Effect of Dietary N-3 Fatty Acids Upon the Phospholipid
`Molecular Species of the Monkey Retina
`
`Don S. Lin, Gregory J. Anderson, William E. Connor, and Martha Neuringer
`
`Purpose. To characterize the molecular species composition of ethanolamine glycerophospho-
`lipids (EGP) in the primate retina and to examine the effects of different dietary fats, the
`authors fed rhesus monkeys diets containing widely ranging amounts of n-3 fatty acids.
`Methods. From birth, infant monkeys were fed either a control soybean oil diet, containing 8%
`of total fatty acids as 18:3(n-3), or a safflower oil-based n-3 fatty acid deficient diet containing
`<0.4% 18:3(n-3). A subset of the n-3 deficient group was later repleted with 1.6% ethyl docosa-
`hexaenoate, 22:6(n-3), starting at 10 months of age. Tissues were taken from all monkeys upon
`termination at 21 to 51 months of age. The diacyl, alkenylacyl, and alkylacyl EGPs were quanti-
`tated by high-pressure liquid chromatography (HPLC).
`Results. Twenty-eight molecular species were identified in the retina of control monkeys. Ether
`phospholipids comprised 36% of the retinal ethanolamine glycerophospholipids. Species con-
`taining polyunsaturated fatty acids in both the sn-1 and sn-2 positions (dipolyenes) were pres-
`ent only in the diacyl subclass and comprised 16% of the total species. Species having n-3 fatty
`acids in the sn-2 position contributed 59%, 36%, and 70% of total species in the diacyl, alkeny-
`lacyl, and alkylacyl subclasses, respectively. In the molecular species of the n-3 fatty acid defi-
`cient monkeys, the major change was the loss of most of the 18:0-22:6(n-3) species and its
`partial replacement with 18:0-22:5(n-6). In contrast, the species 18:l-22:6(n-3) decreased only
`slightly, from 6.2% to 4.8% of total diacyl species. Although the total concentration of dipo-
`lyenes (15% to 20% of the total species) was not affected by diet, their fatty acid compositions
`were changed drastically. The dipolyene species 22:6(n-3)-22:6(n-3) nearly disappeared in the
`n-3 deficient monkeys. Concomitantly, two new species, 22:5(n-6)-22:6(n-3) and 22:5(n-6)-
`22:5(n-6), appeared at 2.6% and 2.0%, respectively. Deficient monkeys given the ethyl ester of
`22:6(n-3) in the diet recovered to a near-normal molecular species composition, except in the
`ether lipids, in which 16:0-20:4 remained low.
`Conclusion. Diets of differing n-3 fatty acid content had profound qualitative and quantitative
`effects on the molecular species of retinal phospholipids, and the replacement of 22:6(n-3) by
`22:5(n-6) in the retinas of n-3 deficient monkeys was asymmetric and functionally incomplete.
`Invest Ophthalmol Vis Sci. 1994;35:794-803.
`
`X he phospholipids of cell membranes represent a het-
`erogeneous population of molecular species that oc-
`cur in characteristic proportions. Different molecular
`species have different metabolic and physical proper-
`
`From the Section of Clinical Nutrition and Lipid Metabolism, Oregon Health
`Sciences University, Portland, and the Division of Neuroscience, Oregon Regional
`Primate Research Center, Beaverton, Oregon.
`Supported by NIH grants DK29930, RR00334 (General Clinical Research
`Centers), RR00163, DK40566 (Clinical Nutrition Research Units), and
`DK40935, arid by a grant from the National Retinitis Pigmentosa Foundation.
`This is publication No. I860 of the Oregon Regional Primate Research Center.
`Submitted for publication July 21, 1993; revised September 27, 1993; accepted
`October 1, 1993.
`Proprietary interest category: N.
`Reprint requests: William E. Connor, M.D., Department of Medicine L465,
`Oregon Health Sciences University, Portland, OR 97201.
