Phospholipid molecular species from isolated bovine rod outer segments incorporate exogenous fatty acids at different rates.

The incorporation of radiolabeled palmitic (16:0), oleic (18:1), and docosahexaenoic (22:6) acids into different molecular species of membrane phospholipids was investigated in isolated bovine rod outer segments (ROS). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) were isolated, and their diacylglyceroacetate and diacylglycerobenzoate derivatives were prepared, separated by HPLC, quantified, and assayed for radioactivity. Maximal incorporation of fatty acids occurred within 15-30 min. The rate of incorporation of the fatty acids into PC was three to six times higher than it was into PS or PE. The rate of incorporation of 22:6 into the molecular species, 22:6-22:6, of PC was ten to 15 times higher than into that of PE or PS, and it was three to four times higher than the incorporation rates of 22:6 into the other 22:6-containing molecular species. Similarly, incorporation of 18:1 into 18:1-22:6 was ten to 30 times more rapid in PC than in PE and PS, but in both PE and PS, 18:1 was incorporated into 18:1-22:6 at a rate of 20 to 25 times higher than the incorporation into the other molecular species analysed. For PC, incorporation of 16:0 was most rapid into 16:0-16:0, but for PE and PS it was most rapid into 16:0-20:4; for all cases, incorporation of 16:0 into these molecular species was four to six times more rapid than into the other 16:0-containing molecular species. These results are further evidence for the presence within a membranous organelle, the ROS, of an active acylation-deacylation system that is selective with regard to phospholipid, molecular species of phospholipid, and fatty acid.     

Metabolism of lipid molecular species in rat rod outer segments

We have studied the metabolism of selected diacyl molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and diacylglycerol (DG) from rat rod outer segments (ROS). Rats were injected intravitreally with [2-3H]glycerol. At 1, 2, 3, 4 and 6 days post-injection, ROS phospholipids and DG were isolated by two-dimensional thin-layer chromatography (TLC), derivatized, and fractionated into molecular species by high-performance liquid chromatography (HPLC). Selected molecular species were quantitated and counted for radioactivity. We found the following. In PC and PE, the specific activities of 22:6-22:6, 18:1-22:6 and 16:0-22:6 were highest at day 1 and then decreased in a nearly exponential manner. In contrast, the specific activities of 18:0-22:6 and 18:0-20:4 were substantially lower than these three molecular species and changed little over the 6-day period. In PS, the specific activities of 22:6-22:6, 18:0-22:6 and 18:1-22:6 were similar and did not reach their maximum until the 3rd or 4th days. In PC, the specific activities of the five molecular species examined were two to three times higher at day 1 than the same species in PE and PS. In PI and DG, the major molecular species were 16:0-20:4 and 18:0-20:4. The specific activities of these two molecular species at day 1 were about ten times higher than those of 20:4-containing species in PE and PC, and showed the most rapid turnover of any of the molecular species examined in this study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unique molecular species composition of glycerolipids of frog rod outer segments

The composition and metabolism of molecular species of glycerolipids, including phosphatidic acid (PA), phosphatidylinositol (PI) and diacylglycerol (DG), were studied in four frog retinal fractions prepared by discontinuous sucrose gradient centrifugation. Six glycerolipid classes were isolated from the lipid extracts of each fraction and converted to their corresponding 1,2-diacylglycerol acetates by acetolysis for quantitation of their molecular species by HPLC. Rod outer segments (ROS) showed a distinctive molecular species composition in all glycerolipid classes except phosphatidylcholine (PC). The relative amounts of dipolyunsaturated species in ROS were higher in phosphatidylethanolamine (PE), phosphatidylserine (PS), and PA, compared to the other retinal fractions. PI and DG of ROS had a similar molecular species composition and contained only small amounts of dipolyunsaturated species. A unique feature of the molecular species of ROS PI and DG was that they had high amounts of species containing docosahexaenoic acid (22: 6 omega 3), while PI and DG from the other retinal membranes consisted mostly of species containing arachidonic acid (20: 4 omega 6). Following in vitro incubation of frog retinas with [2-3H] glycerol, the mass and radioactivity distributions among molecular species were determined following HPLC fractionation. The unique species composition of PS in ROS is determined mainly by selective translocation from the inner segments to ROS, since the dpm %, representative of newly synthesized species composition of the same glycerolipid classes in the other membrane fractions. This suggests that the distinctive species composition of PE and PA in ROS is determined not by selective translocation from the inner segments, but by remodeling processes taking place in the ROS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutral lipids of frog and rat rod outer segments

