Hidalgo Francisco J, Nogales Fátima, Zamora Rosario
Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Avenida Padre García Tejero 4, 41012 Sevilla, Spain.
Anal Biochem. 2004 Nov 1;334(1):155-63. doi: 10.1016/j.ab.2004.08.001.
The Ehrlich reaction was optimized to determine pyrrolized phospholipids produced as a consequence of oxidative stress. The procedure consisted of the treatment of the modified phospholipids with p-(dimethylamino)benzaldehyde at a controlled acidity temperature and the spectrophotometric determination of adducts produced. The extinction coefficient of Ehrlich adducts was calculated by using 1-[1-(2-hydroxyethyl)-1H-pyrrol-2-yl]propan-1-ol (compound 1) as standard and was 56,500 M(-1)cm(-1). The response was linear and reproducible within the range of 0.051-7.65 microM of compound 1. When the assay was applied to determination of pyrrole content in ethanolamine incubated in the presence of 0.25-1mM of 4,5(E)-epoxy-2(E)-heptenal, the complete conversion of the aldehyde into the pyrrole ring was observed and the results obtained were similar to those found when compound 1 was determined by gas chromatography. When phosphatidylethanolamine was incubated in the presence of 0.5-40 mM of 4,5(E)-epoxy-2(E)-heptenal, the phospholipid was pyrrolized similarly to ethanolamine, although there was not a quantitative conversion and the amount of pyrroles produced depended on the pH of the media. Pyrrolized phospholipids were also produced when phosphatidylethanolamine multilamellar vesicles where oxidized in the presence of either Fe(3+)/ascorbic acid or ABAP (2,2'-azobis(2-methylpropionamide) dihydrochloride) and when high-density lipoproteins were incubated in the presence of Cu(2+), thereby confirming that phospholipid pyrrolization is a common consequence of oxidative stress and that Ehrlich adducts may be valid for determining this pyrrolization.