`
`ties and thereby influence membrane fluidity and the
`function and activity of membrane-bound proteins.1'2
`Analysis of the molecular species of retinal phospho-
`lipids, which gives information about the pairing of
`fatty acids in membrane lipids, can provide the foun-
`dation for studies on the biosynthesis of retinal mem-
`brane lipids and their relationship to membrane func-
`tion.
`The phospholipid molecular species of the retinas
`of rainbow trout,3 frog,4'5 cow,6"8 and rat9 have been
`studied by several investigators. Retinal membranes,
`particularly those of photoreceptor outer segments,
`were found to contain the most highly unsaturated
`phospholipids of all vertebrate tissues and to be
`
`794
`
`Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3
`Copyright © Association for Research in Vision and Ophthalmology
`
`000001
`
`

`
`Diet and Retinal Phospholipid Molecular Species
`
`795
`
`unique in containing molecular species with polyunsat-
`urated fatty acids in both the sn-1 and sn-2 positions.
`Docosahexaenoic acid [DHA, 22:6(n-3)] is the major
`polyunsaturated fatty acid of retinal lipids and is pref-
`erentially taken up by photoreceptor cells.10 This fatty
`acid is most concentrated in the ethanolamine and ser-
`ine glycerophospholipids but is also present in the
`choline and inositol glycerophospholipids. No pre-
`vious data are available on the phospholipid molecular
`species composition of the primate retina.
`In our previous studies, we found that rhesus
`monkeys deficient in n-3 fatty acids before and after
`birth had an 80% to 90% decrease in n-3 fatty acids in
`the phospholipids of the brain and retina, combined
`with a compensatory increase in n-6 fatty acids. These
`reciprocal changes involved primarily the interchange
`of 22:5(n-6) for 22:6(n-3). Deficient monkeys had im-
`paired visual acuity by 4 weeks of age1112 and abnor-
`mal electroretinograms by 3 months of age.1314 At
`later ages, they showed changes in behavior, including
`polydipsia.15
`We reported recently that dietary fats with differ-
`ent fatty acid compositions had profound effects on
`the molecular species of brain phospholipids of rhesus
`monkeys.16 In the present study, we characterized the
`phospholipid molecular species composition in the ret-
`ina of nonhuman primates. To assess the effects of
`dietary fats with different amounts and types of n-3
`fatty acids, we analyzed the molecular species of reti-
`nal ethanolamine glycerophospholipids of monkeys
`fed four different diets: control (soybean oil-based),
`n-3 fatty acid deficient (safflower oil-based), 22:6(n-3)-
`enriched safflower oil, or commercial monkey chow.
`
`MATERIALS AND METHODS
`Diets and Animals
`Four groups of rhesus monkeys were studied. The first
`(deficient group, n = 4) was made deficient in n-3 fatty
`acids after birth by feeding a semipurified liquid diet
`with safflower oil as the only dietary fat source from
`the day of birth. Their mothers had been fed a stan-
`dard stock diet (Purina Monkey Chow, Animal Special-
`ties, Hubbard, OR). The second group (control group,
`n = 3) was fed a similar liquid diet with soybean oil as
`the only fat source. These animals were part of a study
`of prenatal n-3 fatty acid deficiency in which their
`mothers had been fed a semipurified safflower oil diet
`throughout pregnancy. However, plasma and red
`blood cell levels of n-3 fatty acids rose to control levels
`by 4 to 8 weeks after birth, and all other biochemical
`and functional parameters, including the overall fatty
`acid composition of the retina, were similar to control
`groups in previous studies in which both mothers and
`offspring received soybean oil diets. Both the deficient
`and control groups were killed at 3 to 4 years of age for
`
`detailed biochemical analyses of tissues. A third group,
`the 22:6(n-3) repletion group (n = 2), was made defi-
`cient in n-3 fatty acids both before and after birth.