Neutral lipids of rod outer segments (ROS) from frog and rat comprise roughly 10 mol% of the total lipids and consist of free sterol, free fatty acids, and 1,2-diglycerides, but contain no detectable sterol esters or triglycerides. Expressed as nmol per mg ROS protein, the levels of both free fatty acids and free sterol are higher in the rat than in the frog, whereas the diglycerides in frogs are about four times higher than in rats. The level of 22:6 omega 3 in the free fatty acids fraction of the frog is twice that of the rat, although the level of 22:6 omega 3 in both the frog and rat free fatty acids is lower than in any of the glycerolipids. The diglyceride fraction from both animals consists almost entirely of two molecular species: in the frog, the C-38 and C-40 type predominate in a molar distribution of 52 and 42% respectively, whereas in the rat, the C-36 and C-38 types are most abundant in a molar distribution of 28 and 60%, respectively. Comparison of the diglyceride fatty acid and molecular species compositions with those of phosphatidylinositol and phosphatidic acid led to the suggestion that ROS diglycerides are derived from phosphatidylinositol.     

Lipids in human lipofuscin-enriched subcellular fractions of two age populations. Comparison with rod outer segments and neural retina

The fatty acid composition and content of total phospholipids, free fatty acids (FFA), diacylglycerols (DG), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) were studied in lipofuscin granules of human donors in two age groups, young (less than 40 yr old) and old (more than 47 yr old), and compared with lipids of the photoreceptor rod outer segments (ROS). Neural retina (NR) and retinal pigment epithelium (RPE) also were studied. In both age groups, the lipid composition of the lipofuscin granules differed from that of the ROS, with a decrease in the proportion of phospholipid and an increase in FFA, suggesting very high phospholipase activity in the lipofuscin granules. In ROS, docosahexaenoic acid (22:6) was the predominant FFA, whereas palmitic acid (16:0), arachidonic acid (20:4) and oleic acid (18:1) were the major fatty acids in the lipofuscin granules. The fatty acid compositions of PC, PE, and PS of lipofuscin granules were different from those of the retina. There was proportionally less 22:6 in lipofuscin, and the amounts of saturated and monounsaturated fatty acyl chains such as 16:0, stearate (18:0), and 18:1 were greater than in retina. Compared to ROS, the lipofuscin granules showed a significant decrease in DG containing 20:4 but not 22:6. With aging, there was a decrease in the amount of total polyunsaturated fatty acyl chains (22:6 and 20:4) in the lipofuscin granules. These results show that the lipid composition of lipofuscin is different from that of ingested ROS, probably because of increased phospholipase and peroxidative activities in lipofuscin, directed toward ingested ROS as well as toward other materials from the RPE and blood.     

Lipids of the retinal pigment epithelium in RCS dystrophic and normal rats

The lipid composition of retinal pigment epithelial cells was determined for normal cells which have full phagocytic ability and for a genetic variant with impaired phagocytic function. Retinal pigment epithelial cells from 9-14-day-old congenic strains of normal (RCS-rdy+) and dystrophic (RCS-rdy/rdy) rats were separated from intact retinas and homogenized in 0.08 M Tris base, pH 7.4. The lipids were extracted using 2:1 chloroform--methanol. Fatty-acid methyl esters identified by gas chromatography were: 16:0, 17:0, 18:0, 18:1, 18:2 omega 6, 20:0, 20:2, 22:0, 20:4 omega 6, 22:4, 22:5, 22:6 omega 3. Major fatty acids for both normal and dystrophic cells were: 16:0, 18:0, 20:4 omega 6, 22:6 omega 3. One- and two-dimensional thin-layer chromatography was used to determine phospholipid composition of pigment epithelial cells at two different age groups. The relative amount of phosphatidylethanolamine was significantly higher in dystrophic RPE cells compared with normal cells (20.7% for 9-11-day-old and 17.3% for 12-14-day-old dystrophic rats). Cells from normal animals contained a higher level of phosphatidylethanolamine in the older age group whereas RPE cells from dystrophic animals contained a lower level of phosphatidylcholine in the older group. Anomalous phospholipid composition of dystrophic pigment epithelial cells may be associated with a change in cellular membranes and a defect in the cellular processes involved in phagocytic function.     

