HPLC Determination of Medium-Chain Fatty Acids in Human Plasma and Their Relationship with Homocysteine Concentration

セミミクロカラム高速液体クロマトグラフ（HPLC）法による簡便で汎用性の高い中鎖脂肪酸（medium-chain fatty acids，MCFAs）類の分析法を確立した．MCFAsは2-(4-hydrazinocarbonylphenyl)-4,5- diphenylimidazole（HCPI）を用いて蛍光ラベル化後，HPLC分析を行った．血漿試料の前処理として，CH3CN及びジメチルホルムアミド混液による簡便な除タンパク操作を行うのみで血漿中のMCFAsを定量可能であった．C6，C8，C10，C12，C14及びC16の6種類のラベル化したMCFAsはCH3CN と水混液によるグラジエント溶出により35分以内に分離・定量することができた．シグナル/ノイズ比が3の場合の検出下限は7.3～13.0 fmol/5 μL injectionと高感度であった．更に本法をヒト血漿に適用し，MCFAsと冠血管疾患の独立危険因子であるホモシステインとの関連性について，血漿濃度レベルに基づいて評価を行った．A simple and rapid semi-micro column HPLC-fluorescence method for the determination of medium-chain fatty acids (MCFAs) is described. The MCFAs involving C6, C8, C10, C12, C 14 and C16 were labeled with 2-(4-hydrazinocarbonylphenyl) -4,5-diphenylimidazole (HCPI). A simple deproteinization with a mixture of CH3CN and dimethylformamide was employed for the extraction of MCFAs in plasma. The separation of HCPI-MCFAs was achieved within 35 min on a semi-micro ODS column (Daisopak-SP-120-5-ODS-BP, 250 x 2.0 mm, i.d.) by gradient elution with a mixture of H2O and CH3CN. The detection limits of MCFAs at a signal-to-noise ratio of 3 were in the range of 0.15-0.26 pmol/5 μL injection. Furthermore, the proposed method was applied to determine MCFAs in human plasma. The relation between the levels of MCFAs and homocysteine, which is considered to be an independent risk factor for atherosclerosis, was estimated