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

著者らは、血液中の亜硝酸塩の測定のための亜硝酸塩保存液の使用の開発に博士A. DejamとMMペルティエの重要な貢献を承諾します。

動物の使用を含むすべてのプロトコルは、NIDDK動物実験委員会によって使用が承認され、ヒトの血液は、健康なドナーからNIHの血液バンクから入手しました。 1.試料の調製<ol><li> 亜硝酸塩保存溶液の調製 <ol><li>蒸留水に890 mMのフェリシアン化カリウムを含む溶液を準備する（K 3のFe（CN）6）および118 mMのNEM（N-エチルマレイミド）。それが存在していない結晶と黄色透明になるまでよく溶かします。 9比（V / V、NP-40 /ソリューション）：1にNP-40（オクチルphenoxylpolyethoxylethanol）を追加します。 ）約一週間のために4℃で発泡や店舗を避けるために、穏やかに混合。 </li></ol></li><li> 収集および血液サンプルの調製 <ol><li>凝固（5 U / ml）を防止するためにヘパリンと溶血を回避するために、少なくとも20 G針を用いて血液を収集します。 4の比率（V：全血サンプルについては、亜硝酸塩は1ですぐに解決策を維持して血液を混ぜます/ v溶液/血液）。 </li><li>血漿および赤血球サンプルについて、遠心分離を4℃で4,000×gで5分間採取した血液は、赤血球（RBC）および血漿を分離しました。上清（血漿）を取り、血漿中亜硝酸塩濃度の決意について上述したように亜硝酸溶液を保存して、それを混ぜます。 </li><li>注意深く切断ピペットチップを使用して、血液細胞の他の種類による汚染を避けるために、管の底部からサンプルピペットRBC試料からの残りの血漿およびバフィーコートを除去し、そして同じで亜硝酸塩保存液を含むチューブにそれを転送します上記のような割合。 </li><li> 1または1：1の比率で冷メタノールで各サンプル（血漿およびRBC）を混ぜる2（V / Vのサンプル/メタノール）およびタンパク質を沈殿させ、15分間4℃で13,000×gで、それらを遠心。 -80℃のドライアイスや店舗に、必要に応じて、上清を取り、亜硝酸塩の測定のためにそれを使用するか、または調製したサンプルを凍結します。 注：亜硝酸急速にREA血液形成硝酸中のオキシヘモグロビン（オキシヘモグロビン）とCTS。オキシヘモグロビンは常に亜硝酸塩を超える大過剰に存在するので、この反応は〜10分の時間枠でネイティブの血液亜硝酸塩がほぼ完全に消滅につながります。内因性の血液亜硝酸塩の大部分を維持するために、亜硝酸塩保存溶液は直ちに採血後の全血試料に添加されます。溶液は急速に、化学的に亜硝酸塩と反応しないヘモグロビンの不活性型の赤血球を溶解し、メトヘモグロビン（メト）にオキシヘモグロビンを酸化します。 </li></ol></li><li> 他のタイプの体液からの試料の調製 <ol><li>適切な容器への試料採取後、亜硝酸塩保存液、deproteinateでよく混ぜ、すぐに測定するか、-80℃で凍結保存します。 </li></ol></li><li> 収集及び組織サンプルの調製 <ol><li>ヘパリン添加生理食塩水で灌流した動物から組織を収集（10 UのHEPARIN / mlで）。所望の組織の物品税約1グラムと均質化、手動ホモジナイザーや自動化されたホモジナイザーを使用して。 </li><li>滑らかなホモジネートを達成するのに必要な亜硝酸塩保存溶液の既知量を加えます。 </li><li>上記のように4℃で13,000×gで遠心分離、その後サンプルは15分間、（2比（v / v）のサンプル/メタノール：1または1）組織を均質化した後、冷メタノールを使用してタンパク質を沈殿させます。 </li><li>上清を収集し、亜硝酸塩レベルを測定します。必要に応じて、-80℃でドライアイスとストアの調製の任意の段階でサンプルを凍結します。 </li></ol></li></ol>ソリューションを減らすの調製<ol><li> 三ヨウ化物（I 3）亜硝酸塩とR-（X）-NO種の決意1,3,4のためのソリューションを削減します <ol><li>水中の138 mMのI 2溶液と一緒に301 mMのKIの溶液を調製します。すべてまで、約30分間マグネチックスターラー上7の比（V / v溶液/酸）：2に氷酢酸と混合結晶を溶解させます。