Estimation of plasmalogens

DETERMINATION OF THE AMOUNT

OF PLASMALOGENS

Three approaches are classically used to quantify plasmalogens (aldehydogenic lipids), namely the estimation of long-chains aldehydes released through an acidic treatment, the determination of the vinyl ether content, and the HPLC separation of intact plasmalogens.

1. Long-chain aldehydes in free or bound forms may be estimated colorimetrically by conversion to nitrophenylhydrazones or by GLC of the dimethyl acetal derivatives.

2. The vinyl ether content of a phospholipid sample can be determined by the iodine uptake method as it was described in its last modification by Gottfried et al (J Biol Chem 1962, 237, 329). The specificity of the determination is based on the more rapid interaction of iodine with vinyl ethers than with olefinic double bonds.

Reagents:

Solution of potassium iodide (3% in water, w/v)
Solution of iodine (7 mg iodine in 10 ml KI solution)
Working solution: mix 1 ml of iodine solution with 9 ml of KI solution

Procedure:

Evaporate an aliquot of lipid solution (50-100 nmol) in a glass tube and dissolve in 0.5 ml methanol. Add 0.5 ml of working solution and mix for 30 min at room temperature.
Add 4 ml of 95% ethanol and read the absorbance at 355 nm against a blank in which 0.5 ml of 3% KI is added to the sample instead of the iodine solution. Run also a control tube in which the lipid sample is omitted. The molar extinction coefficient is about 27 500.

Calculation: µmoles vinyl groups = [(absorbance of iodine control – absorbance of sample) / 27 500] x 5000

Other procedure :

A highly sensitive determination of plasmalogens based on HPLC separation of molecules labeled with radioactive iodine (¹²⁵I) was described (Maeba R et al., Anal Biochem 2004, 331, 169). The method sensitivity is more than 1000-fold higher than the classical determination with nonradioactive iodine.