Rabbit reticulocytes (sixth and seventh day of a bleeding anemia) were prepared and divided into three samples. One sample was hemolyzed with 2 vol of water and the membranes were recovered by centrifugation. The other two samples were incubated in PBS (containing calcium and magnesium) in the presence or in the absence of 4 μmol/L calcium ionophore A23187. After 4 hours, the cells were lysed as described above, the membranes were prepared and the lipids were extracted. Transmethylation of the ester lipids was performed by incubating the lipids with sodium methoxide for 1 hour under argon atmosphere. Aliquots of the transmethylation mixture were subjected to RP-HPLC analysis as described in Materials and Methods. The chromatograms obtained were quantified by peak areas. For calculation of the hydroxy fatty acid/polyenoic fatty acid ratio, the peaks of 13-HODE, linoleic acid, and arachidonic acid (see Fig 4) were quantified.

Comparison of sample 1 and 2.

Comparison of sample 2 and 3. When shorter incubation periods (15 minutes) were used, no significant differences were observed.