`Their mothers were fed a safflower diet throughout
`pregnancy, and the infants received the safflower oil
`liquid diet from birth until 10 months of age, after
`which time their diet was supplemented with 22:6(n-3)
`ethyl ester at 0.25% by weight. These animals were
`killed at 21 months of age. Our previous measure-
`ments of monkey retina and cerebral cortex have
`shown no difference in overall fatty acid composition
`or in the degree of 22:6(n-3) depletion in deficient
`animals in the age range between 10 months and 4
`years. A fourth group of monkeys (chow group, n = 3),
`consuming a standard stock diet (Purina Monkey
`Chow), was included for comparison; they were 2 to 14
`years old at termination. All monkeys were cared for
`according to the National Institutes of Health Guide
`for the Care and Use of Laboratory Animals and the
`ARVO Statement for the Use of Animals in Ophthal-
`mic and Vision Research, as approved by the Animal
`Care Committee of the Oregon Regional Primate Re-
`search Center.
`The composition of the experimental diets has
`been described in detail previously.1213 The fat con-
`tent of the diets was 5% by weight (13.4% of calories)
`for pregnant females and 15% by weight (30% of calo-
`ries) for the offsprings' liquid diets. The fatty acid
`composition of the experimental diets is presented in
`Table 1. Safflower oil contains high levels (76%) of
`linoleic acid [18:2(n-6)] and very low levels (0.3%) of
`linolenic acid [18:3(n-3)], and the resulting high ratio
`of these two fatty acids (255:1) exacerbates the effect
`of the deficient diet by suppressing conversion of lino-
`lenic acid to its longer-chain products. The soybean oil
`control diet provided 53.1% of total fatty acids as lin-
`oleic acid and 7.7% as linolenic acid, for a ratio of
`6.9:1. The 22:6(n-3) repletion diet contained 1.6%
`22:6(n-3) in addition to the low level of linolenic acid.
`The stock diet supplied 2.3% linolenic acid plus 0.7%
`longer-chain n-3 fatty acids, including 0.3% 22:6(n-3)
`derived from fish meal.
`
`Biochemical Analysis
`from bovine
`Ethanolamine glycerophospholipid
`brain, phospholipase C from Bacillus cereus, benzoic
`anhydride, and 4-dimethylaminopyridine were pur-
`chased from Sigma (St. Louis, MO). Chloroform, ace-
`tonitrile, 2-propanol, methanol, hexane, and benzene
`were HPLC grade from Burdick & Jackson (Muske-
`gon, MI), and anhydrous ethyl ether was from Mal-
`linckrodt (Paris, KY).
`At the time of autopsy, the neural retinas were
`rapidly dissected, separated from the retinal pigment
`epithelium, and stored at -70°C until analyzed. Reti-
`nal lipids were extracted by the method of Folch et
`
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`
`

`
`796
`
`Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3
`
`TABLE l. Fatty Acid Composition of Experimental Diets
`Repletion
`Fatty
`Control
`(DHA + Safflower Oil)
`Acids
`(Soy Oil)
`
`Deficient
`(Safflower Oil)
`
`Commercial
`Monkey Chow
`
`16:0
`18:0
`18:1 (n-9)
`18:2 (n-6)
`Total n-6
`18:3 (n-3)
`20:5 (n-3)
`22:6 (n-3)
`Total n-3
`n-6/n-3
`
`10.7
`4.2
`23.7
`53.1
`53.4
`7.7
`
`7.7
`6.9
`
`7.1
`2.5
`13.3
`76.0
`76.5
`0.3
`
`0.3
`255.0
`
`Values are percentage of total fatty acids.
`
`6.0
`1.8
`9.3
`80.1
`80.6
`0.2
`
`1.6
`1.8
`44.8
`
`19.6
`7.3
`26.3
`37.7
`37.7
`2.3
`0.4
`0.3
`3.3
`11.4
`
`al, and butylated hydroxytoluene (5 mg/100 ml) was
`added as an antioxidant. 18 Retinal phospholipids were
`separated by thin-layer chromatography. 19 Ethanol-
`amine glycerophospholipids were extracted from gel
`scrapings with two washes of 5 ml chloroform-metha-
`nol (1:1, vol/vol), followed by one wash with 5 ml chlo-
`roform-methanol-water (65:45:12 by volume) and one
`more with 5 ml chloroform-methanol (1:1, vol/vol). 20
`Molecular species of ethanolamine glycerophospholi-
`pids were analyzed based on the method described by
`Blank et al.21 Briefly, ethanolamine glycerophospholi-
`pids were hydrolyzed with phospholipase C for 4
`hours at room temperature. 22 Diradylglycerols were
`extracted from the hydrolysate by the Bligh and Dyer
`method,23 and benzoate derivatives were prepared by
`reaction with benzoic anhydride and 4-dimethylamino-
`pyridine for 1 hour at room temperature. 24 The reac-
`tion was stopped with concentrated ammonium hy-
`droxide, and the resulting diradylglycerobenzoates
`were extracted with hexane.
