitfall is the limited stability of the reagent, requiring more frequent changes of the reagent solution and, thereby, repeated calibrations46. An assay using carbon monoxide and cuprous chloride (CuCl)-saturated cysteine (3C method) can specifically detect S-NOs with a sensitivity that is comparable with I3-&#160;but still requires treatment with and without HgCl2 to avoid non-specific reactions47. To achieve higher precision in signal analysis, experiments are recorded in the .data format so that offline waveform analysis can be completed with different software other than the bundle program shown in the protocol.
Once the first injection is made, the experiment cannot be stopped. If a pause is needed and/or if any condition is changed (composition of the purge vessel medium, purge gas flow, baseline voltage), the experiment is valid only for the samples injected up to that point. A new standard curve should be performed before measuring more samples. The precision allowed by chemiluminescence-based assays comes with a price. The assays are time-consuming for a number of reasons: 1) automation is not possible as manual sample injection is required in duplicates; 2) most reagents that are used cannot be stocked for long periods; 3) each experiment needs to be started by running standard samples at known concentrations for calibration and cannot be paused unless calibration is repeated; 4) each sample is divided into aliquots containing different mixtures of reagents to block specific reactions in order to finally calculate the concentration of specific metabolites by subtracting others.
This work is focused on the device Zysense NOA 280i, which has a detection threshold of &#60;1 ppb NO in the gas phase (corresponding to up to 1 pM of NO in the liquid phase) and a sampling flow of 10-300 mL/min. Other CLDs are available with a bundle tubing system for the measurement of metabolites in the liquid phase. Ecophysics CLDs have a higher sensitivity threshold, 1 ppb in their most sensitive model but the advantage of not requiring an oxygen tank for ozone generation. On the other hand, their reported sampling flow is 1000 mL/min, which implies a higher consumption of the inert gas tank used for the purge vessel. Other CLD machines are dedicated to exhaled NO analysis for clinical use.
Chemiluminescence is the most sensitive and validated method to specifically assess NO consumption by cell-free Hb in the field of hemolysis. Other techniques are available to measure NO, NO2-, NO3-, S-NOs and mercury-labile nitroso compounds. The direct measurement of NO gas can be obtained either by chemiluminescence or with the use of dedicated electrodes that range from the measurement of the exhaled gas to single-cell electrodes. These measure only NO gas, are less expensive (yet more perishable), require calibration more often, and are less precise compared to a CLD31. Nitrites and NO3- have been historically measured with the Griess reaction, a colorimetric technique that in its original version detects both molecules without the possibility of distinguishing their relative concentration in the sample. Modern variants of the technique involve reverse phase chromatography employing separation of the sample in two columns, allowing two independent reactions with NO3- and NO2-. The sensitivity of those methods is micromolar or slightly less, as compared with 1 nM with CLD. The main pitfalls are time consumption and incompatibility with samples pretreated with mercury and/or ferricyanide14. S-NOs can be detected with colorimetric and fluorometric techniques as well with a sensitivity up to 0.5 &#181;M compared to 100 fM described by using chemiluminescence as shown in the I3- in acetic acid protocol14,41,42. The biotin-switch assay, despite failing to detect S-NOs in the picomolar range, has the advantage of generating biotin-labeled proteins that can be purified and identified with proteomic analysis48. When looked at in detail, each technique shares one common pitfall, which is the extreme reactivity of NO and its derivatives mandating blockade of reactions for the purpose of in vitro measurement. Pre-treatment of samples is critical, and answering the question of whether a certain pre-treatment completely blocks a reaction or does so better than another one has been so far a driving force towards an increase in methodological studies. While the extreme reactivity in vivo of NO has certainly been a drive to research sensitive methods for detection of its metabolites, direct measurement of NO has met significant progress in the past three decades since the first amperometric detector was developed in 1990. More than twenty different probes have been described since then, including nanosensors used to study single-cell NO content49. Electrochemical sensors are the main tools that can detect NO both in vivo and in real-time. In pulmonary medicine, multiple studies have compared electrode-based machines with CLDs. Measurement of exhaled NO is more simple than other assays for metabolite detection, requiring either direct connection of the gas line (via IFD filter) to a closed-system respiratory adjunct or a few breaths in a bag for offline analysis, yet the cost and poorer portability of CLDs standstill. A number of electrochemical devices currently meet the criteria for clinical use, although they have lower sensitivity and reproducibility when compared to CLDs. Furthermore, the concordance in terms of absolute measured values is suboptimal between devices, so it is recommended that patients should be always followed up with the same electrochemical device50. The use of CLDs in settings such as measurement nasal exhaled NO and in research settings like therapeutic low and high-dose inhaled NO is still necessary.
Precise measurements of NO, its derivatives, and NO-binding proteins are necessary at many levels to understand an overlooked and still mostly unknown mechanism of signaling. The fields of application span biology and pathology at both preclinical and clinical levels with specific target areas, including immunobiology, neurosciences, cardiovascular sciences, and infective diseases. Preclinical studies and clinical trials are investigating the use of inhaled NO gas as a therapeutic agent. Deeper knowledge on how exogenous NO gas influences the concentration of NO metabolites and NO-bound proteins at every level, from plasma to cell compartments, will be required. Implementation of high-yield proteomics may likely increase the number of known mediators that are influenced by NO and its derivatives and may mandate new mechanistic investigations requiring precise measurements of NO metabolites.

