Name: measuring nitrite and nitrate, metabolites in the nitric oxide pathway, in biological materials using the chemiluminescence method
Uploaded: 2016-12-25T15:00:00.000+00:00
Duration: 8 min 25 s
Description: Watch this Scientific Journal Video about Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method at JoVE.com

Authors want to acknowledge critical contributions of Dr A. Dejam and M. M. Pelletier in developing the use of nitrite preserving solution for the nitrite measurements in blood.

All protocols including use of animals were approved for use by NIDDK Animal Care and Use Committee and human blood was obtained from NIH Blood bank from healthy donors.
1. Sample Preparation
<ol>
	<li>Preparation of nitrite preserving solution
	<ol>
		<li>Prepare a solution containing 890 mM potassium ferricyanide (K3Fe(CN)6) and 118 mM NEM (N-ethylmaleimide) in distilled water. Dissolve well until it is clear yellow with no crystals present. Add NP-40 (octyl phenoxylpolyethoxylethanol) in a 1:9 ratio (v/v, NP-40/ solution). Mix gently to avoid foaming and store at 4 &#176;C for about a week).</li>
	</ol>
	</li>
	<li>Collection and preparation of blood samples
	<ol>
		<li>Collect blood using at least a 20 G needle to avoid hemolysis with heparin to prevent coagulation (5 U/ml). For the whole blood sample, mix blood with nitrite preserving solution immediately in a 1:4 ratio (v/v solution/blood).</li>
		<li>For plasma and red blood cells samples, centrifuge the collected blood for 5 min at 4,000 x g at 4 &#176;C to separate red blood cells (RBC) and plasma. Take the supernatant (plasma) and mix it with nitrite preserving solution as described above for determination of nitrite levels in plasma.</li>
		<li>Carefully remove remaining plasma and buffy coat from the sample and pipette RBC sample from the bottom of the tube to avoid contamination by other types of blood cells using a cut-off pipette tip, and transfer it into a tube containing nitrite preserving solution in the same ratio as above.</li>
		<li>Mix each sample (plasma and RBC) with cold methanol at a ratio 1:1 or 1:2 (v/v sample/methanol) and centrifuge them at 13,000 x g at 4 &#176;C for 15 min to precipitate proteins. Take the supernatant and use it for nitrite measurement or freeze prepared samples, if needed, on dry ice and store at -80 &#176;C. 
		Note: Nitrite rapidly reacts with oxyhemoglobin (oxyHb) in blood forming nitrate. Since oxyHb is always present in large excess over nitrite, this reaction leads to almost complete annihilation of native blood nitrite with a time frame of ~ 10 min. In order to preserve most of endogenous blood nitrite, nitrite-preserving solution is added to the whole blood samples immediately after the blood draw. The solution will rapidly lyse red blood cells and oxidize oxyHb into methemoglobin (metHb), an inactive form of hemoglobin that does not chemically react with nitrite.</li>
	</ol>
	</li>
	<li>Preparation of samples from other types of body fluid
	<ol>
		<li>After sample collection into appropriate container, mix well with nitrite preserving solution, deproteinate and measure immediately or freeze and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Collection and preparation of tissue samples
	<ol>
		<li>Collect tissues from animals perfused with heparinized saline solution (10 U heparin/ml). Excise about 1 g of desired tissue and homogenize either using a manual homogenizer or an automated homogenizer.</li>
		<li>Add known amount of nitrite preserving solution as needed to achieve smooth homogenate.</li>
		<li>Once tissue is homogenized, precipitate proteins using cold methanol (1:1 or 1:2 ratio v/v sample/methanol) as described above, then centrifuge samples at 13,000 x g at 4 &#176;C for 15 min.</li>
		<li>Collect supernatant and measure nitrite levels. If needed, freeze samples at any stage of preparation on dry ice and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of Reducing Solutions
<ol>
	<li>Tri-iodide (I3) reducing solution for nitrite and R-(X)-NO species determination1,3,4