`Diradylglycerobenzoates were separated into the
`alkenylacyl, alkylacyl, and diacyl subclasses by thin-
`layer chromatography on silica gel G with benzene-
`hexane-ethyl ether (50:45:4; vol/vol/vol). Bands were
`scraped into a 1:1 ethanol and water mixture, and the
`diradylglycerobenzoates were extracted with hexane.
`The samples were then filtered (Millex-HV 0.45 nm
`filter unit, Millipore Corp. Bedford, MA), dried under
`nitrogen, and redissolved in acetonitrile-isopropanol
`(70/30 vol/vol) for HPLC injection.
`Separation of molecular species was accomplished
`with a Perkin-Elmer Model 410 LC BioPump system
`fitted with a jiBondapak C18 precolumn insert and a
`3.9 mm X 30 cm analytical column packed with Nova-
`pak C18 (Water Associates, Milford, MA). Peaks were
`monitored at 230 nm with a Perkin-Elmer LC-235
`diode array detector and quantitated on a Perkin-
`Elmer LCI-100 integrator. Molecular species within
`the diacyl-, alkenylacyl- and alkylacyl-glycerobenzoates
`
`were separated by isocratic elution with acetonitrile-
`isopropanol in the ratios of 70:30, 65:35, and 63:37
`(vol/vol), respectively. Column flow rate was 1 ml/min.
`Identification of molecular species was accom-
`plished by comparison with retention times in control
`samples of bovine brain ethanolamine glycerophos-
`pholipid, as established by Blank et al, 21 and by gas
`chromatographic analysis25 of the collected peaks. The
`elution profile was similar to that obtained by Blank et
`al,21 except that four additional species were identified
`as described in our previous paper. 16 The dipolyunsa-
`turated fatty acid molecular species (dipolyenes) were
`identified by the retention times reported by Louie et
`al20 and Stinson et al 9 and by gas chromatographic
`analysis of the collected peaks from HPLC. The stereo-
`specific position of the fatty acids on the glycerol back-
`bone of the dipolyenes was not determined. Such spe-
`cies are reported arbitrarily with the shortest and/or
`least unsaturated fatty acid in the first position.
`Statistical analysis of diet-induced differences in
`levels of individual molecular species was done by one-
`way analysis of variance, followed by post hoc testing
`with the appropriate ^-statistic. 26 Only molecular spe-
`cies showing a significant overall diet effect by analysis
`of variance (P < 0.05) were subjected to the pair-wise
`post hoc testing. The Bonferroni inequality 27 was used
`to control the overall alpha level of the post hoc test-
`ing. This was done by adding pair-wise P values less
`than 0.05. The sum was constructed by ranking the
`pair-wise P values from lowest to highest, progressively
`adding each P value in turn, starting with lowest, until
`either the sum exceeded 0.05 or until all P values had
`been added. Where the sum exceeded 0.05, the last
`pair-wise P value was discarded, and this last compari-
`son was declared insignificant. In cases where the sum
`only slightly exceeded the traditional level of signifi-
`cance (P = 0.05), the value is nevertheless shown to
`indicate a trend in the data. Thus, the P values listed in
`the tables represent Bonferroni adjusted P values that
`
`000003
`
`

`
`Diet and Retinal Phospholipid Molecular Species
`
`apply to the claims of significant differences indicated
`with appropriate superscripts.