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 with NO and O3 reactions; photomultiplier tube detects emission." class="xfigimg" src="/files/ftp_upload/63107/63107fig01.jpg" data-altupdatedat="1747736610000" 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.remotexs.ntu.edu.sg/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 reaction setup diagram with NaOH trap, purge vessel, gas inlet, and heated jacket." class="xfigimg" src="/files/ftp_upload/63107/63107fig02.jpg" data-altupdatedat="1747736610000" 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.remotexs.ntu.edu.sg/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="Tube prefill process; chemical analysis diagram; NOx compound measurement; reaction steps." class="xfigimg" src="/files/ftp_upload/63107/63107fig03.jpg" data-altupdatedat="1747736610000" 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.remotexs.ntu.edu.sg/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 cardiopulmonary bypass (CPB) during cardiac surgery is shown in Figure 5. It can be assumed that after CPB there is a higher concentration of heme groups in the Hb-Fe2+-O2 status (Figure 5, red) compared to patients receiving NO during CPB from which only a minority of heme groups of cell-free Hb consumes NO (Figure 5, green).
The protocol for assessing NO consumption by cell-free Hb was previously applied to test plasma samples from 50 patients undergoing cardiac surgery and requiring CPB. Blood was collected at baseline and 15 min, 4 h, and 12 h after CPB. The purpose of the study was to investigate the existing relationship between both pulmonary and systemic vascular resistance and the increase in hemolysis observed after the CPB. Cell-free Hb concentration was assessed with a Hb colorimetric assay. Hemolysis peaked 15 min after CPB. The accumulated free Hb was slowly eliminated from plasma within 12 h18 (Figure 6A). NO consumption, instead, peaked at 15 min and reached baseline values in just 4 h (Figure 6B). Linear regression curves of NO consumption by cell-free Hb exhibited a stoichiometric relationship at the 15 min post-CPB timepoint as opposed to baseline, 4 h, and 12 h post-CPB (Figure 6C). The explanation for the observed difference in kinetics lies in the abundance of newly released free Hb that has not yet encountered NO molecules being released by the patient's endothelium. The concentration of oxyHb (scavenging NO and resulting in consumption in the assay) was, in fact, higher at 15 min than at any other timepoint. Plasma collected at baseline and on other timepoints had a relatively higher component of metHb, which had already been oxidized by NO, did not bind NO, and did not cause consumption in the assay. Pulmonary and systemic vascular resistances were measured invasively and peaked at 15 min. For the first time in this specific patient population, NO consumption by free Hb was observed to be temporally coupled with vasoconstriction, which confirmed previous observations in animal models and in patients with sickle cell disease18.
The administration of NO gas during CPB and after surgery increases the relative quantity of circulating metHb vs. oxyHb. When patients undergoing cardiac surgery were treated with NO-gas intra-operatively, and post-operatively, the observed increase of free Hb after CPB (Figure 7A) was not coupled with any increase in NO consumption, as measured with the aforementioned protocol (Figure 7B). The exogenously administered NO gas converts most oxyHb into metHb, which in turn does not scavenge NO in vivo nor consumes NO in vitro (unpublished data).
<img alt="NO consumption vs. free hemoglobin graph; linear correlation analysis; biochemical study result." class="xfigimg" src="/files/ftp_upload/63107/63107fig04.jpg" data-altupdatedat="1747736610000" data-alt="Figure 4"> 
Figure 4: Nitric oxide consumption by purified cell-free oxyhemoglobin. Nitric oxide consumption assay performed by injecting samples of known concentrations of purified cell-free oxyhemoglobin. Regression line is shown in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/63107fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Graph of NO consumption vs. free hemoglobin (µM), CPB study, linear regression analysis." class="xfigimg" src="/files/ftp_upload/63107/63107fig05.jpg" data-altupdatedat="1747736610000" data-alt="Figure 5"> 
Figure 5. Nitric oxide consumption and cell-free hemoglobin concentration from patients undergoing cardiac surgery with cardiopulmonary bypass. Each patient's cell-free hemoglobin level, as measured with a colorimetric kit, was plotted with the nitric oxide (NO) consumption measured through chemiluminescence. Blood samples were drawn 15 min after the end of CPB. Regression lines are shown for cardiac surgery patients not receiving (in red) or receiving (in green) NO during cardiopulmonary bypass (CPB). Hemoglobin is expressed in μM of heme groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/63107fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="NO consumption and free hemoglobin bar charts and scatter plot after CPB; data analysis post-surgery." class="xfigimg" src="/files/ftp_upload/63107/63107fig06.jpg" data-altupdatedat="1747736610000" data-alt="Figure 6"> 
