The authors thank Masayuki Kubo of the US Environmental Protection Agency for his help with the TSP/PM2.5 sampling. This work was supported in part by JSPS KAKENHI Grant Number JP16K15373 and JP18H03039.

1. Collection of Total Suspended Particles, PM2.5, or PM7, and the Hydrolysis Method for PM Proteins
<ol>
	<li>Collect the PM from a prefilter or backup filter.
	<ol>
		<li>Prior to the collection of total suspended particles (TSP), PM2.5, and PM7, weigh the quartz filter. 
		Note: Given that contamination by PM proteins and peptides should not affect the detection of tyrosine nitration (as they lack such a nitro group), we did not treat the quartz filter at a high temperature before the measurement. Additionally, 3-NT content in the quartz filter was under the detection limit, as determined by HPLC-ECD.</li>
		<li>Set the quartz filter on a high-volume air sampler with a particle size selector to collect PM7 at a flow rate of 1,000 L/min continuously for 7 d (Figure 1B). TSP can be collected on a quartz filter without a size selector (Figure 1A).</li>
		<li>Collect PM2.5 using a low-volume air sampler with a size classification unit at a flow rate of 116 L/min continuously for 7 d. Using this unit, particles can be classified as PM4.5-2.5, PM7.3-4.5, PM15-7.3, and TSP-PM15 and collected onto quartz filters for classification. PM2.5 can be collected onto a backup filter (Figure 1C).</li>
		<li>Record the total flow volume at the time of the filter collection. Measure the weight of the TSP, PM2.5, and PM7 on the filters by subtracting the precollection weight from the postcollection weight. Seal the filter in a plastic bag and store it at -30 &#176;C until the next step (see section 2).</li>
	</ol>
	</li>
	<li>Proteolyze the samples.
	<ol>
		<li>Dissolve nonspecific protease cocktail with a 0.1 M acetate buffer (pH 7.4). Put the solution into a dialysis membrane (molecular weight cutoff = 5,000 Da) and immerse it in 1 L of 0.1 M acetate buffer with stirring at 4 &#176;C. Replace the buffer 2x every 12 h, and then perform dialysis overnight in order to remove endogenous 3-NT and nitrite (NO2-). After the dialysis, collect the solution and measure the protein concentration by bicinchoninic acid (BCA) protein assay.</li>
		<li>Punch round circles of 6 mm out of the prefilter or the backup filter and put them into microtubes. Up to five pieces of 6 mm fractions can be used for the analysis (Figure 2).</li>
		<li>Add 300 &#181;L of 0.1 M acetate buffer containing 50 &#181;g of protein of the nonspecific protease cocktail (prepared in step 1.2.1) to the tube to hydrolyze the PM proteins at 50 &#176;C for 16 h with rotation. 
		Note: To subtract endogenous 3-NT from the nonspecific protease cocktail solution, it is necessary to prepare and digest a nonspecific protease cocktail-only sample.</li>
		<li>Rinse the ultrafiltration membrane along with its supplied tube by adding 0.1 M acetate buffer and centrifuging (10,000 x g for 30 sec; the exact speed depends on the membrane), due to contaminating chemicals similar to 3-NT in the membrane. Repeat the rinse 1x.</li>
		<li>Post-proteolysis, centrifuge the samples at 2,500 x g for 10 min. Load the supernatant into the ultrafiltration membrane and centrifuge it at 10,000 x g and 4 &#176;C for ultrafiltration (MWCO = 10,000), until sufficient volume of elution is collected. Store the eluates at 4 &#176;C in the dark. 
		CAUTION: Due to the detection of a similar peak to 3-NT in the spin column, it is necessary to wash it well with clean water or with a buffer such as HPLC water and a 0.1 M acetate buffer prior to use.</li>
	</ol>
	</li>
</ol>
2. Determination of 3-NT Content in TSP, PM2.5, and PM7 via a High-performance Liquid Chromatography and Electrochemical Detection (HPLC-ECD) System
Note: See Figure 3.
<ol>
	<li>Prepare the mobile phase.
	<ol>