`
`RESULTS
`
`Twenty-eight molecular species were identified in the
`retinas of control monkeys (Tables 2 to 4). Retinal eth-
`anolamine glycerophospholipids are composed of
`64%, 31%, and 5% of the diacyl, alkenylacyl, and alkyl-
`acyl subclasses, respectively (Table 5). Dipolyenes were
`found only in the diacyl subclass and comprised 16%
`of the total species. The species with n-3 fatty acids at
`the sn-2 position contributed 59%, 36%, and 70% of
`the total species in the diacyl, alkenylacyl, and alkylacyl
`subclasses, respectively.
`The n-3 fatty acid deficient safflower oil diet had
`its greatest effect upon the molecular species contain-
`
`797
`
`ing 22:6(n-3) in the diacyl subclass of the ethanol-
`amine glycerophospholipids of the retina (Table 2,
`Fig. 1). The deficient diet affected the monopolyene
`molecular species (species containing only one polyun-
`saturated fatty acid), as well as dipolyene molecular
`species (species containing two polyunsaturated fatty
`acids). Most notably, the 22:6(n-3)-22:6(n-3) species
`nearly disappeared, and the 18:0-22:6(n-3) species de-
`creased from 43% to 10% after safflower oil feeding.
`Among the n-3 fatty acid-containing species, 18:1-
`22:6(n-3) was the least affected by the safflower oil
`diet, showing only a slight decrease from 6.2% to
`4.8%, which was not statistically significant. These
`changes were accompanied by reciprocal increases in
`species containing 22:5(n-6), especially 18:0-22:5(n-
`6), which increased from 2.6 to 27.1% (Table 2). New
`dipolyene species, 22:5(n-6)-22:6(n-3) and 22:5(n-6)-
`22:5(n-6), were detected. However, the relative pro-
`
`TABLE 2. Diet-Induced Changes in the Major Molecular Species of Diacyl Ethanolamine
`Glycerophospholipid in Monkey Retina
`Control
`(Soy Oil)
`
`Molecular Species
`
`Deficient
`(Safflower Oil)
`
`Repletion
`(DHA + Safflower Oil)
`
`P Value
`
`Dipolyenes
`20:3(n-6)-22:6
`20:4-22:6
`22:4-22:6
`22:5(n-6)-22:5(n-6)
`22:5(n-6)-22:6
`22:6-22:6
`Unknown
`N-3
`16:0-22:5
`18:0-22:5
`16:0-22:6
`18:0-22:6
`18:1-22:6
`N-6
`16:0-18:2
`18:0-18:2
`16:0-20:3
`18:0-20:3
`16:0-20:4
`18:0-20:4
`18:1-20:4
`16:0-22:4
`18:0-22:4
`18:1-22:5
`16:0-22:5
`18:0-22:5
`N-9/saturated
`16:0-16:0
`17:0-18:1
`16:0-18:1
`18:0-18:1
`18:1-18:1
`
`15.6 ± 3.6
`4.4 ± 1.7
`3.4 ± 0.3
`3.9 ± 1.5
`0.0 ±0.0
`0.0 ±0.0
`2.4 ± 0.7a
`1.5 ± 1.3
`58.9 ± 8.7:1
`0.8 ± 0.3;i
`0.9 ±0.6
`7.8 ± 0.8a
`43.1 ±8.8a
`6.2 ±0.1
`20.7 ± 3.4:1
`0.2 ±0.2
`0.8 ±0.2
`0.8 ±0.8
`0.7 ±0.3
`1.9 ±0.8
`8.3 ± 0.3a
`3.4 ±0.5
`0.2 ±0.3
`1.0 ± 0.5
`0.1 ± 0.1a
`0.6 ± 0.6a
`2.6 ± 1.8a
`4.4 ± 2.6
`0.2 ±0.3
`0.0 ±0.0
`2.1 ±0.9
`1.2 ± 1.1
`0.9 ± 0.5a
`
`17.6 ± 8.0
`2.5 ± 1.1
`2.2 ± 0.4
`5.8 ± 2.5
`2.0 ± 2.4
`2.6 ±3.3
`0.6 ± 1.3a
`2.0 ± 1.4
`16.9 ± 1.6b
`0.3 ± 0.3b
`0.3 ± 0.6
`2.0 ± 0.7b
`9.6 ± 1.4"
`4.8 ± 1.1
`59.7 ± 9.7b
`0.6 ±0.7
`1.0 ±0.7
`0.6 ± 0.4
`1.0 ±0.3
`2.1 ±0.1
`9.6±0.8b
`6.0 ±3.6
`1.4 ±0.9
`3.7 ±2.0
`1.7 ± 0.9b
`5.0 ± 0.9b
`27.1 ±8.2b
`5.4 ± 3.7
`0.8 ± 1.0
`0.0 ±0.0
`2.7 ±2.3
`1.6 ± 1.1
`0.5 ± 0.6a
`
`20.8 ± 2.5
`4.5 ± 1.1
`3.8 ±2.7
`3.6 ±0.8
`0.0 ± 0.0
`0.0 ±0.0
`7.9 ± 4.7b
`1.1 ± 15.