Figure 6: Nitric oxide consumption and cell-free hemoglobin in cardiac surgery patients. (A) Cell-free hemoglobin (Hb) concentration in the plasma of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) (N = 50) was determined at different time points with the use of a commercially available colorimetric determination kit. Data were analyzed by fitting a mixed model with evidence of a fixed effect between time points. Plasma concentration at different time points was compared to baseline with Tukey's multiple comparison test. (B) Nitric oxide (NO) consumption by plasma obtained from the same samples used for quantification of free Hb. Data were analyzed by fitting a mixed model with evidence of a fixed effect between time points. Plasma concentration at different time points was compared to baseline with Dunnett's multiple comparison test. (C) Regression lines for cell-free Hb plasma content matched with NO consumption. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001, **** p &lt; 0.0001, ns not significant. Data are presented as median ± interquartile range. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/63107fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cardiopulmonary bypass effect on free hemoglobin, NO consumption bar graphs; placebo, NO impact." class="xfigimg" src="/files/ftp_upload/63107/63107fig07.jpg" data-altupdatedat="1747736610000" data-alt="Figure 7"> 
Figure 7: Effects of nitric oxide gas administration on nitric oxide consumption in patients undergoing cardiac surgery. (A) Cell-free hemoglobin concentration in the plasma of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) either receiving a placebo (N = 28) or nitric oxide (NO) gas (N = 22) for 24 h since the beginning of CPB. The concentration was determined at different time points with the use of a commercially available colorimetric kit. Data were analyzed by Friedman's test, indicating the presence of an effect between time points in both groups. Plasma concentration at different time points was compared to baseline with Dunn's multiple comparison test. (B) NO consumption by plasma obtained from the same samples used for quantification of cell-free Hb. Data were analyzed by Friedman's test, indicating the presence of an effect between time points in both groups. Plasma concentration at different time points was compared to baseline with Dunn's multiple comparison test. ** p &lt; 0.01, *** p &lt; 0.001. Data are presented as median ± interquartile range. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/63107fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Assay</td>
			<td>Reagent Mixture</td>
			<td>Target Baseline (mV)</td>
		</tr>
		<tr>
			<td>VCl3 in HCl</td>
			<td>5 mL of VCl3 in HCl saturated solution + 100 μL of antifoam agent</td>
			<td>0–5 mV*</td>
		</tr>
		<tr>
			<td>I3– in acetic acid</td>
			<td>5–7 mL of I3– reagent</td>
			<td>0–5 mV*</td>
		</tr>
		<tr>
			<td>NO consumption by cell-free Hemoglobin</td>
			<td>5–7 mL of PBS at pH 7.4 + 100 μL of antifoam agent + 20 μL of expanded DETA-NONOate solution</td>
			<td>70–100 mV**</td>
		</tr>
		<tr>
			<td colspan="3">*ideal value: baseline might be higher due to the quality of the air in the experiment environment</td>
		</tr>
		<tr>
			<td colspan="3">**ensure stability for 20 min before starting the experiment.</td>
		</tr>
	</tbody>
</table>
Table 1: Purge vessel reagent composition and target baseline for various chemiluminescence assays. Purge vessel reagent composition for each described assay. NO = Nitric Oxide; PBS = Phosphate Buffer Saline; DETA-NONOate: (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate. *ideal value: baseline might be higher due to the quality of the air in the experiment environment. **ensure stability for 20 min before starting the experiment. To perform the NO consumption assay, the scale should be changed on the software to capture values between 70 mV and 100 mV (e.g., 0-100 mV), which is the targeted baseline voltage. This adjustment is suggested to have full control on the sample injection, which enables to document the signal moving from baseline following the injection of a sample and to observe the signal returning to baseline. Data recording is not influenced by nor limited to the visualized scale.