		<li>Prepare 0.2 M phosphate buffer (pH 2.5) containing 50 mg/L EDTA. Measure off 98 volumes of the buffer and two volumes of acetonitrile (HPLC grade), pour them into a clean glass bottle, and mix vigorously with a cap.</li>
		<li>Settle the mobile phase at room temperature for several hours until the dissolved air goes flat. 
		Note: If the system is started earlier, the mobile phase can be sonicated and degassed under a vacuum. However, over-degassing will change the ratio between the water and organic phase via evaporation.</li>
	</ol>
	</li>
	<li>The HPLC-ECD system consists of a pump, an HPLC column, a degasser, an ECD, and an injector. Equilibrate the mobile phase and stabilize the system to reduce the background and noise.
	<ol>
		<li>Install the mobile phase and the HPLC column in the HPLC system. Turn on the HPLC pump and stabilize the column with the mobile phase at a flow rate of 500 &#181;L/min at 25 &#176;C column temperature from the day before the start day.</li>
		<li>On the next day, ignite the ECD and set the parameters (the reduction voltage at -900 mV and the excitation voltage at 400 mV). Stabilize it for at least 3 h.</li>
	</ol>
	</li>
	<li>Measure the 3-NT concentration in the PM7.
	<ol>
		<li>Prepare various concentrations of 3-NT standards (5 - 500 nM) and a blank (without the 3-NT standard and nonspecific protease cocktail, to make sure neither the vial nor the buffer is contaminated) in a 0.1 M acetate buffer.</li>
		<li>Run the HPLC data processor.</li>
		<li>Put sample vials in the auto-injector and set the injection volume to 25 &#181;L. Operate the same conditions of mobile phase and flow rate as mentioned in step 2.2.1. 
		Note: If the sample preparation is complete, there should be samples, a standard (5 - 500 nM), a nonspecific protease cocktail-only sample, and a blank vial.</li>
		<li>Make sure that the baseline in the chromatogram is flat and start the sample injection. 
		Note: Generally, 3-NT is detected in 20 - 21 min; however, approximately 30 min is recommended as the running time per sample injection. Otherwise, contaminant peaks will be included in the next run.</li>
	</ol>
	</li>
</ol>
3. Analysis of Chromatograms and Quantification of 3-NT Content in PM7
<ol>
	<li>Check the 3-NT peak in the chromatograms and calculate the content.
	<ol>
		<li>After the data collection, open the chromatogram data file with analysis software.</li>
		<li>Draw a line across the 3-NT peak from the flat baseline and read the peak height from the drawn line. 
		Note: The quantitative limit is approximately 5 nM (125 fmol in a 25 &#181;L injection). 
		CAUTION: The electrode in the ECD becomes depleted slowly, and the limit will be high due to the S/N ratio.</li>
		<li>Prepare a regression line from the 3-NT standard peaks, calculate the slope, and intercept.</li>
		<li>Assign these constants into the following equation and calculate the 3-NT content in the samples as follows. 
		Sample 3 &#8211; NT (nM) = [sample peak &#8211; intercept] / slope&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;Eq. (1) 
		Note: The standard curve should be well represented as a straight line between 5 nM and 500 nM.</li>
	</ol>
	</li>
	<li>Calculate the 3-NT in the air environment and the PM7 from the sample vial.
	<ol>
		<li>Multiply the 3-NT concentration by the reaction volume (and the dilution factor, if any), and evaluate the absolute 3-NT content in PM7 from the circle.</li>
		<li>Measure the collected filter area and calculate the 3-NT content in the total PM7 filter by the following equation. 
		Total PM7 = [3-NT content in the PM7 of the circle] x [ratio of the PM7 filter area to the circle area] x 226.19 (in the case of converting moles to grams)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;Eq. (2)</li>
		<li>Evaluate the 3-NT content in the air environment or PM7 by dividing the total PM7 by the total air flow volume or PM7 weight (recorded in step 1.1.3).</li>
	</ol>
	</li>
</ol>