`46.2 ± 2.5'
`0.0 ± 0.0b
`1.2 ± 0.1
`5.8 ± 0.7<:
`35.0 ± 3.6a
`4.2 ±0.6
`22.7 ± 2.3a
`0.0 ± 0.0
`2.0 ± 1.4
`1.0 ± 0.3
`1.4 ±0.3
`1.6 ±0.2
`7.1 ±0.3a
`5.5 ± 1.4
`0.9 ±0.2
`1.8 ±0.6
`0.4±0.1a
`0.0 ± 0.0a
`1.1 ± 0.3a
`8.1 ± 2.1
`1.7 ± 2.0
`0.0 ±0.0
`1.4 ± 0.3
`2.7 ±0.4
`2.3 ± 0.0b
`
`0.042
`
`0.039
`0.049
`
`0.022
`0.002
`
`0.001
`
`0.042
`
`0.061
`0.001
`0.004
`
`0.030
`
`Values are mean ± SD (mol %). Values with unlike superscripts within a given row are different at the indicated P value, which reflects a
`Bonferroni adjustment for all possible pairwise comparisons after ANOVA.
`
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`Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3
`
`TABLE 3. Diet-Induced Changes in the Major Molecular Species of Alkenylacyl Ethanolamine
`Glycerophospholipid in Monkey Retina
`
`Molecular
`Species
`
`N-3
`16:0-22:5
`18:0-22:5
`16:0-22:6
`18:0-22:6
`18:1-22:6
`N-6
`16:0-18:2
`18:0-18:2
`16:0-20:3
`18:0-20:3
`16:0-20:4
`18:0-20:4
`18:1-20:4
`16:0-22:4
`18:0-22:4
`18:1-22:5
`16:0-22:5
`18:0-22:5
`N-9/Saturated
`16:0-16:0
`17:0-18:1
`16:0-18:1
`18:0-18:1
`18:1-18:1
`
`Control
`(Soy Oil)
`
`36.2 ± 3.4a
`1.1 ± 0.4a
`1.6±0.5a
`7.2 ± 0.5a
`20.9 ± 2.la
`5.3 ±2.8
`55.8 ± 4.2a
`0.5 ±0.4
`1.6 ±0.2
`1.5 ±0.9
`1.4 ± 0.4
`11.7 ± 1.2a
`30.9 ±4.2
`4.7 ±0.9
`0.7 ± 0.7a
`2.0 ± 0.8
`0.9 ± 0.2a
`0.0 ± 0.0a
`0.0 ±0.0
`7.2 ± 1.5
`0.1 ±0.2
`1.1 ±0.8
`4.0 ± 1.0
`1.4 ± 1.0
`0.6 ± 0.9a
`
`Deficient
`(Safflower Oil)
`
`Repletion
`(DHA + Safflower Oil)
`
`13.7 ± 3.2b
`0.2 ± 0.2b
`0.0 ± 0.0b
`2.1 ± 0.3b
`6.9 ± 1.0b
`4.5 ±2.3
`78.6 ± 5.2b
`0.9 ± 1.1
`1.3 ±0.9
`1.1 ±0.2
`1.3 ±0.7
`7.0±2.2b
`31.6 ±3.9
`4.5 ± 0.5
`2.9 ± l.lb
`5.3 ± 3.4
`4.9 ± 1.0b
`5.1 ±0.9b
`12.8 ± 1.9
`7.0 ± 5.4
`0.5 ± 0.8
`1.3 ± 1.6
`3.4 ±2.9
`1.2 ± 1.2
`0.7 ± 0.7a
`
`38.4 ± 0.6a
`0.0 ± 0.0b
`1.9±0.7a
`5.6 ± 0.5*
`24.5 ± 2.la
`6.5 ± 1.5
`48.1 ±4.2a
`0.0 ± 0.0
`2.7 ± 1.1
`1.4 ± 0.3
`2.0 ±0.4
`7.4 ± 0.6b
`22.8 ± 1.6
`6.0 ± 1.6
`1.3±0.2ab
`2.8 ±2.1
`1.9±0.1a
`0.0 ± 0.0"
`0.0 ±0.0
`11.1 ± 2.3
`0.0 ±0.0
`3.6 ± 1.7
`2.0 ±0.4
`2.8 ±0.6
`2.8 ± 0.4b
`
`P Value
`
`0.000
`0.007
`0.005
`0.004
`0.000
`
`0.001
`
`0.047
`
`0.021
`
`0.003
`0.000
`
`0.038
`
`Values are mean ± SD (mol %). Values with unlike superscripts within a given row are different at the indicated P value, which reflects a