Rabbit photoreceptor outer segments contain high levels of docosapentaenoic acid

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.     

Lipids of frog retinal pigment epithelium: comparison with rod outer segments, retina, plasma and red blood cells.

The glycerolipid and fatty acid compositions of frog retinal pigment epithelium (RPE) were determined and compared with rod outer segments (ROS), retina, plasma, and red blood cells (RBC). The glycerolipid class composition of RPE was similar to RBC and ROS or retina, with phosphatidylcholine and phosphatidylethanolamine being the major components. The fatty acid composition of RPE differed substantially from that of plasma or RBC; the former contained much higher levels of C-20 and C-22 polyunsaturated fatty acids (PUFAs), such as 20:4n-6 and 22:6n-3, but less C-18 mono-, dienoic, and trienoic acids. The difference between RPE and ROS or retina with respect to fatty acid profile was also dramatic; RPE had relatively less 22:6n-3, but more 20:4n-6 and 18:2n-6, than ROS or retina. These results suggest that frog RPE cells may selectively take up C-20 and C-22 PUFAs from the circulation, but preferentially deliver 22:6n-3 to the ROS and retina. Fatty acid analyses show that 20:4n-6 and 22:6n-3 were unevenly distributed among RPE glycerolipids; phosphatidic acid, diglyceride, triglyceride, and phosphatidylserine are relatively more enriched in 22:6n-3 compared with 20:4n-6. This information might imply that these two PUFAs are metabolized differently inside the frog RPE cells.     

Enrichment of polyunsaturated fatty acids from rat retinal pigment epithelium to rod outer segments.

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA, 22:6n-3), are enriched in phospholipids of vertebrate rod outer segments (ROS). Retinal ROS can incorporate 22 carbon (C-22) PUFA from the plasma pool where C-20 PUFA are predominant. In this study, we analyzed the fatty acid composition of retinal pigment epithelium (RPE) and ROS from rats fed different fatty acid supplements to determine whether this enrichment is at the photoreceptor-RPE boundary or the RPE-choriocapillaris boundary. Long Evans rats were raised from birth for 13-14 weeks on a diet supplemented with 10% (wt/wt) hydrogenated coconut oil (COC; 0.2% 18:2n-6, no 18:3n-3), safflower oil (SAF; 73.8% 18:2n-6, 0.1% 18:3n-3), or linseed oil (LIN; 16.4% 18:2n-6, 52.2% 18:3n-3). These diets were chosen because they increased plasma levels of 20:3n-9, 20:4n-6, and 20:5n-3, respectively. These three fatty acids served as metabolic markers. Plasma levels of 22:6n-3 were reduced by the COC and SAF diets. The RPE incorporated 20:3n-9, 20:4n-6, and 20:5n-3 from the plasma. However, the levels of 20:3n-9 and 20:5n-3 were very low in ROS and 20:4n-6 was not significantly elevated in the ROS of the SAF diet group. The relative amount of total C-20 PUFA in phospholipids in RPE was similar to that found in plasma and was about 4-16 times (depending on different lipid classes) that in the ROS. In contrast, C-22 PUFA (22:6n-3 and 22:5n-6) showed a step-wise, average 3-5 fold increase in concentration from the plasma to the RPE to the ROS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of linolenic acid and docosahexaenoic acid in rat retinas and rod outer segments.