<ol><li>Castillo, J. A., Staton, S. J. R., Taylor, T. J., Herckes, P., Hayes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+the+feasibility+of+bioaerosol+analysis+as+a+novel+fingerprinting+technique%5BTitle%5D)+AND+%22Analytical+and+Bioanalytical+Chemistry%22%5BJournal%5D)">Exploring the feasibility of bioaerosol analysis as a novel fingerprinting technique.</a> Analytical and Bioanalytical Chemistry. 403 (1), 15-26 (2012).</li>
<li>Franze, T., Weller, M. G., Niessner, R., Pöschl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+nitration+by+polluted+air%5BTitle%5D)+AND+%22Environmental+Science+%26+Technology%22%5BJournal%5D)">Protein nitration by polluted air.</a> Environmental Science & Technology. 39 (6), 1673-1678 (2005).</li>
<li>Abe, R. Y., Akutsu, Y., Kagemoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+amino+acids+as+markers+for+biological+sources+in+urban+aerosols%5BTitle%5D)+AND+%22Environmental+Chemistry+Letters%22%5BJournal%5D)">Protein amino acids as markers for biological sources in urban aerosols.</a> Environmental Chemistry Letters. 14 (1), 155-161 (2016).</li>
<li>D'Amato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allergenic+pollen+and+pollen+allergy+in+Europe%5BTitle%5D)+AND+%22Allergy%3A+European+Journal+of+Allergy+and+Clinical+Immunology%22%5BJournal%5D)">Allergenic pollen and pollen allergy in Europe.</a> Allergy: European Journal of Allergy and Clinical Immunology. 62 (9), 976-990 (2007).</li>
<li>Bolzacchini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gas-phase+reaction+of+phenol+with+NO3%5BTitle%5D)+AND+%22Environmental+Science+%26+Technology%22%5BJournal%5D)">Gas-phase reaction of phenol with NO3.</a> Environmental Science & Technology. 35 (9), 1791-1797 (2001).</li>
<li>Shiraiwa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiphase+chemical+kinetics+of+the+nitration+of+aerosolized+protein+by+ozone+and+nitrogen+dioxide%5BTitle%5D)+AND+%22Environmental+Science+%26+Technology%22%5BJournal%5D)">Multiphase chemical kinetics of the nitration of aerosolized protein by ozone and nitrogen dioxide.</a> Environmental Science & Technology. 46 (12), 6672-6680 (2012).</li>
<li>Gruijthuijsen, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitration+enhances+the+allergenic+potential+of+proteins%5BTitle%5D)+AND+%22International+Archives+of+Allergy+and+Immunology%22%5BJournal%5D)">Nitration enhances the allergenic potential of proteins.</a> International Archives of Allergy and Immunology. 141 (3), 265-275 (2006).</li>
<li>Kubo, M., Ogino, K. Analytical Procedures for Nitrative/Nitrosative Stress. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKubo%2C+M+%22Studies+on+Pediatric+Disorders%22">Studies on Pediatric Disorders</a>. Tsukahara, H., Kaneko, K. , Humana Press. 149-158 (2014).</li>
<li>Thomson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3-nitrotyrosine+modified+proteins+in+atherosclerosis%5BTitle%5D)+AND+%22Disease+Markers%22%5BJournal%5D)">3-nitrotyrosine modified proteins in atherosclerosis.</a> Disease Markers. 2015, 708282(2015).</li>
<li>Kuhn, D. M., Sakowski, S. A., Sadidi, M., Geddes, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitrotyrosine+as+a+marker+for+peroxynitrite-induced+neurotoxicity%3A+the+beginning+or+the+end+of+the+end+of+dopamine+neurons%3F%5BTitle%5D)+AND+%22Journal+of+Neurochemistry%22%5BJournal%5D)">Nitrotyrosine as a marker for peroxynitrite-induced neurotoxicity: the beginning or the end of the end of dopamine neurons?</a> Journal of Neurochemistry. 89 (3), 529-536 (2004).</li>
<li>Teixeira, D., Fernandes, R., Prudêncio, C., Vieira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3-Nitrotyrosine+quantification+methods%3A+Current+concepts+and+future+challenges%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">3-Nitrotyrosine quantification methods: Current concepts and future challenges.</a> Biochimie. 125, 1-11 (2016).</li>
<li>Hitomi, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disposition+of+protein-bound+3-nitrotyrosine+in+rat+plasma+analysed+by+a+novel+protocol+for+HPLC-ECD%5BTitle%5D)+AND+%22Journal+of+Biochemistry%22%5BJournal%5D)">Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD.</a> Journal of Biochemistry. 141 (4), 495-502 (2007).</li>
<li>Ogino, K., Wang, D. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomarkers+of+oxidative%2Fnitrosative+stress%3A+an+approach+to+disease+prevention%5BTitle%5D)+AND+%22Acta+Medica+Okayama%22%5BJournal%5D)">Biomarkers of oxidative/nitrosative stress: an approach to disease prevention.</a> Acta Medica Okayama. 61 (4), 181-189 (2007).</li>
<li>Tsikas, D., Mitschke, A., Suchy, M. -T., Gutzki, F. -M., Stichtenoth, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+3-nitrotyrosine+in+human+urine+at+the+basal+state+by+gas+chromatography-tandem+mass+spectrometry+and+evaluation+of+the+excretion+after+oral+intake%5BTitle%5D)+AND+%22Journal+of+Chromatography.+B%2C+Analytical+Technologies+in+the+Biomedical+and+Life+Sciences%22%5BJournal%5D)">Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography-tandem mass spectrometry and evaluation of the excretion after oral intake.</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 827 (1), 146-156 (2005).</li>
<li>Ito, T., Ogino, K., Nagaoka, K., Takemoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+of+particulate+matter+and+ozone+with+3-nitrotyrosine+in+the+atmosphere%5BTitle%5D)+AND+%22Environmental+Pollution%22%5BJournal%5D)">Relationship of particulate matter and ozone with 3-nitrotyrosine in the atmosphere.</a> Environmental Pollution. 236, 948-952 (2018).</li>
</ol>

