Improvement and validation of ¹²⁵I-high-performance liquid chromatography method for determination of total human serum choline and ethanolamine plasmalogens.

Background: Serum plasmalogens (Pls) have gained interest in several clinical symptoms such as metabolic syndrome/atherosclerosis or Alzheimer's disease possibly because of their antioxidant properties. We have developed a highly sensitive and simple method to determine plasmenylcholine (PlsCho; choline plasmalogen) and plasmenylethanolamine (PlsEtn; ethanolamine plasmalogen) separately, using a radioactive iodine and high-performance liquid chromatography ((125)I-HPLC method). The present study reports the improvement and validation of (125)I-HPLC method by introducing a quantitative standard (QS) and online detection with a flow γ-counter.

Methods: 1-Alkenyl 2,3-cyclic glycerophosphate was prepared as QS from l-α-lyso plasmenylcholine by enzymatic treatment with phospholipase D. Online detection with a flow γ-counter was investigated to be available to quantify Pls. The method validation was carried out in terms of selectivity, sensitivity, linearity, precision, accuracy and recovery.

Results: Linearity was established over the concentration range 5-300 μmol/L for Pls and QS with regression coefficients >0.99. The accuracy and reliability were satisfactory. The method has been applied to the determination of human serum Pls from healthy subjects and the elderly with dementia or artery stenoses.

Conclusions: The improved (125)I-HPLC method is useful as an autoanalytical system for a routine diagnostic test of human serum Pls.