Edward A. Dratz

A reinvestigation of the fatty acid content of bovine, rat and frog retinal rod outer segments

Abstract 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 whereas the higher density band has a lower PUFA 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 the purest ROS band. We find that the highest purity ROS band from bovine, frog and rat retinal homogenates have weight-percents of docosahexaenoic acid (22:6ω3) of 50·7±1·4, 50·9±0·6, 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 22:6ω3 of ≈51·5) and similar phospholipid: rhodopsin ratios. The more dense band has a significantly higher 280 nm/Δ500 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 the more dense band has intact plasma membranes. Homogenization of whole retinas introduces considerable non-ROS membrane contamination into the more dense ROS band. We have developed specific procedures (i.e. use of 0·1 m m 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 PUFA content.

Oxidative damage of retinal rod outer segment membranes and the role of vitamin E.

Highly purified bovine rod outer segment membranes show loss of structural integrity under an air atmosphere. Obvious ultrastructural changes are preceded by increases in absorbance below 400 nm. These changes are inhibited by Ar or N2 atmospheres and appear to be due primarily to oxidative damage to the polyunsaturated fatty acids of the membrane lipids. Loss of polyunsaturated fatty acids, formation of malonaldehyde and fluorescent products characteristic of lipid oxidation accompany the spectral alterations. The elevated ultraviolet absorbance can largely be removed from the membranes by gentle extraction of the lipids using phospholipase C and hexane without changing the visible absorbance of rhodopsin. We have found a large seasonal variation in the endogenous level of alpha-tocopherol (vitamin E) in the bovine rod outer segment preparations. For much of the year we find that the rod outer segment membranes contain higher levels of alpha-tocopherol than have been previously reported in biological membranes. Rod outer segments which are low in endogenous tocopherol can be protected from oxygen damage by adding exogenous tocopherol. The rod outer segments are extremely susceptible to oxygen damage due to the unusually high content of polyunsaturated fatty acids in the membrane lipids. The presence of tocopherol inhibits oxygen damage but does not eliminate it. The tocopherol in the rod outer segments is consumed in air, thus complete protection from peroxidation in vitro requires an inert atmosphere as well as high levels of tocopherol. This work suggests that extensive precautions against oxidative degradation should also be employed in studies of other membrane systems where important deleterious effects of oxygen may be less obvious.

The structure of rhodopsin and the rod outer segment disk membrane

Abstract Rhodopsin is the photoreceptor protein of rod cells in the vertebrate retina. Physical studies of the photoreceptor membrane provide a low-resolution structure and show that rhodopsin has a large fraction of its mass embedded in the lipid bilayer. The complete amino acid sequence of bovine rhodopsin has recently been obtained and this, combined with other information, leads to testable predictions of the basic structural features of vertebrate rhodopsin.

The role of antioxidants in the retina and retinal pigment epithelium and the nature of prooxidant-induced damage

Abstract The vertebrate retina has several features that make it vulnerable to damage from autoxidation. The photoreceptor membranes contain high levels of polyunsaturated fatty acids; abundant mitochondria are present which may leak activated oxygen species; and light exposure of the retina may cause photoxidation. These features are analyzed in detail, and the various antioxidant mechanisms of the vertebrate retina are surveyed. The interplay among oxidant stress and antioxidant defenses is illustrated by review of situations where these variables are either artificially manipulated or changed naturally. Vitamin E deficiency damages the retina in a number of well-defined vertebrate model systems, and a lipid autoxidation mechanism for this damage is widely assumed. The retina is quite sensitive to damage by elevated or prolonged light exposure; however, a free-radical role in light damage to the retina has not been established. An alternative mechanism for damage due to vitamin E deficiency and light is considered, which involves elevated vitamin A levels and vitamin A toxicity. Evidence is reviewed that the primate retina requires both vitamin E and selenium. The puzzling role of ocular melanin in light damage and protection is reviewed. Possible contributions of autoxidative damage to aging of the human retina are discussed.

