This study was supported by grant no. CA-81301 from the National Cancer Institute. We thank Bob Carlson for editorial assistance, Dr. Peter Villalta and Xun Ming for mass spectrometry assistance in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, and Dr. Adam T. Zarth and Dr. Anna K. Michel for their valuable discussions and input. The Analytical Biochemistry Shared Resource is partially supported by National Cancer Institute Cancer Center Support Grant CA-77598

1. Materials and general procedures
<ol>
	<li>Synthesize NNN as previously described15. Obtain norcotinine, P450 2A6 Baculosomes, NADPH regeneration system, 0.5x&#160;reaction buffer, and all other chemicals or solvents from commercial sources in reagent grade.</li>
	<li>Record NMR spectra on a 500 MHz spectrometer. Report chemical shifts as parts per million (ppm). Use residual solvent peaks as internal references for 1H-NMR (7.26 ppm CDCl3) and 13C-NMR (77.2 ppm CDCl3). 
	NOTE: Peak splitting used the following abbreviations: s = singlet, d = doublet, dd = doublet of doublets, dt = doublet of triplets, dq = doublet of quartets, ddd = doublet of doublet of doublets, and m = multiplet.</li>
	<li>Perform high resolution mass spectrometry (HRMS) for selected compounds on an LTQ Orbitrap Velos and report data as m/z.</li>
	<li>Use polygram pre-coated silica gel TLC plates (40 mm x 80 mm, 0.2 mm thick) with 254 nm fluorescent indicator for thin-layer chromatography (TLC). Visualize TLC plates by UV lamp irradiation.</li>
	<li>Perform flash chromatography on a 60 &#197;, 70-150 mesh silica gel using a 1.5 cm x 15 cm glass column.</li>
</ol>
2. Preparation of nitrosamide positive control (N&#8242;-nitrosonorcotinine, NNC)
<ol>
	<li>Dry, in an oven overnight (16 h, ~140 &#176;C), a 25 mL, round-bottom flask containing a magnetic stir bar. In the morning, cool the flask under a stream of N2 while using an oil bubbler to keep the N2 flow rate under one bubble per second. 
	NOTE: After cooling, no precautions to keep the flask under N2 are required.</li>
	<li>In a fume hood, add norcotinine (31.8 mg, 0.196 mmol), acetic anhydride (5 mL), and acetic acid (1 mL) to the cooled flask. Stir this mixture continuously while cooling to 0 &#176;C with a water-ice bath. 
	Caution: Acetic anhydride and acetic acid are corrosive.</li>
	<li>Add NaNO2 (33.3 mg, 0.483 mmol) in one portion and continuously stir the mixture for 2.5 h. Monitor reaction progress by TLC (100% EtOAc, Rf = 0.19, see step 1.4). 
	NOTE: Over this time, the mixture will become increasingly yellow with occasional bubbling.</li>
	<li>Quench the reaction by pouring the mixture into ice-cold H2O (18 mL). Immediately extract the aqueous solution with 18 mL of CH2Cl2 in a 100 mL separatory funnel. Extract the aqueous layer at least 2 additional times with CH2Cl2 (9 mL each).</li>
	<li>Dry the pooled organics over ~100 mg of MgSO4 for 2 min. Filter and concentrate the solution by rotary evaporation to yield a crude, yellow oil. Heat the water bath to 30 &#176;C during evaporation to remove the residual acetic acid. 
	NOTE: The protocol can be paused here if the compound is dissolved in CH2Cl2 and the solution stored in the dark at 2 - 8 &#176;C.</li>
	<li>Purify the crude compound by column chromatography (see step 1.5) using silica gel as the stationary phase and 100% EtOAc as the eluent16. 
	Caution: NNC and related nitrosamides should be handled with care as they are assumed to be human carcinogens.</li>
	<li>Dissolve the pure compound in CDCl3 and use NMR (see step 1.2) to confirm the structure and determine the molarity of this solution17. Store this solution in the dark at 2 - 8 &#176;C until needed. 
	NOTE: This solution will be used as the positive control in the in vitro assay (step 3.5). NMR spectra and chemical shifts are available in the Supporting Information.</li>