	<ol>
		<li>Prepare a solution of 301 mM KI together with 138 mM I2 solution in water. Mix with glacial acetic acid in 2:7 ratio (v/v solution/acid) on a magnetic stirrer for ~ 30 min until all crystals are dissolved. Preferably keep in dark bottle, as iodide is light sensitive, and use within one week from preparation. 
		Note: This solution will reduce nitrite into NO and it will also release NO from R-SNO, R-NNO and Fe-NO functional groups (R-(X)-NO), but nitrate will not be affected. The signal from the above NO-based functional groups can be separated from true nitrite signal by treatment of half of the sample with acidified sulfanilamide (AS) and comparing AS-treated and untreated signal. AS irreversibly forms diazonium cation with nitrite and this complex cannot be reduced by I3 solution. For AS treatment, prepare 290 mM solution of sulfanilamide in 1 M HCl and add it to an aliquot of sample in ratio 1:9 (v/v AS/sample). Measure the CL signal in AS-treated and untreated parts of the sample. Calculate the true nitrite signal as a difference of AS-treated and untreated part of sample. Nitrite can be also measured by using a selective nitrite-reducing solution of ascorbic / acetic acid mixture as described in part 2.2. However, due to the usually small amount of R-(X)-NO species present, measurements using AS-untreated samples are in most cases a very good approximation of total nitrite content.</li>
	</ol>
	</li>
	<li>Ascorbic acid / acetic acid (4A) solution for selective determination of nitrite2
	<ol>
		<li>Prepare 500 mM ascorbic acid in water. Mix this solution with glacial acetic acid in a ratio 1:7 (v/v, ascorbic acid / acetic acid) to prepare the reaction mixture. 
		Note: This solution is specific for nitrite, will not release NO from any R-(X)-NO species or nitrate. However, the solution of ascorbic and acetic acid must be prepared freshly every day before the measurements, as ascorbic acid easily oxidizes in solution. 
		Completion of nitrite reduction depends on ascorbic acid concentration; and at least 50 mM ascorbic acid is recommended for complete reduction of plasma nitrite. However, it is recommended to perform a few pilot experiments with different concentrations of ascorbic acid and nitrite standards in the expected nitrite concentration range prior to final sample measurements. Keep in mind that ascorbic acid slightly depletes during the measurements, so frequent changes of reaction mixture in the glass reaction chamber are recommended.</li>
	</ol>
	</li>
	<li>Vanadium(III) chloride reducing solution for nitrate determination9
	<ol>
		<li>Prepare 51 mM solution of VCl3 in 1 M HCl. Filter solution through 200 &#956;m filter and store in a dark bottle, use within two weeks. 
		Note: This solution will reduce nitrate, nitrite and all R-(X)-NO species, so if comparable amounts of nitrite or other R-(X)-NO species are present together with nitrate, the final nitrate content has to be calculated as the difference between the CL signals obtained with VCl3 and I3 reducing solutions.</li>
	</ol>
	</li>
</ol>
3. Chemiluminescence (CL) Analyzer Setup and Measurements
<ol>
	<li>Standard solution
	<ol>
		<li>Prepare 1 &#956;M solution of sodium nitrite (NaNO2). The same solution can be used for nitrite and nitrate determinations.</li>
	</ol>
	</li>
	<li>Using NO analyzer 
	Note: Currently there are two commercially available NO analyzers that are sensitive enough for biological research purposes &#8212; Sievers NOA and Ecophysics CLD 88Y. They both operate on the same principle; the main difference is that CLD 88Y uses oxygen from the room air to make ozone (O3), while NOA requires an external oxygen tank for this purpose. The procedure below describes the set up for the Sievers NOA.
	<ol>
		<li>Instrument setup
		<ol>
			<li>Perform initial set up of the NO analyzer according to the manufacturer&#39;s recommendations as seen on Figure 1.</li>