The authors have nothing to disclose.

This article describes a quantification method to evaluate 3-NT in airborne PM collected on quartz filters using highly sensitive HPLC-ECD techniques.
In general, 3-NT measurement methods have been developed as biomarkers of oxidative stress in human diseases. Antibody-based methods (e.g., the enzyme-linked immunosorbent assay) are considered as semi-quantitative because there is no strict assay validation and it is difficult to assess the test&#39;s reliability. HPLC with electrochemical detection (ECD) and mass spectrometry-based assays have adequate sensitivity for the quantification of 3-NT13. Although they are sensitive, mass spectrometry-based methods like GC-MS or GC-MS/MS require the derivatization of amino acids, and the process of derivatization often results in the formation of artifacts14.
Compared to both the GC and LC techniques, HPLC-ECD is relatively less expensive and has a sufficient sensitivity to measure 3-NT. Additionally, the derivatization step in GC-MS and LC-MS often requires additional time.&#160;Although the method presented here requires 16 hours for the protein digestion step, it can, nevertheless, be carried out overnight (as described above, about 30 minutes of hands-on time is required per sample).Automatic repeat measurement by using an autosampler may provide a high-throughput measurement system.Previous studies have reported immunological methods to measure 3-NT in PM2,7; however, such methods could not detect 3-NT in the winter season due to the low levels of 3-NT in the PM15.
The HPLC-ECD method has additional advantages over other standard methods; for example, (i) an extraction process is not required in this method, (ii) it is completely&#160;detergent-free, (iii) it has minimal sample requirement, and, importantly, (iv) it has high sensitivity. Particles that are collected onto filters are frequently separated by sonication for further investigations, including the quantification of various components. Detergents are also used to isolate PM-bound proteins from filters. However, these additional steps increase the risk of contamination, sample loss, and underestimation due to extraction efficiency, and furthermore, most detergents are incompatible with LC/MS. In the present HPLC-ECD method, HPLC samples can easily be separated from the filter by using a simple hollow punch and without the requirement of an additional sample preparation process. The area of the round-shaped sample is 28.3 mm2, which is very small compared to the size of the original quartz filter (8 x 10 inch = 203.2 x 254 mm = 51,600 mm2).
The HPLC-ECD condition has been reviewed carefully to avoid interference to the 3-NT signal, as described previously12.&#160;A strong acidic pH of mobile phase and an adequate concentration of acetonitrile is important for detection.Using these conditions, other compounds, including nitro base, can be separated from nitrotyrosine.The present condition is suitable for the detection of 3-NT from samples with a different physical property (e.g., particulate matter and plasma).
To evaluate 3-NT in the air, the measurement of the filter weight and the elimination of the background are critical steps. In general, over approximately 100 mg of PM7 are collected over a period of four to seven days; however, several factors affect the filter weight before and after the PM7 collection, including humidity and static electrical charge. To avoid these effects, the stabilization of the filter weight for long periods is necessary under subequal temperature and humidity. The location where the electronic balance is installed should be maintained in a stable environment. For sample preparation, it is important to correct the background effects from the nonspecific protease cocktail and the ultrafiltration membrane, which often show a similar peak to 3-NT.
