JP5731860B2 - 腹膜組織の傷害検出方法 - Google Patents
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Description
以上、腹膜組織の傷害の検出の重要性ついて説明したが、他の組織や臓器でも同様に早期に傷害を検出することで、予後が大きく異なってくる。
(1)腹膜透析患者から採取された腹膜透析液の排液を検体とし、該検体のタウリン濃度およびヒポタウリン濃度を測定し、健常値に比較したタウリン濃度の上昇およびヒポタウリン濃度の低下を指標として腹膜透析患者における腹膜組織の傷害を検出する傷害検出方法。
したがって生体から採取した検体においてタウリンおよびヒポタウリンの濃度を測定し、それぞれの健常値と比較することにより、臓器または組織の傷害を簡便に評価することができる。ここで、本発明における「健常値」とは、臓器または組織の傷害を生じる前の健常時に採取した検体のタウリンおよびヒポタウリン濃度測定値、もしくは臓器または組織の傷害を生じていない健常人から採取した検体のタウリンおよびヒポタウリン濃度測定値を意味する。
実験1)組織傷害動物モデルの作成
SDラット(雄、5週齢、n=6)に対し、健常群(コントロール群)には腹膜透析液(pH5)を、組織傷害群には20mMメチルグリオキサール添加腹膜透析液(pH5)を、1回/日、21日間連続腹腔内投与した(MGO投与群)。22日目に組織および臓器の傷害を解析するために、腹膜機能をPETで評価した。PETは以下の手順で行った。まず、2.5%グルコース含有腹膜透析液を腹腔内に注液した後、注液直後および90分後に排液の一部を回収し、グルコース濃度を測定した。得られた測定値からD/D0グルコースを算出し、腹膜機能を評価した。この結果、MGO投与群では腹膜機能が低下しており、機能の面から腹膜傷害が確認された(図1)。
腹腔排液中のタウリン量およびヒポタウリン量は、飛行時間型質量分析法(TOFMS)法で解析した。
検体の前処理として、内部標準物質の終濃度が10μMとなるように調整したメタノール溶液900μLに腹腔排液100μLを添加して、検体中の酵素を失活させて代謝反応を止めた。さらに精製水400μLとクロロホルム1mLを加え、1分間攪拌し、4℃にて2300×g、5分間の遠心分離を行い、2液に分離した上層の水層を限外濾過チューブ(5kDa、ミリポア社性ウルトラフリーMC PBCC遠心フィルターユニット)に250μL×3本に分注し、4℃、9100×g、120分間の限外濾過を行い、タンパク質を除去した。得られた濾液を凍結乾燥させ、再度精製水25μLに溶解して測定に供した。
キャピラリー電気泳動は、Fused silica capillary (内径:50μm、外径:350μm、全長:100cm)で行った。検体は加圧法で50mbarで15秒間注入し、緩衝液は1M蟻酸(pH1.8)を使用し、20℃で加電圧+30kVで解析した。
続いて飛行時間型質量分析計(アジレント CE−TOF−MS Dシステム、アジレント社)で質量を分析した。正イオンモードで、キャピラリー電圧4000V、フラグメンター電圧75V、スキマー電圧50V、OctRFV電圧125Vで行った。乾燥ガスは窒素を用い、300℃、圧力10psingで行い、スキャンレンジはm/z 50−1000で解析した。質量校正はレゼルピン(m/z:83.0703)で行った。
TOFMSで検出されたピークは、自動積分ソフトウエアMasterHands Ver.1.0.6.8(慶應義塾大学製)で自動抽出し、質量荷電比(m/z)、泳動時間(migration time:MT)とピーク面積を計測した。ピーク以外のノイズを除去した後、得られたm/zとMTの値をもとにピークの同定と定量を行った。
Claims (1)
- 腹膜透析患者から採取された腹膜透析液の排液を検体とし、該検体のタウリン濃度およびヒポタウリン濃度を測定し、健常値に比較したタウリン濃度の上昇およびヒポタウリン濃度の低下を指標として腹膜透析患者における腹膜組織の傷害を検出する傷害検出方法。
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