Docosahexaenoate-containing molecular species of glycerophospholipids from frog retinal rod outer segments show different rates of biosynthesis and turnover

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Abstract
We have studied the de novo synthesis and subsequent turnover of major docosahexaenoate-containing molecular species in frog rod outer segment (ROS) phospholipids following intravitreal injection of [2-3H]glycerol. On selected days after injection, ROS were prepared and phospholipids extracted. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) were isolated and converted to diradylglycerols with phospholipase C. Diradylglycerols were derivatized with benzoic anhydride and resolved into diacylglycerobenzoates and ether-linked glycerobenzoates. The diacylglycerobenzoates were fractionated into molecular species by HPLC, quantitated, and counted for radioactivity. Label was incorporated into ROS phospholipids by day 1 and was followed up through the eighth day. The dipolyenoic species 22:6-22:6 from PC showed a 3-5 times higher radiospecific activity than the same species from either PE or PS. In PC, the specific activities of 16:0-22:6 and 18:0-22:6 were 3-5 times lower than the specific activity of 22:6-22:6. In contrast, for PE, the specific activities of 16:0-22:6 and 18:0-22:6 were 2-5 times higher than that of 22:6-22:6. The specific activities of 18:0-22:6 and 22:6-22:6 were similar in PS. Specific activities of the docosahexaenoate-containing species began approximating an exponential decline 6-8 days postinjection and continued through the 31st day. The rate of decline was determined by calculating the half-life of each molecular species, which was used as a measure of the turnover of the species. The species 22:6-22:6-PE and 18:0-22:6-PE showed a 2-3 times slower turnover of the species. The species 22:6-22:6-PE and 18:0-22:6-PE showed a 2-3 times slower turnover rate than the corresponding species from either PC or PS. The turnover rates for 22:6-22:6, 18:1-22:6, and 16:0-22:6 were similar in PC but were 1.5 times faster than 18:0-22:6. In PE, 18:1-22:6 and 16:0-22:6 had similar rates but were 2-3 times faster than 22:6-22:6 and 18:0-22:6. For PS, 22:6-22:6 and 18:0-22:6 turned over at the same rate. The percent distribution of radioactivity in the molecular species of PC and PE was quite different from the relative mass distribution at day 1. However, percent dpm approach the mole percent by 31 days. In PS, percent dpm and mole percent were the same at all time points. These results indicate that the molecular species composition of PC and PE in frog retinal ROS is determined by a combination of factors, which include rate of synthesis, rate of degradation, and selective interconversions. In contrast, PS composition appears to be determined at the time of synthesis.

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