	<li>With a pure NNC solution, confirm the accurate parent mass and determine product ion masses by direct infusion on a high-resolution mass spectrometer (HRMS) under parameters described in steps 1.3 and 4.3. 
	NOTE: Product masses will be used for detecting this compound in the in vitro metabolism assay (step 4.3).</li>
</ol>
3. An example nitrosamine-P450 in vitro incubation
<ol>
	<li>Remove P450 2A6 Baculosomes, Vivid-NADPH-Regeneration System, and 10 mM NADP+ Stock Solution from a -80 &#176;C freezer and let them thaw on ice. Once thawed, dilute the Baculosomes and NADPH-Regeneration System 1:10 and 1:50, respectively, into a single 1 mL tube with 0.5X reaction Buffer. Similarly, add 3 &#956;L of the NADP+ Stock Solution to 97 &#956;L of 0.5X Reaction Buffer. 
	NOTE: NADP+ is light sensitive. Keep this solution protected by wrapping its container in aluminum foil. Enzyme solutions are sensitive to freeze-thaw cycles; limit this to no more than 2 cycles. Prepare aliquots as necessary for future experiments.</li>
	<li>For each incubation, add 50 &#956;L of enzyme solution (containing 5 pmol P450) to a 1 mL tube containing 40 &#956;L of 4 &#956;M NNN solution made with Reaction Buffer. Pre-incubate this new mixture for 2 min at 37 &#176;C using a water bath and then add 10 &#956;L of diluted NADP+ solution. Incubate the full system for 1 - 30 min at 37 &#176;C. 
	NOTE: Using 5-min intervals for incubation times (e.g. 5, 10, 15 min, etc.) will provide a sufficient nitrosamide formation time course.</li>
	<li>After the desired incubation time, quench by first adding 10 &#956;l of 3.0 N ZnSO4 and then 10 &#956;L of 3.0 N Ba(OH)2. Vortex this solution and a white precipitate will form. Centrifuge the sample at 8000 x g for 4 min to pellet the precipitate. 
	NOTE: The procedure must not pause at this step as the formed nitrosamides have limited stability in aqueous solutions. Long pauses may result in false negatives.</li>
	<li>Immediately pipette the supernatant and analyze 2 &#956;L by liquid chromatography positive nanoelectrospray-ionization high-resolution tandem mass spectrometry (LC-NSI+-HRMS/MS, Steps 4.1 - 4.3).</li>
	<li>For positive control incubations, evaporate an aliquot containing 100 fmol of synthesized NNC solution into a 1 mL tube under a stream of N2. To this vial, add the full enzyme system from step 3.2 and proceed as described for steps 3.3 - 3.4.</li>
	<li>For negative control incubations, perform the experiment as described (steps 3.2 - 3.4) except replace the 50 &#956;L enzyme solution with 0.5X reaction buffer.</li>
</ol>
4. Example parameters for nitrosamide detection by LC-NSI+-HRMS/MS
<ol>
	<li>For the LC component, apply the following multi-step gradient in step 4.2 to a UPLC system using a commercial, hand-packed18 C18 (5 &#956;m), 100 mm x 75 &#956;m, 15 &#956;m orifice capillary column.</li>
	<li>Using 5 mM NH4OAc as solvent A and MeCN as solvent B, load the sample onto the column by running at 5% B at 1 &#956;L/min from 0 - 5 min, and then slowing the flow rate to 0.3 &#956;L/min afterwards. Next, run a gradient from 5 to 20% B over 4 min, followed by a ramp to 55% B over 10 min, and then re-equilibration to 5% B.</li>
	<li>Perform the LC-NSI+-HRMS/MS on a LTQ Orbitrap. Monitor for NNC by both full scan and MS2 fragmentation. Perform full scan at a resolution of 60,000 and extract the accurate parent mass (192.07670) at a mass tolerance of 5 ppm. Use MS2 fragmentation to isolate parent ions (2.0 amu) and fragment by collision-induced dissociation (CID) with a collision energy of 25 eV, resolution of 15,000, and scan time of 30 ms. Extract the accurate masses for product ions of NNC (m/z 192 m/z 134.04739 and 162.07874) at a mass tolerance of 5 ppm.</li>
</ol>

