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 border="0" cellpadding="0" cellspacing="0" width="812">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:351px;">potassium ferricyanide; K3Fe(CN)6</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">702587</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEM; N-ethylmaleimide</td>
			<td style="width:135px;">Sigma</td>
			<td>4260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40; 4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sulfanilamide; AS&#160;</td>
			<td style="width:135px;">Sigma</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HCl</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">H1758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">acetic acid, glacial</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">A9967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ascorbic acid&#160;</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">A7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">potassium iodide; KI</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">60399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">iodine; I2</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">207772</td>
			<td>light sensitive, toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">sodium nitrite; NaNO2</td>
			<td style="width:135px;">Sigma</td>
			<td>563218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">vanadium(III) chloride; VCl3</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">208272</td>
			<td>ligt sensitive, toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMac</td>
			<td style="width:135px;">Miltenyi</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sievers NOA 280i</td>
			<td style="width:135px;">GE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLD 88Y</td>
			<td style="width:135px;">&#160;Ecophysics&#160;</td>
			<td style="width:119px;"></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

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...