Efficient recovery of Sph-1-P and LPA after the repeated phase separations under alkaline and acid conditions.

(A) After the initial phase separation of [³²P]Sph-1-P, [³H]Sph-1-P, [³H]LPA, [³H]Sph, [³H]C₆-Cer, or [¹⁴C]sphingomyelin (SM) under alkaline conditions, the resultant aqueous upper phase was phase-separated again under the acidic conditions. The radioactivities of each radiolabeled lipid into the final lower chloroform phase were counted by liquid scintillation counting. The percentage of recovery of each radiolabeled lipid into the final lower chloroform phase was calculated as 100 × the radioactivity in the final lower chloroform phase/the radioactivity originally added. (B) [³H]Sph-1-P (left lane) or [³H]LPA (right lane), recovered in the final lower chloroform phases, was analyzed by TLC autoradiography. Note that no bands other than [³H]Sph-1-P and [³H]LPA were detected.