This method can be applied to other particles, including those in indoor environments. The nitration of proteins may increase their allergenic potential2. Therefore, this method may help to evaluate environmental cleanliness and prevent nitrosative stress.
In this study, we report a highly sensitive measurement method for atmospheric 3-NT of air sampler filters with easy-to-handle and inexpensive apparatus. The generation of 3-NT in the atmosphere is associated with environmental pollutants such as O3, NO2, PM proteins, and meteorological elements which affect human allergenicity. In conclusion, the method developed in the present investigation may help to assess the atmospheric reaction of O3, various pollutants, and PM proteins under various meteorological conditions. With these endeavors of developing HPLC-ECD for an estimation of 3-nitrotyrosine in the atmosphere, we anticipate better environmental cleanliness, improving human health.

Aerosol or airborne PM contains proteins from various biological origins, including viruses, prokaryotes, fungi, plants (pollen), and animals (insects, human)1.&#160;The ratio of protein content in PM is estimated to be nearly 5%, which has been implicated to play a major role as an airborne allergen2. Recent reports suggest that urban ambient aerosol of various sizes contain amino acids with ratios ranging between 0.5% and 2%3. In addition, chemical modifications of PM proteins have been suggested to be generated by the reactions of proteins with various pollutants, such as O3, nitrogen dioxide (NO2), and sulfur dioxide (SO2)4.
3-NT&#8212;a protein modification&#8212;is generated by the nitration of tyrosine residues.&#160;A higher concentration of O3 and NO2 has been shown to promote the nitration of protein molecules in pseudo-atmospheric space5,6.&#160;The nitration of tyrosine residues in the polypeptide chain enhances the allergenic potential of pollen proteins7.Thus, the quantification and assessment of airborne 3-NT is an important aspect in addressing the concerns of environmental health.
3-NT is also known as a biomarker of oxidative and nitrosative stress8.&#160;An emerging body of evidence has shown a significant association of 3-NT content with several human diseases9,10.&#160;Owing to its detrimental effects, the detection and quantitative estimation of 3-NT in a biological sample hold great relevance in determining the condition of an individual&#39;s health. In recent years, diverse methods have been introduced to estimate the 3-NT content, including enzyme-linked immunosorbent assays, HPLC, and LC/MS11. In previous studies, we have reported a detection method using the HPLC-ECD technique bestowed with several advantages compared to previous methods. For example, the HPLC-ECD does not require an extraction or derivatization procedure which makes it a relatively simple assay system12.We have confirmed that this method is applicable to detect 3-NT from biological samples (plasma from human and rat).
Although many investigations have emphasized the detection of 3-NT in biological samples, its detection in nonbiological samples remains elusive, and hence the present study has been pursued. In fact, to assess the harmful effect of airborne 3-NT, a quantitative method that is useful to detect 3-NT directly from airborne particles is required; therefore, we applied the existing HPLC-ECD method to detect 3-NT from PM2.5 collected on a glass-fiber filter.&#160;By using the technique, 3-NT could be detected directly from small pieces (6 mm-diameter round holes) of the sample from a PM collection filter.We further evaluated the content of 3-NT for various PM sizes and measured the detection limit of 3-NT.The present article proposes a high-sensitive and high-throughput method to detect 3-NT directly from both nonbiological and biological samples.

