Exp. Eye Res. (1979) 28, 387-397
A Reinvestigation of the Fatty Acid Content of Bovine, Rat and Frog Retinal Rod Outer Segments W. L. STONE,C. C.FARNSWORTHASD
E. ~.DRATZ
Division of Natural Sciences, University of California Santa Cruz, Cd;f. 95064, U.S.A. (Received 23 May 1978, Xezo York) Continuous-sucrose gradient centrifugation of whole retina homogenates from bovine, frog and rat were found to yield two major bands of rhodopsin-containing material having distinctly different polyunsaturated fatty acid (PUFA) contents. The lowest density band has the highest purity rod outer segment (ROS) membranes and the highest PUFA content nhereas the higher density band has a lower PUPA content and contains non-ROS membrane impurities. It follows, that in ROS preparations from whole retinal homogenates in which these two bands are pooled, the measured PUFA content will be lower than that of t~he purest ROS band. We find that the highest purity ROS band from bovine, frog and rat retinal homogenates have weight-percents of docosahexaenoic acid (226~3) of 50.7&1.4, 56910.6, and 46.2*3.6, respectively, which is equivalent, to 47.1, 46.9, and 43.3 mol-percent. Two ROS bands were also isolated from homogenates of semipurified ROS removed from retinas by shaking (i.e. the shakate preparation). Both shakate bands have similar PUFA contents (with weight-percent, 22 :6~3 of ~51.5) and similar phospholipid : rhodopsin ratios. The more dense band has a significantly higher 280 nm/r1500 nm ratio which probably reflects some nonrhodopsin protein retained in the ROS by the intact plasma membrane. The lowest density band has ROS with disrupted plasma membranes whereas t,he more dense band has intact plasma membranes. Homogenization of whole retinas introduces considerable non-ROS membrane contamination into the more dense ROY band. We have developed specific procedures (i.e. use of 0.1 mar CaEDTA and an inert argon atmosphere) that inhibit ROS lipid autoxidation and loss of PUFA. The results indicate that if adequate levels of vitamin E are present the maintenance of antioxidant conditions during preparation procedure is not a highly critical factor in determining ROS PUPA content. retinal rod outer segments; lipid autoxidation; Key words: fatty acid composition; membrane antioxidants; cc-tocopherol.
1. Introduction The fatty acid composition of lipids from purified vertebrate retinal rod outer segment rnenlbranes (ROS) has been published by numerous laboratories (cf. Daemen, 1973). These data show a surprising degree of variability for ROS membranes from identical or similar species. For example, the values reported for the weight-percent of tlocosahexaenoic acid, 22 16~3, vary from 22:; (Poincelot and dbrahamson, 1970) to 4t(q{, (Hendricks, Klompmakers, Daemen and Bonting, 1976) of the fatty acids in bovine ROS. Similarly, in frog ROS the 226~3 values vary from 1576 (Ralza cntesbima; Mason, Fager and Abrahamson, 1973) to 44qd (Rnna temporwia; Kagan, Shvedova, Novikov and Kozlob, 1975). A number of different factors could contribute to the variability in ROS PIJFA content. For example, differences in preparation procedures could yield ROS membranes containing varying amounts of non-ROS membrane contaminants having a PUPA content different from that of pure ROS membranes. It is also possible that differences in precautions against lipid autoxidation could contribute to some of the Reprint
requests to : E. A. Dratz.
Q 1979 Academic Press Inc. (London) Limitetl
OOl4-4835/79/040387+11 $01.00/O 387
38s L
IT.
L.
STOKE.
c’. (1. FAI!Ns~~oJ:‘1’lI
ASD
I