Docosahexaenoic acid (22:6 omega 3) is uniquely enriched in photoreceptor outer segment phospholipids, comprising up to one-half of the fatty acids of phosphatidylethanolamine and phosphatidylserine. The current study was designed to investigate the incorporation of 22:6 omega 3 into outer segment phospholipids over 12 days and to determine whether the retina contained the enzymes necessary for elongation and desaturation of the major dietary precursor of 22:6 omega 3, the essential fatty acid linolenic acid (18:3 omega 3). Sprague-Dawley rats were injected intravitreally with [14C]22:6 omega 3 or [14C]18:3 omega 3 and kept in cyclic light (12 hr light/12 hr dark) for 2 hr to 12 days. Phospholipids from rod outer segments and the remaining retinal debris were separated by two-dimensional thin-layer chromatography. [14C]22:6 omega 3 radioactivity was initially highest in phosphatidylcholine and rapidly decreased from 45% of total phospholipid labeling at 2 hr to 26% by 1 and 3 days in ROS, while phosphatidylethanolamine labeling increased from 49 to 68% by 3 days and phosphatidylserine labeling increased from 3 to 14% over 12 days. Phenacyl derivatives of total fatty acids were separated by HPLC. A substantial conversion of [14C]18:3 to [14C]20:5, [14C]22:5 and [14C]22:6 was noted after 1 days, with increasing conversion to [14C]22:6 over the 12-day period. When only one eye was injected with [14C]18:3 omega 3, negligible radioactive fatty acids were detected in the contralateral eye from 1 to 12 days post-injection demonstrating that conversion of 18:3 to 22:6 occurred primarily within the injected eye. All enzymes for elongation and desaturation of 18:3 to 22:6 appear to be present in the eye. However, the conversion of 22:5 to 22:6 by delta-4 desaturase is evidently rate-limiting and may affect phospholipid replacement during photoreceptor outer segment renewal if this pathway proves to be essential for the supply of 22:6 during disk membrane formation.     

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

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 mm 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.     

Uptake of 22-carbon fatty acids into rat retina and brain.

Rat retina accumulates high levels of 22-carbon (C22) polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA, 22:6 omega 3), in rod outer segment (ROS) phospholipids (PL). However, plasma, the source of retina lipids, is enriched in 20-carbon (C20) fatty acids instead of C22 PUFA. This suggests that the retina has a mechanism(s) for selective uptake of C22 PUFA from the blood. It is not known if the selective uptake is specific for the carbon number alone, or if the number of double bonds is also important. To address this question, the following study was carried out using erucic acid (22:1 omega 9) as a metabolic marker molecule. Albino rats were raised from birth on a diet containing 10% (by weight) of either rapeseed oil (43% 22:1 omega 9) or blended canola oil (0.4% 22:1 omega 9). At 4 months of age, plasma, liver, adrenal gland, brain and retina were collected, lipids were extracted, and fatty acids were determined. In those rats fed rapeseed oil, 22:1 omega 9 was incorporated into the lipids of plasma (2.3%), liver (0.6%), and adrenal gland (17.6%), indicating that this fatty acid was absorbed, transported, and metabolized by the rats. However, 22:1 omega 9 was not incorporated into the lipids of retinal ROS or brain. Our results suggest that both the carbon number and degree of unsaturation are important determinants in the selective uptake of C22 fatty acids from plasma into both the brain and the retina.     

Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding.

The vertebrate retina conserves docosahexaenoic acid (22:6n-3) during n-3 fatty acid deficiency. The mechanism of conservation is not known, although recycling of this fatty acid between the retinal pigment epithelium (RPE) and retina is one possibility. We examined the role of the RPE in conservation of 22:6n-3 by quantitating the fatty acids and phospholipid molecular species (PLMS) in frog RPE before and after light-stimulated shedding of rod outer segments (ROS). RPE cells were dissociated with brush agitation and purified by a discontinuous ficoll density gradient. One hour after the light-induced shedding of ROS, the phagocytosed ROS tip and opsin content of RPE had increased. Simultaneously, the levels of 22:6n-3 and 22:6(n-3)-containing PLMS were increased in the RPE. Within 8 hr following the shedding event, 22:6n-3 in the RPE had returned to the dark level. These findings indicate that the phagocytosed ROS tips contain 22:6n-3 and that the RPE metabolizes these ROS tips and eliminates 22: 6n-3 from the cell. Thus, the RPE is intimately involved in the metabolism of 22: 6n-3 in the retina. The recycling of 22: 6n-3 from the RPE to the retina is a possible means of conserving this important fatty acid in the retina.     

Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes

PURPOSE: In recent years, detergent-resistant membranes (DRMs) have been isolated in in vitro models of lipid rafts, from photoreceptor outer segments (ROS), and the localization of a specific complement of photoreceptor proteins has been demonstrated. However, surprisingly little is known about the lipid composition of these important membrane domains. The present study provides the first characterization of phospholipids and fatty acids from ROS-derived DRMs. METHODS: Bovine ROS membranes were incubated with 1% Triton X-100 at 4 degrees C and subjected to density gradient centrifugation to isolate DRMs from the parent membranes. Lipids of ROS and DRMs were separated by two-dimensional, thin-layer chromatography and converted to methyl esters, and fatty acids were analyzed by gas chromatography. Proteins of ROS and DRMs were analyzed by SDS-PAGE and Western blot analysis. RESULTS: The DRMs represented 8% and 3%, respectively, of total ROS lipid and protein. In general, DRMs were enriched in saturated fatty acids when compared with ROS membranes. Relative to ROS, DRMs were enriched in free fatty acids (FFAs) and a specific phosphatidylcholine (PC) fraction that was almost devoid of polyunsaturated fatty acids (PUFAs). DRMs contained less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Ceramide (CM) from ROS contained PUFAs but no saturated fatty acids; the converse was true of CM from DRMs. Docosahexaenoic acid was diminished in DRM PS and was not detected in the FFAs, but was equally abundant in ROS and DRM PE. ROS-derived DRMs were dramatically enriched in caveolin-1, contained significant amounts of transducin-alpha and c-Src, and were relatively devoid of arrestin. CONCLUSIONS: The relatively saturated lipid environment observed in DRMs is likely to promote the localization of signaling proteins modified with saturated fatty acyl chains. Based on the lipid composition of DRMs, the authors conclude that they would not efficiently support phototransduction.     

角膜伤口修补白内障摘除联合人工晶体植入术

目的：评价角膜伤口修补、白内障摘除联合人工晶体植入术的临床疗效。方法：对36例角膜穿通伤患者施行角膜伤口修补的同时行白内障摘除联合人工晶体植入术。人工晶体前膜、后发性白内障给予YAG激光切除。角膜中央疤痕严重影响视力者行穿透性角膜移植术。对所有病例进行3－12个月的随访。结果：术后末次随访时裸眼视力0．1－0．3者8例（22．8％）,0．4－0．5者14例（40％）,0．6－0．8者13例（37．1％）。无因人工晶体植入术引起的严重并发症。结论：只要能准确地把握好手术适应症,在角膜伤口修补的同时行人工晶体植入术能取得较好的疗效。     

Rod outer segment disc membranes are capable of fusion.

The retinal rod outer segment (ROS) is maintained at a constant length through the formation of new discs and the phagocytosis of old discs by the pigment epithelium. The ROS is composed of approximately 50 mol% of phosphatidylethanolamine (PE), an unusually high PE content for biologic membranes. Because this lipid is highly fusogenic, due to its low head group hydration, the ability of ROS disc membranes to fuse with PE large unilamellar vesicles (LUV) was examined. The initial rates of fusion of discs with LUV were measured by following the relief of self-quenching of octadecylrhodamine B chloride (R18)-labeled disc membranes. Fusion was initiated by reducing the pH of the mixture to 7.2. The initial rates of fusion of disc membranes with transphosphatidylated PE (trans-PE LUV) were measured as a function of temperature. The ROS discs fused readily with these LUV. Fusion was confirmed by density-gradient centrifugation. The initial rates of fusion increased with increasing temperature. Subsequently, the initial rates of fusion between disc membranes and disc lipid vesicles were examined using the R18 mixing assay. Fusion of the two membranes was confirmed by sucrose density-gradient centrifugation. Calcium and EGTA had no significant effect on disc membrane-trans-PE LUV fusion or on disc membrane-disc lipid vesicle membrane fusion. Papain proteolysis of the disc membranes enhanced initial rates of fusion between disc membrane and PE LUV but inhibited disc membrane-disc lipid vesicle fusion.     

Light activation of phosphatidylethanolamine N-methyltransferase in rod outer segments and its modulation by association states of transducin.