好ましくはヨウ化物は、光に敏感であるように、暗いボトルに保管し、準備から1週間以内に使用します。 注：このソリューションは、NOに亜硝酸塩を削減し、それはまた、R-SNO、R-NNOとFe-NO官能基（R-（X）-NO）からNOを放出しますが、硝酸塩は影響を受けません。上記でNO系官能基からの信号は、酸性化スルファニルアミド（AS）と試料の半分を処理することにより真の亜硝酸塩信号から分離し、AS処理および未処理の信号を比較することができます。 AS不可逆的に亜硝酸とジアゾニウム陽イオンを形成し、この複合体はI 3溶液によって低減することができません。治療としては、1 M HClにスルファニルアミドの290 mM溶液を準備し、比1に試料のアリコートに追加：9（V / V AS /サンプル）。サンプルのAS-処理および未処理の部分でCL信号を測定します。サンプルのAS-処理および未処理部分の差として真の亜硝酸塩信号を計算します。亜硝酸塩はまた、選択NITを用いて測定することができます儀式低減部分2.2で説明したように、アスコルビン/酢酸混合物の溶液を。しかし、現在の通常小さいR-量（X）-NO種に、AS-未処理試料を用いた測定は、ほとんどの場合、総亜硝酸塩の含有量の非常に良好な近似です。 </li></ol></li><li> 亜硝酸塩2の選択的決意のためのアスコルビン酸/酢酸（4A）ソリューション <ol><li>水の中の500mMアスコルビン酸を準備します。反応混合物を調製するための7（V / V、アスコルビン酸/酢酸）：比1氷酢酸を用いてこの溶液を混合します。 注：このソリューションは、亜硝酸塩に特異的である、任意のR-（X）-NO種または硝酸塩からNOを放出しません。アスコルビン酸は容易に溶液中で酸化するようしかし、アスコルビン及び酢酸の溶液は、毎日測定の前に新たに調製しなければなりません。 亜硝酸還元の完了は、アスコルビン酸の濃度に依存します。少なくとも50 mMのアスコルビン酸は完全なrのために推奨されていますプラズマ亜硝酸塩の排気行程。しかし、前に最終的なサンプル測定に予想される亜硝酸塩濃度範囲内アスコルビン酸及び亜硝酸規格の異なる濃度のいくつかのパイロット実験を行うことをお勧めします。アスコルビン酸はわずかガラス製反応容器内の反応混合物のように頻繁な変更が推奨され、測定中に枯渇することに注意してください。 </li></ol></li><li> 硝酸決意9用の塩化バナジウム還元溶液 <ol><li> 1 M HCl中のVCl 3の51 mM溶液を準備します。暗いボトルで200μmのフィルターとストアを介してフィルタソリューションは、2週間以内に使用します。 このソリューションは、硝酸塩、亜硝酸塩、すべてのR-（X）-NO種を削減する、亜硝酸塩または他のR-（X）-NO種の匹敵する量が一緒に硝酸で存在しているので、もし、最後の硝酸塩含量を計算しなければならない注： VCl 3とIで得られたCL信号の差として3ソリューションを減少させます。 </li></ol></li></ol> 3.化学発光（CL）アナライザのセットアップと測定<ol><li> 標準溶液 <ol><li>亜硝酸ナトリウム（NaNO 2を）の1μM溶液を調製します。同じ溶液は、亜硝酸塩と硝酸塩の測定のために使用することができます。 </li></ol></li><li> NOアナライザを使用していません 注： - SieversのNOAとEcophysics CLD 88Y現在の生物学的研究目的のために十分な感度である2つの市販されているNOアナライザはありません。これらは両方とも同じ原理で動作します。主な違いは、CLD 88Y、オゾンを作るために室内空気からの酸素を使用することである（O 3）、NOAは、この目的のために外部の酸素タンクを必要とします。手順は以下のSievers NOAのためのセットを記述します。 <ol><li> 機器の設定 <ol><li>製造業者の推奨に従ってNO分析装置の初期セットアップを実行します図1に見られるように。 </li><li>機器に接続されたオープン・O 2のタンク。 「分析」を選択し、前面パネルのメインメニューから「入力します」。次の画面で選択し、「開始」を押し「入力してください。」 図1に見られるような機器に（1 M NaOHを含む）は、酸トラップを接続します。 </li><li>光電子増倍管は、-12℃以下の温度まで冷却し、反応室は6 Torrまで排気されるまで待ちます。これは、安定した平らなベースラインに達するのに約30分かかります。ベースラインが1以内に安定化する必要がある - 2 mVの、公称値は、その安定性よりも重要です。 注：硝酸塩が測定された場合、冷却（酸と水と蒸気用としてコールドトラップを提供しています4°Cに設定）水浴及び加熱水浴（主反応室、95°C）をオンにします。 VCl 3溶液中のNOへの硝酸塩の減少率は、温度依存性であり、そしてそれは部屋テンペで非常に遅いですratureは、温度のように実質的な増加は、反応を観察するために必要とされます。 </li><li>彼タンクを開き、 図1に見られるような酸トラップするガラス反応容器を接続します。適切な還元溶液との反応チャンバを満たす最小の泡立ちを減少させ、酸トラップへの反応チャンバを接続します。試行錯誤を使用して、機器の吸引速度（SieverのNOAのための通常〜200ミリリットル/分）を一致させるために彼バブリング速度を調整します。 </li><li>楽器を制御するコンピュータの電源を入れおよびLabVIEWベースのリキッドソフトを起動します。機器および取得ソフトウェア、プレスの間の通信を有効にするには、画面を読み取るまで「データメニュー」の「出力が有効になって」と、その後、「クリア」を押し、データ画面に戻って戻るために「決定」。 </li><li>画面上の安定したベースライントレースを待ってセプタムを通してソリューションを減らすにサンプルおよび亜硝酸標準を注入開始。常に安定したベースライン後方に到達するのを待ちますピークえー。これは2分かかることがあります。液体ソフトウェアは、注射のオプションに &quot;マーク&quot;時間があり、注射に注釈を付け、それがデータファイル（filename.data）に役立つ補完するものとして、別のファイル（filename.info）にこれらのコメントをエクスポートします。 </li><li>これは「filename.data」として知られている永続的なファイルに一時ファイルからデータを書き込みます - サンプルおよび標準が測定されると、液体のソフトウェアメニューのオプションを「停止」を選択します。押すと「中止」オプションは、永続的なファイルに取得したデータを書き込むことなく測定を終了します。 </li><li> 、実験を終了し、反応容器の上部にコックをオフにすることによって、反応容器からマシンを切断し、酸トラップと反応容器間のチューブを切断する（ 図1を参照）。今NOAアナライザを停止 - を押して「クリア」、「分析」に行き、「スタンバイ」と「確認」スタンバイ状態にマシンを置きます。定期的に使用した場合、器具は、数週間待機モードのままにすることができます。酸素の供給をオフにします。次いで、反応室、切断の還元溶液を洗い流し、ガラス反応容器からヘリウムタンクを閉じて反応容器を洗浄終了。 </li></ol></li><li> 標準およびサンプル注入 <ol><li>よく洗浄したハミルトンシリンジを用いて反応混合物に1μM硝酸溶液50μlを注入します。ピークの良好な分離を達成するために注射間2分 - 少なくとも1のを待ちます。各データポイントに重複を取得するために50μlの注入を繰り返します。各注入後に脱イオン水で注射器を洗浄します。 </li><li>標準曲線のためのデータの完全なセットを取得するには、100の重複注射、1μM亜硝酸溶液を150μlの（亜硝酸塩や硝酸塩の高濃度が予想される場合、追加の200μlを必要とするかもしれません）に進みます。それぞれのケースでは、信号がベースラインに戻って低下するまで待つように世話をしてから1獲得 - 2マイルをピークの良好な分離を達成するために、ベースラインのn個。 注：亜硝酸塩標準実験中の任意の時点で測定することができます。また、機器の機能の独立したチェックとして機能するようにしかし、データ測定の前に、標準曲線を取得することが好ましいです。 データが収集される場合、試料の量が十分に顕著なピークをもたらすはずで注入しました。しかし、光電子増倍管が唯一〜800 mVの最大線形であるため、ピークのいずれも〜700 mVでより高くてはならないことを心に留めておくべきです。これは、量注入されたサンプルの減少又は脱イオン水で試料を希釈することによってのいずれかで達成することができます。各点は、二重に、少なくとも測定されるべきです。 </li></ol></li></ol></li></ol>