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<li>Chowdhury, G., Calcutt, M. W., Nagy, L. D., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidation+of+methyl+and+ethyl+nitrosamines+by+cytochrome+P450+2E1+and+2B1%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.</a> Biochemistry. 51 (50), 9995-10007 (2012).</li>
<li>Chowdhury, G., Calcutt, M. W., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxidation+of+N-nitrosoalkylamines+by+human+cytochrome+P450+2A6%3A+sequential+oxidation+to+aldehydes+and+carboxylic+acids+and+analysis+of+reaction+steps%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Oxidation of N-nitrosoalkylamines by human cytochrome P450 2A6: sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps.</a> J Biol Chem. 285 (11), 8031-8044 (2010).</li>
<li>Carlson, E. S., Upadhyaya, P., Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+nitrosamide+formation+in+the+cytochrome+P450-mediated+metabolism+of+tobacco-specific+nitrosamines%5BTitle%5D)+AND+%22Chem+Res+Toxicol%22%5BJournal%5D)">Evaluation of nitrosamide formation in the cytochrome P450-mediated metabolism of tobacco-specific nitrosamines.</a> Chem Res Toxicol. 29 (12), 2194-2205 (2016).</li>
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<li>Pauli, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Importance+of+purity+evaluation+and+the+potential+of+quantitative+%281%29H+NMR+as+a+purity+assay%5BTitle%5D)+AND+%22J+Med+Chem%22%5BJournal%5D)">Importance of purity evaluation and the potential of quantitative (1)H NMR as a purity assay.</a> J Med Chem. 57 (22), 9220-9231 (2014).</li>
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The authors have nothing to disclose.

Elucidating the metabolism of nitrosamines is a critical component to understanding their carcinogenicity. Since the involved cytochrome P450s and other metabolic enzymes are polymorphic, further application of this knowledge could potentially identify high risk individuals1,4. New data indicates that further oxidation of &#945;-hydroxynitrosamines, the presumed major metabolites of nitrosamines involved in DNA binding, to nitrosamides is possible; however, this ...

N-nitrosamines are a large class of carcinogens found in the diet, tobacco products, and the general environment; they can also be formed endogenously in the human body1. More than 300 N-nitroso compounds have been tested and &#62;90% were evaluated as carcinogenic in animal models2,3. To exhibit their carcinogenicity, these compounds must first be activated by cytochrome P450s1,2,3. Research shows that cytochrome P450s readily oxidize nitrosamines to &#945;-hydroxynitrosamines (Figure 1), which are highly reactive compounds with half-lives of ~5 s before spontaneously decomposing to alkyldiazohydroxides. The latter can alkylate DNA after the loss of H2O and N2. The resulting DNA adducts, if unrepaired, can cause mutations that, if in critical onco- or tumor suppressor genes, lead to cancer development1. For this reason, much effort has been expended to acquire a full understanding of the metabolic pathways, DNA adducts, and downstream metabolites of cytochrome P450 oxidation of carcinogenic nitrosamines. This knowledge has potential application in individual cancer risk assessment4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg" /> 
Figure 1: General and proposed metabolism of nitrosamines. 
Nitrosamines (1) are oxidized by P450s to &#945;-hydroxynitrosamines (2) that spontaneously decompose to alkyldiazohydroxides (3). These compounds can bind to DNA to form DNA adducts. It is hypothesized that 2 are further oxidized by P450s to nitrosamides 4. These can directly bind to DNA to form novel DNA adducts or be hydrolyzed to 3 to form known DNA adducts. R1 and R2 represent any alkyl group. <a href="//cloudflare.jove.com/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although the &#945;-hydroxynitrosamine hypothesis is solidly supported by extensive data, there are a few inconsistencies; a major one is the short half-life of &#945;-hydroxynitrosamines5,6. It is known that these compounds are produced at the endoplasmic reticulum membrane and later alkylate nuclear DNA. Given their lifetime of a few seconds, it is puzzling how these intermediates survive the required travel though the cytosol. One hypothesis is that a portion of the &#945;-hydroxynitrosamines are processively oxidized to nitrosamides7,8, which are quite stable in comparison9. This would presumably occur via retention of &#945;-hydroxynitrosamines in the cytochrome P450 active site. Precedent for this type of oxidation has been seen with nicotine10, alcohols11, and simple alkylnitrosamines12,13. Additionally, nitrosamides are direct-acting carcinogens2,3. Based on their reactivity9, these compounds are believed to produce DNA adducts identical to those resulting from &#945;-hydroxynitrosamines along with new, unexplored DNA adducts (Figure 1). Thus, this hypothesis not only explains the transport through the cytosol, but also the formation of DNA damage products.
In this paper, a general protocol for assessing the in vitro cytochrome P450-mediated conversion of nitrosamines to nitrosamides is described. The previously reported conversion of N&#8242;-nitrosonornicotine (NNN) to N&#8242;-nitrosonorcotinine (NNC) by cytochrome P450 2A6 is showcased as an example14. Application of this protocol to a wide range of substrate-enzyme systems will help determine the importance of nitrosamides in overall nitrosamine metabolism.