<table><tbody><tr><td>Quartz filter, backup quartz filter</td><td>PALL life sciences</td><td>7204</td><td>Tissuquatrz 2500QAT-UP, 8x10in</td></tr><tr><td>High-volume air sampler</td><td>SIBATA SCIENTIFIC TECHNOLOGY</td><td>HV-1000F</td><td></td></tr><tr><td>Particle size selector for SPM</td><td>SIBATA SCIENTIFIC TECHNOLOGY</td><td>080130-061</td><td>SPM (suspended particulate matters) is defined in Japan that particles with 10 um of 100% cut-off diameter.&amp;nbsp; SPM is almost identical to PM7.</td></tr><tr><td>Low-volume air sampler</td><td>SIBATA SCIENTIFIC TECHNOLOGY</td><td>AH-600F</td><td></td></tr><tr><td>Size classification unit for PM2.5</td><td>SIBATA SCIENTIFIC TECHNOLOGY</td><td>AH-600</td><td></td></tr><tr><td>Quartz filters for classification (PM4.5&amp;ndash;2.5, PM7.3&amp;ndash;4.5, PM15&amp;ndash;7.3, and TSP-PM15)</td><td>Tokyo Dylec</td><td>AHQ-630</td><td></td></tr><tr><td>Non-specific protease cocktail</td><td>Roche</td><td>11459643001</td><td>Pronase from Streptomyces griseus&amp;nbsp;</td></tr><tr><td>3-Nitrotyrosine</td><td>SIGMA</td><td>N7389</td><td></td></tr><tr><td>Ultrafiltration membranes</td><td>Millipore</td><td>UFC5010BK</td><td>AMICON ULTRA 0.5 mL - 10kDa cutoff</td></tr><tr><td>ECD</td><td>Eicom</td><td>HTEC-500, PEC-510</td><td>standalone HPLC-ECD unit (HTEC-510) and Pre-electrolysis cell (PEC-510), includes pump</td></tr><tr><td>HPLC column</td><td>Eicom</td><td>SC-5ODS</td><td></td></tr><tr><td>Data processor</td><td>Eicom</td><td>EPC-300</td><td></td></tr><tr><td>Injector</td><td>Hitachi High-Tech Science&amp;nbsp;</td><td>L-7200</td><td>Auto sampler</td></tr><tr><td>Analyzing software</td><td>eDAQ Japan</td><td>ES280&amp;nbsp;</td><td>PowerChrom software</td></tr></tbody></table>

detection of 3-nitrotyrosine in atmospheric environments via a high-performance liquid chromatography-electrochemical detector system