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane.

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography. The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.

Effects of antioxidant nutrient deficiency on the retina and retinal pigment epithelium of albino rats: a light and electron microscopic study

Abstract Male Sprague-Dawley rats were maintained, from weaning, on diets deficient either in vitamin E, selenium, chromium and sulfur amino acids (−E−Se−S−Cr), or only in vitamin E and selenium (−E−Se+S+Cr). Control animals (+E+Se+S+Cr) received all four nutrients. After 24–26 weeks on their respective diets, both deficient groups showed a dramatic accumulation of an autofluorescent pigment, similar to lipofuscin, in the retinal pigment epithelium. This increased autofluorescence was correlated with a large increase in the number of electron-dense inclusion bodies observed in the pigment epithelium by transmission electron microscopy. Accompanying the build-up of autofluorescent pigment was the development of an irregularity and an overall increase in retinal pigment epithelium cell height. There was also an increase in the number of lipid droplets in the retinal pigment epithelium, particularly in the periphery of the eye. In the eyes of deficient animals, cells were occasionally seen which appeared to have detached from Bruchs membrane and migrated into the region of the photoreceptor outer segments. Photoreceptor outer segment phagocytosis by the retinal pigment epithelium appeared to be decreased as a consequence of dietary antioxidant deficiency, since a reduction of over 75% in the number of phagosomes per unit retinal pigment epithelium cell length was seen in some areas of the eye in both deficient groups. These changes in the retinal pigment epithelium were accompanied by a pronounced loss of photoreceptor cells, particularly from the central retina (20–34% fewer cells than supplemented controls). The disk membranes of the photoreceptor outer segments of deficient animals were often swollen, disoriented and vesiculated, and areas were frequently seen where outer segment debris had accumulated at the interface between the photoreceptors and retinal pigment epithelium. Many of the changes in the retina and retinal pigment epithelium were more severe in the fully deficient (−E−Se−S−Cr) group, than in the group deficient only in vitamin E and selenium.

Occurrence of 4-hydroxyalkenals in rat tissues determined as pentafluorobenzyl oxime derivatives by gas chromatography-mass spectrometry

Malondialdehyde measurements have been the major tool for studying relationships between lipid peroxidation and tissue pathology. Recently, we presented a novel gas chromatography-mass spectrometry method for direct detection of phospholipid peroxides with picogram sensitivity based on transesterification of phospholipids or triglycerides to form pentafluorobenzyl esters. Under some circumstances the reactive primary oxidation products break down. Therefore, we developed a convenient, high sensitivity method to detect more stable secondary lipid oxidation products, the 4-hydroxyalkenals. The method accomplishes a facile extraction of 4-hydroxynonenal from tissues by forming pentafluorobenzyl oxime derivatives to displace aldehydes from Schiff base linkages. 4-hydroxynonenal was found in heart, liver, adrenal, and testis from rats and was detected to the 10-100 pg level by the current method.

Thermotropic behavior of retinal rod membranes and dispersions of extracted phospholipids

SummaryHigh sensitivity, differential scanning calorimetry studies of vovine retinal rod outer segment (ROS) disk membranes and aqueous dispersions of the extracted ROS phospholipids have been performed. ROS disk membranes were found to exhibit a broad peak of excess heat capacity with a maximum at less than about 3°C, ascribable to a gel-to-liquid crystalline phase transition of traction of the phospholipids. A similar thermotropic transition was observed for aqueous dispersions of the total extracted and purified ROS phospholipids. Comparison of the results obtained for the dispersion of total ROS phospholipids to those of the purified head group fractions. suggests that the thermotropic behavior reffects a gel-to-liquid crystalline transition, leading to lateral phase separation, involving those phosphatidylcholine (PC) molecules containing saturated fatty acylchains, possibley together with the highest melting ROS phosphatidylethanolamine (PE) and phosphatidylserine (PS) components. The interpretation of the thermal behavior of the ROS disk membranes depends on whether the transition is assumed to derive from the ROS PC and/or PE/PS fractions, and whether the transbilayer arrangement of the ROS phospholipids is assumed to be symmetric or asymmetric. The calorimetric data can be simply explained in terms of an asymmetric distribution of the major ROS disk membrane phospholipids (G.P. Miljanich et al.,J. Membrane Biol.60:249–255, 1981). In this case, the transition would arise from the PE/PS fractions in the outer ROS disk membrane monolyer, and the anticipated transition from the PC in the inner monolayer would be broadened due to interaction with cholesterol. For the ROS membranes at higher temperatures, two additional, irreversible transitions are observed at 57 and 72°C, corresponding to the thermal denauturation of opsin and rhodopsin, respectively.

The asymmetric transmembrane distribution of phosphatidylethanolamine, phosphatidylserine, and fatty acids of the bovine retinal rod outer segment disk membrane

SummaryThe transmembrane distribution of the major aminophospholipids in the bovine retinal rod outer segment disk membrane, phosphatidylethanolamine and phosphatidylserine, was determined using a novel pair of permeable and impermeable covalent modification reagents. The values for the percentages of phosphatidylethanolamine and phosphatidylserine in the outer monolayer were calculated from a simple expression which takes into account the leakage of impermeable reagent into the disk lumen as monitored by the extent of labeling of lysine entrapped in the lumen. We infer from our results that at least 73 to 87% of the disk phosphatidylethanolamine and 77 to 88% of the disk phosphatidylserine are in the outer disk membrane monolayer. The fatty acid composition of the inner aminophospholipids is slightly more saturated than the outer aminophospholipids. Calculations using the lateral surface areas occupied by the disk membrane lipids suggest that 65 to 100% of the disk phosphatidylcholine is on the inner membrane surface. Since the disk phosphatidylcholine is also somewhat more saturated than the phosphatidylethanolamine and phosphatidylserine of the outer monolayer, the total inner membrane monolayer fatty acid composition is more saturated than that of the outer monolayer fatty acid composition.

Consecutive action of phospholipase A2 and glutathione peroxidase is required for reduction of phospholipid hydroperoxides and provides a convenient method to determine peroxide values in membranes

The purpose of this study was to investigate the ability of selenium-dependent glutathione peroxidase to reduce phospholipid hydroperoxides in membrane bilayers and to develop a method to measure the peroxide content of phospholipids. Phospholipid hydroperoxides were synthesized by photooxidation of 1-palmitoyl 2-linoleoyl phosphatidylcholine and characterized by gas chromatography-mass spectrometry. Phospholipid hydroperoxides in phosphatidylcholine bilayers showed no detectable reactivity with Se-dependent glutathione peroxidase (the reaction is at least 65,000 times slower than with an available hydroperoxide). However, after the phospholipid hydroperoxides were preincubated with phospholipase A2, the free fatty acid hydroperoxides became available as a substrate for Se-dependent glutathione peroxidase. The enzyme assay can be used for convenient determination of peroxide values in phospholipids at the 1 nmole level and free fatty acid hydroperoxides can be distinguished from phospholipid hydroperoxides by omitting phospholipase A2. The accuracy of the enzymatic method was confirmed using an improved colorimetric chemical assay to measure peroxide values of phospholipid hydroperoxides to the same sensitivity. The chemical assay was not linear in the presence of high levels of lipid, but at low levels of lipid the peroxide values of phospholipid hydroperoxides measured by both methods agreed to within 1%. Since high levels of lipid inhibited the chemical assay, the enzyme assay is more accurate for determination of peroxides in membranes and tissues. The possible role of phospholipase deficiencies as a causal factor in degenerative diseases thought to be due to lipid peroxidation, such as Neuronal Ceroid Lipofuscinosis (Battens disease), is discussed.