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博士アランシェクターは、心血管疾患の治療のための亜硝酸塩の使用のために国立衛生研究所に発行されたいくつかの特許上の共同発明者として記載されています。彼は、NIHの臨床開発のためにこれらの特許のライセンスが、ありませんその他の報酬に基づくロイヤルティを受け取ります。

プロトコル内の重要なステップオリジナルサンプルを調製希釈又は他の方法で治療するために使用される（水を含む）全ての溶液のアリコートを保存する必要があり、可能な亜硝酸または（より頻繁に）硝酸塩汚染を確認しました。私たちは、最も汚染が水から来て、多くの化学物質はまた、内因性の硝酸決意を妨害するいくつかのロットでの硝酸塩汚染のかなりの量が含...

亜硝酸塩、およびより少ない硝酸塩を拡張するために、血液中の濃度は、身体の全体的な状態のNO代謝を反映していません。亜硝酸塩の血液中の濃度は、ほとんどの臓器や組織は、高ナノモルまたは低マイクロモルの範囲であるだけで、硝酸塩は、通常、はるかに高い量で存在する - マイクロモルの範囲内で。亜硝酸塩の疾患の進行に起因するレベルまたは食習慣の変化の変化は非常に小さく、非常に感度の高い方法を用いて測定することができます。そのため、それらの非常に異なるレベルと異なる代謝過程で、亜硝酸塩と硝酸塩のレベルの独立した決意が不可欠です。亜硝酸塩と硝酸塩が一緒に測定されている」 の NO x決意」いわゆるませんが、非常に小さな値となっています。 種々の生物学的試料中の亜硝酸塩を定量化するためのいくつかの方法が開発されている - 最も一般的には元々あっても現代modificatioと1879年に記載されたグリース反応に基づいて、最も古いものですNS、グリース」の方法によって達成亜硝酸塩のための感度限界は、低マイクロモルの範囲です。三ヨウ化物還元溶液と組み合わせた化学発光（CL）は、現在、亜硝酸塩濃度1-8,10,11の低いナノモル範囲の定量化を可能にする、最も敏感な方法であると考えられています。溶液を還元バナジウム（III）塩化物と組み合わされ、同じCL法は、ナノモル範囲9の精度と、硝酸塩の敏感な測定のために使用することができます。 CLは、遊離ガスのNOを検出します。従って、亜硝酸塩、硝酸塩、Rニトロソチオール（R-SNO）、R-ニトロソアミン（R-NNO）、または金属-NO化合物（後の原稿の「R-（X）-NO」と称する）に変換されなければなりませんCLを介して元の量を定量化するために、NOガスを解放しません。 NOへの変換は、NO代謝物の性質に応じて、いくつかの異なる還元性溶液を用いて達成されます。変換後、遊離NOガスは、キャリアガス（彼は、Nによって反応容器からパージされていませんオゾン（O 3）（NO 2）その活性化状態で二酸化窒素を形成するために、NOと組み合わされCL分析装置の反応チャンバ内に2またはAr）。基底状態に戻ると、NO 2が *赤外領域で発光し、放出された光子は、CL装置の光電子増倍管（PMT）によって検出されます。放出される光の強度は、適切な較正曲線を使用して、元の種の濃度の計算を可能にする反応チャンバ内のNO濃度に正比例します。 我々のプロトコルでは、我々最も使用される臨床現場における亜硝酸塩と硝酸塩の最初の存在CLベースの決意 - 血液および血漿中、およびその後、我々は、組織試料中のこれらのイオンを決定する方法について説明します。我々はまた、血液やそのコンパートメント、血漿および赤血球などの亜硝酸塩反応性環境でオリジナルの生理的亜硝酸塩濃度を維持する方法を詳細に説明します。

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

measuring nitrite and nitrate, metabolites in the nitric oxide pathway, in biological materials using the chemiluminescence method