<table><tbody><tr><td>Norcotinine</td><td>AKoS GmbH (Steinen, Germany)</td><td>CAS 17708-87-1, AKoS AK0S006278969</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma-Aldrich</td><td>242845</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>431311</td><td></td></tr><tr><td>Barium Hydroxide</td><td>Sigma-Aldrich</td><td>433373</td><td></td></tr><tr><td>D-Chloroform</td><td>Sigma-Aldrich</td><td>151823</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Sigma-Aldrich</td><td>34998</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methylene Chloride</td><td>Sigma-Aldrich</td><td>34856</td><td></td></tr><tr><td>Sodium Nitrite</td><td>Sigma-Aldrich</td><td>237213</td><td></td></tr><tr><td>ViVid CYP2A6 Blue Screening Kit</td><td>Life Technologies</td><td>PV6140</td><td></td></tr><tr><td>Zinc Sulfate</td><td>Sigma-Aldrich</td><td>221376</td><td></td></tr><tr><td>0.5 mL tubes</td><td>Fisher</td><td>AB0533</td><td></td></tr><tr><td>100 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z510424</td><td></td></tr><tr><td>125 mL Erlenmeyer flask</td><td>Sigma-Aldrich</td><td>CLS4980125</td><td></td></tr><tr><td>125 mL Separatory Funnel</td><td>Sigma-Aldrich</td><td>Z261017</td><td></td></tr><tr><td>25 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z278262</td><td></td></tr><tr><td>500 MHz NMR Spectrometer</td><td>Bruker</td><td></td><td></td></tr><tr><td>Allegra X-22R Centrifuge</td><td>Beckman-Coulter</td><td></td><td></td></tr><tr><td>LC vials</td><td>ChromTech</td><td>CTC&amp;ndash;0957&amp;ndash;BOND</td><td></td></tr><tr><td>LTQ Orbitrap Velos</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Magnetic Stir bar</td><td>Sigma-Aldrich</td><td>Z127035</td><td></td></tr><tr><td>NMR tube</td><td>Sigma-Aldrich</td><td>Z274682</td><td></td></tr><tr><td>P1000, P200, and P10 pipettes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Rotary evaporator</td><td>Sigma-Aldrich</td><td>Z691410</td><td></td></tr><tr><td>RSLCnano UPLC system</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Shaking Water Bath</td><td>Fisher</td><td>FSSWB15</td><td></td></tr><tr><td>Stir plate</td><td>Sigma-Aldrich</td><td>CLS6795420</td><td></td></tr><tr><td>PicoFrit Column</td><td>New Objective</td><td>PF3607515N5</td><td></td></tr><tr><td>Luna C18, 5 um</td><td>Phenomenex</td><td>535913-1</td><td></td></tr></tbody></table>

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.

Based on the work of White et al.19, norcotinine was nitrosated to NNC cleanly and in high yield (80 - 92%) to produce a standard for the in vitro experiment. Structural evidence for a successful reaction was obtained from spectroscopic analyses including 1H-NMR, 13C-NMR, COSY, and HSQC (Supporting Information) along with HRMS which confirmed the parent mass [M + H]+ within 5 ppm of the theoretical value (<strong c...

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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.

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 ...

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Research

JoVE Journal

Cancer Research

10.0K Views.  University of Minnesota. α-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. 

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

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

Biochemistry

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Medicine

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.

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).

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

Environment

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 ...

Chemistry

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 ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Biology

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 ...

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

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

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

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

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

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

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

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

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

Summary

Explore More Videos

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by ³²P-postlabeling

Analytical Techniques for Assaying Nitric Oxide Bioactivity

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

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

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

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

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

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements