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Humans with retinitis pigmentosa and dogs with progressive rod-cone degeneration (prcd) have lower than normal blood levels of long-chain polyunsaturated fatty acids, including docosahexaenoic acid (DHA), the major fatty acid found in retinal rod outer segments (ROS). In addition, prcd-affected dogs have lower levels of DHA in their ROS than control animals. The present study was designed to determine whether mice that are heterozygous for the rds mutation and transgenic mice heterozygous for a specific rds/peripherin mutation (P216L) have lower DHA levels in their ROS and other tissues than do control mice.Wild-type (rds(+/+)) mice, mice with the rds(-/-) (null) and rds(+/-) mutations, and mice with the P216L rds/peripherin mutation on the rds(+/-) background were maintained in the vivarium under identical husbandry conditions, and tissues were removed from each group for analysis at approximately 2 months of age. Fatty acid compositions of total lipids from plasma, red blood cells, liver, and ROS were determined by gas-liquid chromatography. ROS purity from each group was determined by SDS-PAGE with silver staining. The morphologic status of retinas representing each genotype was analyzed by light and electron microscopy.There was no difference between rds(+/-), P216L on rds(+/-), and rds(+/+) (control) animals in the fatty acid composition of plasma, expressed as relative mole percent or as nanomoles fatty acid per milliliter of plasma. Small but statistically significant differences were found in 18:0 and C-22 polyunsaturated fatty acids of red blood cells. In the liver, the control animals had higher levels of 20:4n-6. In contrast, the ROS of control animals had levels of DHA that were 1.4 times that of ROS from either rds(+/-) or P216L on rds(+/-) mice of the same age. The reduction in DHA was not accompanied by an increase in 22:5n-6, which always occurs in neural tissues of animals deprived of n-3 fatty acids. SDS-PAGE of the three ROS membrane preparations showed that they were of identical purity.Mice heterozygous for the spontaneous rds/peripherin mutation or mice carrying the P216L mutation on this heterozygous background have a statistically significant reduction of DHA in their ROS membranes. The authors propose that reduction in DHA is an adaptive response to metabolic stress caused by the mutation.

Previous studies have shown that persons with retinitis pigmentosa and Usher's syndrome have lower blood levels of long-chain polyunsaturated fatty acids (PUFAs). In this study, the fatty acid composition of phospholipids from plasma and red blood cells (RBCs) was compared in persons with Usher's syndrome type I; Usher's syndrome type II; or no retinal disease (control subjects).Blood was drawn from fasting volunteers and separated into plasma and RBCs by centrifugation. Lipids were extracted and phospholipids were obtained by thin-layer chromatography. Fatty acid methyl esters were prepared and analyzed by gas-liquid chromatography.There were no differences in plasma or RBC phospholipid fatty acid composition between control subjects (n = 54) and persons with Usher's syndrome type II (n = 20). However, all 20- and 22-carbon PUFA levels from RBCs of persons with Usher's syndrome type I were lower than those from control subjects and persons with Usher's Syndrome type II. Likewise, plasma levels of 20:3n-6, 20: 5n-3, and 22:6n-3 were lower in Usher's syndrome type I compared with the control group. In contrast, plasma levels of 18:1n-9 and RBC levels of 16:0 and 18:1n-9 were higher in the group with Usher's syndrome type I.Plasma and RBCs from Usher's syndrome type I, but not type II, have lower levels of long-chain PUFAs than plasma and RBCs from control subjects.

Results of a previous study show abnormal plasma lipids in progressive rod-cone degeneration (prcd)-affected dogs, with lower docosahexaenoic acid (DHA; 22:6n-3) and cholesterol levels but no differences in other plasma fatty acids, lipids, triglycerides, and fat-soluble vitamins. There is also an increase of the DHA precursor 22:5n-3, so that the ratio of 22:5n-3 to 22:6n-3 is higher in affected than in normal dogs. Because DHA is the predominant esterified fatty acid in rod outer segment (ROS) phospholipids, these findings suggest a possible causal association between abnormal plasma lipid levels and retinal degeneration. In the current study, dietary supplements rich in 22:6n-3 were used to determine whether plasma, liver, and rod outer segment phospholipid composition can be altered to modify the prcd disease phenotype.prcd-affected and normal control dogs were given DHA-enriched supplements for short (7- and 25-day) and long (21-week) periods, and the fatty acid composition of plasma, liver, and rod outer segment phospholipids were examined. In the long-term study, electroretinography and morphology were used to assess modification of the retinal degeneration phenotype.Administration of DHA-enriched supplements resulted in increases in plasma DHA and n-3 polyunsaturated fatty acids and in decreases in some n-6 fatty acids in normal and prcd-affected dogs. Similar increases in DHA and n-3 fatty acids were observed in the liver, but affected dogs had significantly higher levels at all supplementation time points examined. In contrast, the ROS of affected dogs had statistically lower (approximately 20%) DHA levels, and these levels could not be increased with dietary supplementation. The disease phenotype could not be modified by DHA-enriched supplements.Regardless of the sustained three- to fourfold elevation in plasma and liver DHA that occurs as the result of supplementation, the ROS DHA levels remain unchanged, and the prcd disease phenotype is not modified by the dietary manipulation. These findings could be the result of a reduction in the synthesis of DHA-containing phospholipids in the retinas of affected dogs; or, alternatively, there could be a reduction in DHA uptake, transport, or storage within the retinal pigment epithelium-photoreceptor complex.

To understand the difference between macular and peripheral regions, tissue samples of human retina, retinal pigment epithelium, and Bruch's membrane/choroid were dissected and analyzed for lipid composition.To facilitate dissections and enhance the recovery of tissues, eyecups were prefixed for 1 hour in 10% formalin (pH 7). Lipids were extracted from 4-mm trephined punches of tissues and analyzed by high-performance liquid chromatography. After separation of neutral lipids and phospholipids, total fatty acids in both lipid classes were quantitated.The major phospholipid classes in retina, retinal pigment epithelium, and Bruch's membrane/choroid were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl serine; the major fatty acids were palmitic (16:0), stearic (18:0), and oleic (18:1). Although the three tissues had similar total fatty acid and phospholipid components, their relative compositions were different. Neutral lipid/phospholipid ratios in retinal pigment epithelium and Bruch's membrane/choroid were almost three times higher than in the retina.This study provides information about the lipid composition of macular and peripheral regions of the human retina, retinal pigment epithelium, and Bruch's membrane/choroid. The methodology employed enabled study of lipids in small amounts of tissue, which will be of value in investigating the biochemical aspects of age-related macular degeneration.

The nucleic acid and protein contents of the various subcellular fractions of rat spleen homogenates were determined at varying intervals of time following a single intravenous injection of sheep red blood cell membranes. Small differences were recorded in the NA content of the different fractions, as compared with controls. The microsomal fraction was the only one to exhibit significant increases in NA content. The protein content of the various subcellular fractions did not show significant deviations from control values. Antibodies could be eluted from microsomes of spleens of immunized rats by sodium deoxycholate and ribonuclease treatment. A quantitative difference was found in the immune response of the two strains of rats used (R.V.H. and Wistar rats).

It is possible that a genetic defect observed in the prcd poodle involves the abnormality of an enzyme which functions in phospholipid or lipoprotein metabolism. In our studies thus far, we have been unable to detect any defect in retinal phospholipid biosynthesis, but we have noted a decrease in plasma levels of 22:6w3 which may be a result of an enzyme defect in liver biosynthesis of 22:6w3 from its dietary precursor, linolenic acid, or some defect in the blood lipoprotein transport of this essential fatty acid. If 22:6w3 is essential to the normal elaboration and functioning of the photoreceptor outer segment, it is possible that decreased access to this fatty acid due to lower blood levels of 22:6w3 could cause photoreceptor abnormalities. Further studies are needed to confirm the possible defect in delta-4 desaturase activity and possible dietary modification of the course of this prcd retinal degeneration.

The effect of light on the biosynthesis and the assembly of rod photoreceptor outer segment membranes was analyzed in vitro using retinas from Xenopus laevis. The number of open discs at the base of the outer segment was used as a morphologic index to evaluate relative differences in rates of membrane assembly. Assembly was stimulated in vitro by light, as evidenced by a greater number of open discs that accumulated in light-exposed retinas than that in retinas maintained under the same conditions but in darkness. Quantitative autoradiography indicated, however, that unlike membrane assembly, the incorporation of 3H-leucine or 3H-mannose into proteins by rods was nearly identical in light-stimulated and dark-maintained retinas. Likewise, the synthesis of opsin, a protein destined for the rod outer segment, was not affected by light. We conclude from these studies that the enhanced rate of outer segment membrane assembly that occurs during the early light phase of the diurnal cycle takes place in the absence of an increase in the rate of biosynthesis of opsin, the major outer segment membrane protein.

Docosapentaenoic acid (22:5 omega 6), a minor constituent (less than 4%) of photoreceptor outer segment membranes in all vertebrate species examined to date, comprises 23% of the fatty acids in total lipids from rabbit outer segment membranes. This fatty acid is a significant constituent of each of the three major phospholipid classes in these membranes. The levels of docosahexaenoic acid (22:6 omega 3), the major polyunsaturated fatty acid of most other vertebrate outer segments, was 20%. The sum of 22:5 omega 6 and 22:6 omega 3 in rabbit outer segment membrane lipids is similar to the amount of 22:6 omega 3 usually found in membranes from other vertebrate species.

Previous evidence suggests that lipid peroxidation may initiate photoreceptor damage induced by constant light exposure. In order to investigate the role of the antioxidant vitamin E in light damage, Long-Evans (pigmented) rats were atropinized and exposed to constant fluorescent light (Vita-Lite) of 10-20 foot candles for intervals up to 5 days. Following light exposure, retinal rod outer segments (ROS) were prepared and their lipids extracted. Retinas processed in parallel for morphological examination showed progressive ROS deterioration and selective loss of photoreceptor cells at 3 and 5 days of constant light. Similar to previous observations in undilated albino rats, constant illumination resulted in the specific loss of docosahexaenoic acid (22:6 omega 3) in the ROS. A novel finding in this study was an increase in the content of vitamin E relative to lipid phosphorus, stearic acid, and docosahexaenoic acid in the ROS of constant light-exposed animals.