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

The HPLC-ECD principle is simple. Samples containing the target are separated by the HPLC column and mobile phase, and the separated target compound is reduced and/or oxidized in the electrochemical detector.
In the HPLC-ECD system, a 3-NT peak is detected in approximately 22 minutes. ECDs (PEC-510 and HTEC-500) are connected in tandem in the HPLC line. The first ECD reduces the hydroxyl groups in the compounds, and the next oxidizes the reduced chemicals. ECDs have a highly sensitive detection range of 10-15 M. The detection limit of the HPLC-ECD system was 1.13 pg/m3, which was calculated as the mean basal signal (mV) of 10 parts x 3.
Representative chromatograms are shown in Figure 4. By analyzing a pure 3-NT standard, a definitive peak with a stable baseline can be detected (Figure 4A). 3-NT in PM7 was also measured using 6 mm, round-shaped samples (Figure 4B).
Regarding the accuracy of 3-NT detection capability, when a 3-NT standard was injected in HPLC, the peak corresponding to 3-NT was detected at a retention time of 20 minutes. In PM samples, 3-NT was separated from other substances, and the independent peak was detected at the same retention time as the standard. A typical standard curve is shown in Figure 5, where linearity is observed between 5 and 40 nM. Linearity can be seen up to 500 nM (data not shown).
Table 1 shows the 3-NT contents in the atmosphere, calculated as pg/m3 air. The highest concentration of 3-NT in a PM2.5 prefilter was observed in #3 (PM7.3-4.5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58371/58371fig1.jpg" /> 
Figure 1: Collection scheme for TSP, PM7, and PM2.5. (A) The total suspended particles (TSP) are collected directly onto a collection filter (at a flow rate of 1,000 L/min). (B) When PM7 is collected, large particles are excluded by a size selector (at a flow rate of 1,000 L/min). (C) PM2.5 is collected through four prefilters (at a flow rate of 116 L/min). A quartz filter is commonly used as a backup filter for PM7 and PM2.5 or as a collection filter for TSP, as shown in this figure. <a href="https://www.jove.com/files/ftp_upload/58371/58371fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58371/58371fig2.jpg" /> 
Figure 2: Preparation of a 6 mm-round circle. The collection filter was cut using a hollow punch tool with a 6 mm diameter. Then, the sample was stored in a 1.5 mL microtube. <a href="https://www.jove.com/files/ftp_upload/58371/58371fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58371/58371fig3.jpg" /> 
Figure 3: Schematic diagram of the HPLC-ECD system. 3-NT in an injected sample was separated in the HPLC column through which mobile phase flows. The 3-NT was reduced to aminotyrosine, oxidized to iminotyrosine, and detected electrochemically in the detector. <a href="https://www.jove.com/files/ftp_upload/58371/58371fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58371/58371fig4.jpg" /> 
Figure 4: Typical chromatograms in the HPLC-ECD system. (A) Pure 3-NT analyzed by the HPLC-ECD system. (B) 6 mm circle sample from a PM7 filter used to detect 3-NT. A 3-NT peak is indicated by an arrow. <a href="https://www.jove.com/files/ftp_upload/58371/58371fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58371/58371fig5.jpg" /> 
Figure 5: Standard curve of pure 3-NT samples. The results at a lower concentration are indicated in another graph. <a href="https://www.jove.com/files/ftp_upload/58371/58371fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Subject</td>
			<td>Days of 
			Collection</td>
			<td>Spot</td>
			<td>3-NT 
			(pg/m3 air)</td>
		</tr>
		<tr>
			<td>PM2.5</td>
			<td>Prefilter #1</td>
			<td>27</td>
			<td>4</td>
			<td>8.25 &#177; 0.37</td>
		</tr>
		<tr>
			<td></td>
			<td>Prefilter #2</td>
			<td>27</td>
			<td>4</td>
			<td>29.66 &#177; 1.94</td>
		</tr>
		<tr>
			<td></td>
			<td>Prefilter #3</td>
			<td>27</td>
			<td>4</td>
			<td>74.37 &#177; 4.09</td>
		</tr>
		<tr>
			<td></td>
			<td>Prefilter #4</td>
			<td>27</td>
			<td>4</td>
			<td>32.70 &#177; 0.75</td>
		</tr>
		<tr>
			<td></td>
			<td>Backfilter</td>
			<td>7</td>
			<td>5</td>
			<td>4.21 &#177; 0.91</td>
		</tr>
		<tr>
			<td>PM7</td>
			<td>Backfilter</td>
			<td>7</td>
			<td>1</td>
			<td>89.16 &#177; 6.36</td>
		</tr>
		<tr>
			<td>TSP</td>
			<td>Filter</td>
			<td>7</td>
			<td>1</td>
			<td>10.34 &#177; 2.09</td>
		</tr>
	</tbody>
</table>
Table 1: 3-NT in the atmosphere measured by the HPLC-ECD system. The 6 mm circular spots from each filter were measured using the HPLC-ECD system. Prefilters #1 - #4 were collected in the same period. Note that the concentrations of 3-NT in the air are influenced by various factors, such as the collected date, year, or location. The data (n = 3 - 10) are represented as the mean &#177; the standard error.

