2018/04/11

薬品を生物に投与し,組織への取り込み量を測定するような実験

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何らかの薬品を生物に投与し,組織への取り込み量を測定するような実験には,ある程度決まった方法論がある。このページでは,実験の基本的な手順や,このような実験を計画するときの注意点 についてまとめる。
 
多くの論文では,単純に目的物質を定量するだけでなく
 
  • 回収率 recovery rate
  • 検出限界 quantification limit
  • 同じサンプルから定量した際の誤差 intra- and inter-day variation
 
なども算出している。
参考にした文献の内容は以下のとおり。
 
1. 漢方薬の成分である baicalin をラットに投与し(i.v. 単回投与),脳内の量を 逆相 HPLC で測定。
3. 食品中のソルビン酸,安息香酸 benzoic acid を抽出し,逆相 HPLC で定量。
投与法

経口投与および静脈注射 i.v. infusion がよく使われる。単回投与 single dosage の方が解釈が簡単になりそう。
 

静脈注射
  • 目的物質は水溶性か,脂溶性か?
  • 水溶性の場合,水に溶かすか,緩衝液に溶かすか?
  • 脂溶性の場合,溶媒は生体に影響を与えないか?
  • 目的物質の投与量はどれぐらいになるか? 達成したい血中濃度と血液量から算出することになる。
  • ラットの血液量は,約 70 mL/kg body weight である(2)。
  • Blood に血液量の一覧があります。
  • どれぐらいの液量を注入することになるか? 目的物質の溶解度と投与量から計算。
 
 
経口投与
  • 動物がその餌をコントロールの餌と同じぐらい食べるか?
  • 食べる量が変わったとしたら,それによる影響は?
  • どれぐらいが血液中に移行するか? 最大濃度,時間など。
 
 
組織からの抽出
  • どんな溶媒で抽出するか?
  • 回収率の検討

> 文献 3 では抽出に水蒸気蒸留を用いており,抽出を繰り返して回収率と留出パターンを求めている(3)。
: 安息香酸標準液(0.1 g/kg)を試料に,100 mL ごとの画分で 600 mL まで流出液を採取する。
: 画分 1, 2, 3... に含まれる安息香酸の量を測定する。
: 1 回目で 85%, 2 回目で 95% のように,回収率が上がっていき,やがて上限に達する。
検出限界 Quantification limit

> 検量用標準液の最低濃度(安息香酸 0.1 µg/ml)測定時の標準偏差の3倍を検出限界値としている(3)。
> "The quantification limit ... defined as a signal-to-noise ratio of 3:1 was 10 ng/ml (1).


  1. Zhang et al. 2005a. A chromatographic method for baicalin quantification in rat thalamus. Biomed Chromatgr 19, 494-497.
  2. Rat Biomethodology, Laboratory of Animal Resource Center, The University of Texas at San Antonio.
  3. 山上ほか 2010a. 高速液体クロマトグラフィーによる食品中のソルビン酸,安息香酸分析法について. 山梨衛環研年報, 54, 48-51.

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