			<li>Open O2 tank connected to the instrument. Choose &#34;analysis&#34; and &#34;enter&#34; from the main menu on the front panel. On the next screen choose &#34;start&#34; and press &#34;enter.&#34; Connect the acid trap (containing 1 M NaOH) to the instrument as seen in Figure 1.</li>
			<li>Wait until photomultiplier cools to temperature below -12 &#176;C and the reaction chamber is evacuated to 6 Torr. It takes about 30 min to reach a stable flat baseline. The baseline needs to stabilize within 1 - 2 mV, its nominal value is less important than its stability. 
			Note: If nitrate is measured, turn on the cooling water bath (set at 4 &#176;C, which serves as cold trap for acid and water vapors) and heating water bath (the main reaction chamber, 95 &#176;C). The rate of the reduction of nitrate into NO in VCl3 solution is temperature-dependent, and it is very slow at room temperature, so substantial increase of temperature is needed to observe the reaction.</li>
			<li>Open the He tank and connect the glass reaction chamber to acid trap as seen on Figure 1. Fill the reaction chamber with appropriate reducing solution, reduce the bubbling to minimum and connect reaction chamber to the acid trap. Using trial and error, adjust the He bubbling rate to match the instrument suction rate (usually ~ 200 ml/min for Siever&#39;s NOA).</li>
			<li>Turn on the computer controlling the instrument and start Labview-based Liquid software. To turn on communication between the instrument and the acquisition software, press &#34;enter&#34; when in &#34;data menu&#34; until screen reads &#34;output enabled&#34;, then press &#34;clear&#34; to return back to data screen.</li>
			<li>Wait for a stable baseline trace on the screen and start injecting samples and nitrite standards into reducing solution through the septum. Always wait to reach a stable baseline after the peak. This can take up to 2 min. The Liquid software has an option to &#34;mark&#34; times of injection and annotate the injection and it will export these comments into a separate file (filename.info) as a useful complement to the data file (filename.data).</li>
			<li>Once samples and standards are measured, choose &#34;stop&#34; option in the Liquid software menu &#8212; this will write data from a temporary file into a permanent file known as &#34;filename.data&#34;. Pressing &#34;abort&#34; option will terminate measurement without writing acquired data into a permanent file.</li>
			<li>To terminate experiment, disconnect the machine from the reaction vessel by turning off the stopcock on the top of reaction vessel and disconnect the tubing between acid trap and reaction vessel (see Figure 1). Now stop NOA analyzer &#8212; press &#34;clear&#34;, go to &#34;analysis&#34;, put machine on &#34;standby&#34; and &#34;confirm&#34; standby. If used regularly, the instrument can be left in standby mode for several weeks. Turn off supply of oxygen. Then flush the reducing solution from the reaction chamber, disconnect and close the He tank from the glass reaction vessel and finish cleaning reaction vessel.</li>
		</ol>
		</li>
		<li>Standards and sample injections
		<ol>
			<li>Inject 50 &#181;l of 1 &#181;M nitrite solution into reaction mixture using a well-washed Hamilton syringe. Wait for at least 1 - 2 min between injections to achieve good separation of peaks. Repeat injection of 50 &#181;l to get duplicates to each data point. Wash syringe with deionized water after each injection.</li>
			<li>To acquire full set of data for standard curve, continue with duplicate injections of 100, 150 &#181;l (additional 200 &#181;l might be needed if high concentrations of nitrite or nitrate are expected) of 1 &#181;M nitrite solution. In each case, take care to wait until the signal drops back to the baseline and then acquire 1 - 2 min of baseline to achieve good separation of peaks. 
			Note: Nitrite standards can be measured at any time during experiments. However, it is preferable to acquire the standard curve before data measurement, as it also serves as an independent check of instrument functionality. 