Supplemental Figure 1: Peaks following NO detection in the VCl3 in HCl assay. The peaks which follow sample administration in the VCl3 in HCl are shown on the CLD bundle software. Calculation of the area under the peak for each injection is shown for convenience and explained in Supplemental Figure 2. In this experiment, standards of different concentrations have been administered for the calibration curve, followed by plasma samples. Dilution is a useful solution to calculate metabolites from samples that yield NO at the high end or even outside of the calibration curve. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/Supplemental%20Figure%201.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 2: User-assisted automated calculation of the area under the peak. When performing the NO consumption assay by cell-free hemoglobin, it may be necessary to set peaks manually in order to provide the software with the most precise limits to measure the area under the peak generated by each sample injection. (A) Representative full view of a recorded experiment. It is reached by clicking on Process in the main menu and by selecting and opening the recorded file of interest. The yellow line represents the signal threshold (mV) that constitutes the straight segment of the area under the peak. By clicking on it, the user can drag it towards the signal area. The x and y-axis can be changed to zoom on the peak of interest for precise measurement by either clicking on the lower left buttons or by typing in the desired minimum and maximum value for each. (B) Threshold positioning. The user can select the optimal baseline threshold by dragging the yellow line to the signal midpoint right prior to the signal downslide caused by the sample injection. To continue limiting the area, the user shall click on the Set Peaks Manually button. (C) Start and End Cursors positioning. By clicking on the Set Start Cursor button, a green line appears on the screen. The green line should be placed (by dragging and releasing) at the very beginning of the downslide caused by the sample injection. The same button changes its label to Set End Cursor by clicking on which a red line appears on the screen. The red line should be placed at the point where the signal reaches the baseline again after the deflection caused by NO consumption from the injected sample. Altogether, the threshold line (yellow, B), the green line, and the red line precisely outline the area generated by the sample injection. (D) Yielding the area under the peak. By clicking on the Integrate button (C), the calculated area under the peak is displayed on the top right of the screen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/Supplemental%20Figure%202.pdf" target="_blank">Please click here to download this File.</a>
Supplemental Figure 3: Representative spreadsheet with calibration curve and results. The calculated areas under the peaks generated from nitric oxide (NO) consumption by injection of samples with a known concentration of cell-free hemoglobin (expressed as [µM] of heme) are shown on the top left (on yellow background). The values are plotted to build the calibration curve and its resulting linear equation y= mx + q. The slope (m) is the number of oxyHb heme groups (µM) required to decrease the signal detected by the photomultiplier tube by 1 mV. Calculated values from triplicate injections (Run N 1,2,3) of samples from one patient obtained at four different time points (marked by different background colors) were entered in the spreadsheet. The average of each triplicate was used to yield the value of NO consumption caused by each sample by solving the linear equation: <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/Supplemental%20Figure%203.pdf" target="_blank">Please click here to download this File.</a>
<img alt="NO consumption calculation formula, NO=(Area-q)/m=Area-113.84/85.882, chemical analysis." src="/files/ftp_upload/63107/63107eq01.jpg" data-altupdatedat="1747736610000" data-alt="Equation 1">
Supplemental File 1: Reviewing the peaks using the bundled software. This file includes a screenshot taken from the bundled software and the actions allowed for each button. For every peak (including false peaks caused by artifacts), the user can use the buttons indicated in the file to confirm, or disregard, or delete, or name a specific peak, as needed. The user can also record a peak that was not previously detected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63107/Supplemental%20File%201.docx" target="_blank">Please click here to download this File.</a>

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 ...

chemiluminescence-based-assays-for-detection-nitric-oxide-its

Nanyang Technological University

Research

JoVE Journal

Medicine

3.7K Views.  Massachusetts General Hospital. Dit protocol stelt onderzoekers in staat om vakkundig een chemiluminescentiedetector te gebruiken voor het meten van stikstofmonoxidemetabolieten zonder de begeleiding of kosten van het in dienst nemen van toegewijd personeel ter plaatse. Chemiluminescentie-gebaseerde test is de meest gevoelige methode om minimale veranderingen in de niveaus van stikstofmonoxide en zijn metabolieten in een bepaald biologisch monster te detecteren. Geïnhaleerd stikstofmonoxide is een therapeutisch gas dat wor...

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

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

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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