Phosphatidylethanolamine N-Methyltransferase (PE N-MTase) is the enzyme responsible for the synthesis of phosphatidylcholine from phosphatidylethanolamine by successive transfer of methyl groups. This enzyme is present in bovine rod outer segments (ROS) and it is the only pathway for the synthesis of phosphatidylcholine in the outer segment of rod photoreceptor cells. In dark-adapted ROS membranes PE N-MTase activity is stimulated by 100% when ROS membranes are incubated under light condition. To determine whether the retinal G protein, transducin (Gt), intervenes in the regulation of PE N-MTase in these membranes, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and guanosine 5'-O-(2-thiodiphosphate (GDPbetaS) on the enzyme activity were examined. In dark, GTPgammaS which induces dissociation of Gt, stimulates the enzyme activity mimicking the stimulation by light. On the contrary, GDPbetaS stabilizes the inactive state of Gt, inhibiting the stimulation by light of PE N-MTase without affecting basal activities. In addition, adenosine 5'-diphosphate (ADP)-ribosylation by cholera and pertussis toxin was studied. ADP-ribosylation of ROS membrane with pertussis toxin, which stabilizes transducin in its inactive state, prevents the light-induced increase in PE N-MTase activity. On the contrary ADP-ribosylation with cholera toxin stimulates the enzyme activity. Our findings therefore suggest that light-stimulated effect of PE N-MTase activity is transducin-mediated.     

Synthesis of docosahexaenoyl-, arachidonoyl- and palmitoyl-coenzyme A in ocular tissues

The synthesis of long-chain acyl coenzyme A (CoA) was studied in the cornea, lens, vitreous, retina and pigment epithelium (PE) in the rat using [14C]-labeled palmitic, arachidonic and docosahexaenoic acids as substrates. Except for retina and PE, the ocular tissues studied showed relatively little enzyme activity with the fatty acid substrates. In addition, the enzyme activities were studied in homogenates and microsomal fractions from retina, pigment epithelial cells and choroid of frog, bovine and human eyes. Long-chain acyl CoA synthetase from the microsomal fraction exhibited three- to fivefold greater activity than homogenates in retina and PE. The enzyme activity was highest with palmitic acid, followed by arachidonic acid and docosahexaenoic acid. There were significant differences in enzyme activity between the species. The apparent Km (microM) and Vmax [nmol min-1 (mg protein)-1] values for the enzyme in bovine retinal microsomes were 7.91 +/- 0.39 (S.E.) and 21.6 +/- 1.04, respectively, for palmitic acid substrate and 5.88 +/- 0.25 and 4.58 +/- 0.21, respectively, for docosahexaenoic acid substrate. These values for bovine pigment epithelial microsomes were 13.0 +/- 0.27 and 36.9 +/- 1.18, respectively, for palmitic acid and 15.8 +/- 0.40 and 13.2 +/- 0.56, respectively, for docosahexaenoic acid. The synthesis of acyl CoA may play a central role in controlling the availability of free arachidonic acid for eicosanoid formation and in the retention of polyunsaturated fatty acid families (18:2, n-6 and 18:3, n-3) within cells of ocular tissues, particularly retina and retinal PE.     

Low docosahexaenoic acid levels in rod outer segment membranes of mice with rds/peripherin and P216L peripherin mutations.

PURPOSE. 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. METHODS. 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. RESULTS. 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. CONCLUSIONS. 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.     

Plasma lipid abnormalities in the miniature poodle with progressive rod-cone degeneration.

The miniature poodle with progressive rod-cone degeneration (prcd) is a model for human retinitis pigmentosa (RP). Since previous studies from several laboratories have shown abnormalities in plasma lipids in human RP, we examined the plasma lipids of prcd-affected animals. Fasting blood was drawn on three separate occasions from affected and control miniature poodles and on one occasion from normal Irish setters and those affected with a different inherited retinal degeneration (rod-cone dysplasia). Plasma phospholipids from prcd-affected animals had significantly lower levels of docosahexaenoic acid (22:6 omega 3) and cholesterol, compared to control miniature poodles. No differences were observed in plasma levels of phospholipids, vitamin E, or vitamin A, and no lipid differences were found between control and affected Irish setters. The ratios of 22:5 omega 3 to 22:6 omega 3 and of 22:4 omega 6 to 22:5 omega 6 were significantly elevated in prcd-affected poodles compared to controls. Since the conversion of 22:5 omega 3 to 22:6 omega 3 and of 22:4 omega 6 to 22:5 omega 6 is catalysed by a delta 4-desaturase, these results are consistent with a defect in desaturase activity in the prcd-affected poodle.