Determination of choline and ethanolamine plasmalogens in human plasma by HPLC using radioactive triiodide (1-) ion ( 125I 3−)

Abstract

For the purpose of developing highly sensitive and convenient determination of plasmalogens, the high-performance liquid chromatography (HPLC) method using radioactive iodine ( 125I) was investigated. Radioactive triiodide (1-) ion ( 125I 3 −), which is an actual iodine form capable of reacting with vinyl ether bond (–CH 2–O–CH=CH–) of plasmalogens, could be safely and efficiently produced by oxidizing a commercial radioactive sodium iodine (Na 125I) with hydrogen peroxide (H 2O 2) under acid condition (pH 5.5–6.0), which is called iodine-125 reagent. I 3 − specifically reacted with plasmalogens at the molar ratio of 1:1 in methanol, and 1 or 2 mol of plasmalogens was involved in the binding with iodine per iodine atom, resulting in the formation of stable iodine-binding phospholipids. The HPLC system with Diol column and acetonitrile/water as a mobile phase was available for separating iodine-binding phospholipids from nonbinding free iodine and for separately eluting iodine-binding phospholipids derived from choline and ethanolamine plasmalogens. Using iodine-125 reagent (1.85 MBq/ml), plasmalogens were detectable at high sensitivity of 10,000–15,000 cpm/nmol, which is more than 1000-fold higher sensitivity than the classical determination with nonradioactive iodine. Plasmalogen concentrations in human plasma were measured with the HPLC system and determined as, on average, 129.1 ± 31.3 μM ( n=8) in a 1.2 content ratio of choline to ethanolamine plasmalogens, a concentration that nearly agrees with the value reported previously.