			When data are collected, amount of sample injected should lead to well pronounced peaks. However it should be kept in mind that the photomultiplier is linear only up to ~ 800 mV, so none of the peak should be higher than ~ 700 mV. This can be achieved either by decreasing of amount injected sample or diluting the sample with deionized water. Each point should be measured at least in duplicate.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Piknova, B., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Nitrite+in+Blood+Samples+Using+the+Ferricyanide-Based+Hemoglobin+Oxidation+Assay.">Measurement of Nitrite in Blood Samples Using the Ferricyanide-Based Hemoglobin Oxidation Assay.</a> Methods Mol Biol. 704, 39-56 (2011).</li><li>Nagababu, E., Rifkind, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+plasma+nitrite+by+chemiluminescence+without+interference+of+S-,+N-nitroso+and+nitrated+species.">Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species.</a> Free Radic Biol Med. 42, 1146-1154 (2007).</li><li>Pinder, A. G., Rogers, S. C., Khalatbari, A., Ingram, T. E., James, P. E., Hancock, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+nitric+oxide+and+its+metabolites+in+biological+samples+by+ozone-based+chemiluminescence.">The measurement of nitric oxide and its metabolites in biological samples by ozone-based chemiluminescence.</a> Methods in Molecular Biology, Redox-Mediated Signal Transduction. 476, 11-28 (2008).</li><li>Pelletier, M. M., Kleinbongard, P., Ring-wood, L., Hito, R., Hunter, C. J., Schechter, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+blood+and+plasma+nitrite+by+chemiluminescence:+pitfalls+and+solutions.">The measurement of blood and plasma nitrite by chemiluminescence: pitfalls and solutions.</a> Free Radic Biol Med. 41, 541-548 (2006).</li><li>Mac Arthur, P. H., Shiva, S., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+circulating+nitrite+and+S-nitrosothiols+by+reductive+chemiluminescence.">Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.</a> J Chromatogr B. 851, 93-105 (2007).</li><li>Bryan, N. S., Grisham, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+detect+nitric+oxide+and+its+metabolites+in+biological+samples.">Methods to detect nitric oxide and its metabolites in biological samples.</a> Free Radic Biol Med. 43, 645-657 (2007).</li><li>Hendgen-Cotta, U., Grau, M., Rasaaf, T., Gharinin, P., Kelm, M., Kleinbongard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+gas-phase+chemiluminescence+and+flow+injection+analysis+for+measurement+of+nitric+oxide+pool+in+biological+matrices.">Reductive gas-phase chemiluminescence and flow injection analysis for measurement of nitric oxide pool in biological matrices.</a> Method Enzymol. 441, 295-315 (2008).</li><li>Yang, B. K., Vivas, E. X., Reiter, C. D., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+for+the+sensitive+and+specific+measurement+of+S-nitrosothiols,+iron-nitrosyls+and+Nitrite+in+biological+samples.">Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls and Nitrite in biological samples.</a> Free Radic Res. 37, 1-10 (2003).</li><li>Sm&#225;rason, A. K. 1., Allman, K. G., Young, D., Redman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+serum+nitrate,+a+stable+end+product+of+nitric+oxide,+in+women+with+pre-eclampsia.">Elevated levels of serum nitrate, a stable end product of nitric oxide, in women with pre-eclampsia.</a> Br J Obstet Gynaecol. 104 (5), 538-543 (1997).</li><li>Beckman, J. S., Congert, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Dilute+Nitric+Oxide+in+Solution+with+an+Ozone+Chemiluminescent+Detector.">Direct Measurement of Dilute Nitric Oxide in Solution with an Ozone Chemiluminescent Detector.</a> Methods: A companion to Methods in Enzymology. 7, 35-39 (1995).</li><li>Bates, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+measurements+by+chemiluminescence+detection.">Nitric oxide measurements by chemiluminescence detection.</a> Neuroprotocols: A companion to Methods in Neuroscience. 1, 141-149 (1992).</li></ol>

Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. He receives royalties based on NIH licensing of these patents for clinical development but no other compensation.

Critical Steps within the Protocol
Aliquots of all solutions (including the water) used to prepare, dilute or otherwise treat original samples have to be saved and checked for possible nitrite or (more often) nitrate contamination. We found that most contamination comes from water and many chemicals used to treat sample (ferricyanide in particular) also contain significant amount of nitrate contamination in some lots that interferes with the endogenous nitrate determination. We therefore check al...

Nitrite, and to a less extend nitrate, levels in blood reflect overall state of body NO metabolism. Nitrite concentrations in blood and most organs and tissues are only in high nanomolar or low micromolar range, nitrate is usually present in much higher amounts &#8212; in micromolar range. Changes in nitrite levels due to disease progression or changes in dietary habits are quite small and can be only measured using a very sensitive method. Because of their very different levels and different metabolic processes, separate determination of nitrite and nitrate levels is essential. So-called &#34;NOx determination&#34; where nitrite and nitrate are measured together has very little value.
Several methods for quantifying nitrite in various biological samples have been developed&#160;&#8212; the most common being the oldest one, based on the Griess reaction that had been originally described in 1879. Even with modern modifications, the sensitivity limit for nitrite attainable by Griess&#39; method is in low micromolar range. Chemiluminescence (CL), combined with tri-iodide reducing solution, is currently considered the most sensitive method, allowing quantification in the low nanomolar range of nitrite concentrations1-8,10,11. The same CL method, combined with vanadium(III) chloride reducing solution, can be used for sensitive measurements of nitrate, with precision in the nanomolar range9.
CL detects free gas NO. Therefore, nitrite, nitrate, R-nitrosothiols (R-SNO), R-nitrosoamines (R-NNO), or metal-NO compounds (later in manuscript referred as &#34;R-(X)-NO&#34;), must be converted into free NO gas in order to quantify their original amounts via CL. Conversion to NO is achieved using several different reducing solutions, depending on the nature of the NO metabolite. After conversion, free NO gas is purged from the reaction vessel by a carrier gas (He, N2 or Ar) into the reaction chamber of CL analyzer where ozone (O3) is combined with NO to form nitrogen dioxide (NO2) in its activated state. With return to the ground state, NO2* emits in infrared region and emitted photon is detected by photomultiplier (PMT) of CL instrument. The intensity of emitted light is directly proportional to NO concentration in reaction chamber, which allows calculation of the concentration of the original species using proper calibration curves.
In our protocol, we first present CL-based determination of nitrite and nitrate in the most used clinical settings &#8212; in blood and plasma, and then we discuss how to determine these ions in tissue samples. We also explain in detail how to preserve the original physiological nitrite concentration in nitrite-reactive environments, such as blood and its compartments, plasma and red blood cells.

<table><tbody><tr><td>potassium ferricyanide; K&lt;sub&gt;3&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>702587</td><td></td></tr><tr><td>NEM; N-ethylmaleimide</td><td>Sigma</td><td>4260</td><td></td></tr><tr><td>NP-40; 4-Nonylphenyl-polyethylene glycol</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>sulfanilamide; AS&amp;nbsp;</td><td>Sigma</td><td>S9251</td><td></td></tr><tr><td>HCl</td><td>Sigma</td><td>H1758</td><td></td></tr><tr><td>acetic acid, glacial</td><td>Sigma</td><td>A9967</td><td></td></tr><tr><td>ascorbic acid&amp;nbsp;</td><td>Sigma</td><td>A7506</td><td></td></tr><tr><td>potassium iodide; KI</td><td>Sigma</td><td>60399</td><td></td></tr><tr><td>iodine; I&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>207772</td><td>light sensitive, toxic</td></tr><tr><td>sodium nitrite; NaNO&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>563218</td><td></td></tr><tr><td>vanadium(III) chloride; VCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>208272</td><td>ligt sensitive, toxic</td></tr><tr><td>GentleMac</td><td>Miltenyi</td><td></td><td></td></tr><tr><td>Sievers NOA 280i</td><td>GE</td><td></td><td></td></tr><tr><td>CLD 88Y</td><td>&amp;nbsp;Ecophysics&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

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.

Figure 2 shows representative results collected from standards and five different samples. As shown in this figure, photomultiplier voltage increases immediately after nitrite-containing solution (standards or samples) is injected into reducing solution (injections times are indicated by red arrows below the curve) and returns to the baseline value once all nitrite present in the injected solution was reduced. It is also clear from this figure that accurate volume measure...

Watch this Scientific Journal Video about Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method at JoVE.com

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

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 ...

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22.4K Views.  NIDDK, NIH. 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.

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

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

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Environment

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 ...

Medicine

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

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Bioengineering

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 ...

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.

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 ...

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 ...

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

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

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

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

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

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

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