Watch this Scientific Journal Video about Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System at JoVE.com

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

detection-3-nitrotyrosine-atmospheric-environments-via-high

Research

JoVE Journal

Environment

7.4K Views.  Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in milieu-onderzoek over Nitrapyrin en 3-nitrotyrosine. Het belangrijkste voordeel van deze techniek is dat het een unieke en zeer gevoelige methode is voor het detecteren van 3-nitrotyrosine op luchtmonsterfilters. Om totale zwevende deeltjes of deeltjes van minder dan zeven micron groot te verzamelen, weegt u eerst een binder 3 kwartsfilter en bevestigt u het op een luchtmonsternemer met een hoog volume. Bedek het filter me...

Video: Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently than plants3, but also synthesize advanced biofuels2-4. Generally, atmospheric CO2 is not a sufficient source for supporting maximal algal growth5. On the other hand, the high concentrations of CO2 in industrial exhaust gases have adverse effects on algal physiology. Consequently, both cultivation conditions (such as nutrients and light) and the control of the flue gas flow into the photo-bioreactors are important to develop an efficient &ldquo;flue gas to algae&rdquo; system. Researchers have proposed different photobioreactor configurations4,6 and cultivation strategies7,8 with flue gas. Here, we present a protocol that demonstrates how to use models to predict the microalgal growth in response to flue gas settings. We perform both experimental illustration and model simulations to determine the favorable conditions for algal growth with flue gas. We develop a Monod-based model coupled with mass transfer and light intensity equations to simulate the microalgal growth in a homogenous photo-bioreactor. The model simulation compares algal growth and flue gas consumptions under different flue-gas settings. The model illustrates: 1) how algal growth is influenced by different volumetric mass transfer coefficients of CO2; 2) how we can find optimal CO2 concentration for algal growth via the dynamic optimization approach (DOA); 3) how we can design a rectangular on-off flue gas pulse to promote algal biomass growth and to reduce the usage of flue gas. On the experimental side, we present a protocol for growing Chlorella under the flue gas (generated by natural gas combustion). The experimental results qualitatively validate the model predictions that the high frequency flue gas pulses can significantly improve algal cultivation.

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype &#34;flue gas to algal cultivation&#34; systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

Flue gas from power plants can promote algal cultivation and reduce greenhouse gas emissions1. Microalgae not only capture solar energy more efficiently ...

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

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

Trace explosive vapors of TNT and RDX collected on sorbent-filled thermal desorption tubes were analyzed using a programmed temperature desorption system coupled to GC with an electron capture detector. The instrumental analysis is combined with direct liquid deposition method to reduce sample variability and account for instrumentation drift and losses.

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive ...

Chemistry

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Biochemistry

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 ...

Biology

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Bioengineering

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 ...

Medicine

Carbon and Nitrogen Analysis of Environmental Samples

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Elemental Analysis is a method used to determine elemental composition of a material. In environmental samples such as soils, scientists are particularly interested in the amounts of two ecologically important elements, nitrogen and carbon. Elemental analysis by the flash combustion technique works by oxidizing the sample with a catalyst through combustion in a high-temperature chamber. The products of combustion are then reduced to N2 and CO2 and detected with a thermal conductivity detector.
Unlike other methods for total nitrogen determination (Kjeldahl method) and total carbon determination (Walkley-Black, Heanes or Leco methods), the flash combustion technique does not use toxic chemicals and is therefore much safer to use.
This video will demonstrate combustion-based elemental analysis using the Flash EA 1112 instrument from Thermo Fisher Scientific.

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
Elemental Analysis is a method used to determine elemental composition of a ...

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

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