一酸化窒素（NO）は、血管の恒常性とも神経伝達物質の主要な調節因子分子の一つです。酵素的に生成NOは、様々なオキシヘムタンパク質および他のまだあまり知られていない経路との相互作用によって亜硝酸塩と硝酸塩に酸化されます。逆のプロセス、NOへの亜硝酸塩と硝酸塩の減少は10年で、哺乳類で発見されていた、あると考えられている心血管、代謝と筋肉の障害の全範囲を防止または緩和するのいずれかの可能な経路の一つとして注目を集めていますNOレベルの低下に関連すること。血液、体液、および様々な組織 - 異なる身体区画にNO及びその代謝産物の量を推定することが重要です。その簡単なアクセシビリティのために血液は、NO代謝物の推定のために使用される好ましいコンパートメントです。その寿命が短い（数ミリ秒）および低サブナノモル濃度、血液のNOの直接の信頼性の高い測定のために&lt;em&gt;の生体内存在の偉大な技術的困難インチしたがって、NO可用性は、通常、その酸化物、亜硝酸塩と硝酸塩の量に基づいて推定されていません。これら二つの代謝産物は、常に別々に測定されています。生物学的液体および組織におけるそれらの濃度を決定するためのいくつかの十分に確立された方法があります。ここでは、それぞれ、三ヨウ化物またはバナジウム（III）塩化物溶液によって亜硝酸塩や硝酸塩還元後にNOの分光光度検出に基づいて、化学発光法（CL）のためのプロトコルを提示します。亜硝酸塩と硝酸塩の検出感度はNO代謝経路の変化を決定するために現在利用可能な最も感度の高い方法としてCLを設定し、低ナノモル範囲です。私たちは、サンプル中のそれぞれの量を決定するために収集し、どの時点で亜硝酸塩と硝酸塩の存在の元の量を維持するために、生物学的流体および組織からのサンプルを調製する方法を詳細に説明します。 CL技術の限界もEXPありますlained。

図2は、基準および5の異なるサンプルから収集代表的な結果を示しています。この図に示すように、光電子増倍管電圧が上昇するが、亜硝酸塩含有溶液（標準またはサンプル）を溶液に還元中に注入された直後にベースライン値に戻る注入で一度全て亜硝酸存在（注射時間は赤い曲線の下の矢印で示されています）溶液を還元しました。また、正確な量?...

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

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

化学発光法を用いて、生物学的材料に、一酸化窒素経路に亜硝酸塩と硝酸塩、代謝物を測定します

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

measuring-nitrite-nitrate-metabolites-nitric-oxide-pathway-biological

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Signaling Networks of G Protein-coupled Receptors

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22.4K ビュー.  NIDDK, NIH. Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

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

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

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

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

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

チトクローム p450 によるニトロソアミンのIn Vitro代謝 Nitrosamide 形成を検出するための一般的な方法

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

がん研究

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.

NOの窒素同位体分析のための自動化、高解像度モバイル収集システム<sub&gt; X</sub

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

環境学

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

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

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

窒素酸化物ガスで害虫駆除のため研究所くん蒸処理の手順

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

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.

一酸化窒素の生物活性をアッセイするための分析手法

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 ...

医学

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

異なる動物モデルにおけるNADPHオキシダーゼ活性の生物発光イメージング

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

免疫・感染学

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3&#8722; for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3&#8722; transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3&#8722; added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3&#8722; via persulfate oxidation. Third, the NO3&#8722; is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography&#8211;mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography&#8211;mass spectrometry.

N2Oへの逐次変換による14NH4+/154+ 1415NH4++分析に基づくアンモニウムへの非シミラトリー硝酸還元の電位率の測定

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has ...

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

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

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

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

無傷の動脈の内皮層における一酸化窒素とスーパーオキシドのエン顔検出

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

生物学

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.

自動酸化およびニトロソ化化合物からの一酸化窒素およびその誘導体の検出のための化学発光ベースのアッセイ

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遺伝的にコードされた蛍光一酸化窒素の応用（•NO）シングル細胞におけるNO•信号のリアルタイムイメージングのためのプローブ、geNOps、

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

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

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

硝酸塩および亜硝酸塩測定のためのラット骨格筋ホモジネートの調製

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

化学発光法を用いて、生物学的材料に、一酸化窒素経路に亜硝酸塩と硝酸塩、代謝物を測定します

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