`Bonfenoni adjustment for all possible pairwise comparisons after ANOVA.
`
`portion of total dipolyene molecular species was not
`significantly affected by the deficient diet.
`The molecular species compositions of the alkenyl-
`acyl and alkylacyl ethanolamine glycerophospholipids
`are shown in Tables 3 and 4, respectively. Interest-
`ingly, there were no dipolyene fatty acid species found
`in these subclasses regardless of dietary background.
`For the monopolyene fatty acid species, the two sub-
`classes were affected by diet in a manner similar to the
`diacyl group, except for the unexpected result that the
`16:0-20:4 alkenylacyl species was significantly reduced
`in the safflower oil group.
`Feeding 22:6(n-3)-supplemented safflower oil to
`n-3 fatty acid-deficient monkeys almost restored their
`retinal phospholipid molecular species composition to
`the pattern found in control monkeys (Tables 2 to 4).
`However, some abnormalities remained. In the alke-
`nylacyl subclass, 16:0-20:4(n-6) remained at the lower
`level induced by safflower oil feeding. In other words,
`this species did not return to control levels when the
`n-3 fatty acid deficiency was alleviated. In the alkylacyl
`subclass, a minor component of the ethanolamine gly-
`cerophospholipids, the reversal was the least pro-
`nounced. In fact, the differences in total n-3 and total
`n-6 molecular species between the deficient and reple-
`
`tion diets did not reach statistical significance. In addi-
`tion, feeding 22:6(n-3) had some effects that went
`beyond reversal of n-3 fatty acid deficiency. In the
`diacyl subclass, the proportion of 22:6(n-3)-22:6(n-3)
`was increased over that found in control animals. An-
`other curious effect of the 22:6(n-3)-supplemented
`diet was a small but significant rise in the proportion
`of 18:1-18:1 in the diacyl and alkenyl subclasses and
`18:0-18:2 in the alkylacyl subclass, compared to both
`control and deficient groups.
`The ratio of sn-1 18:0 species to sn-1 16:0 species,
`for a given sn-2 fatty acid, was different in the differ-
`ent subclasses of ethanolamine glycerophospholipid.
`For instance, in both the diacyl and alkenylacyl sub-
`classes, the predominant fatty acid paired with sn-2
`22:6(n-3) was 18:0. In the alkylacyl subclass, on the
`other hand, sn-1 18:0 and 16:0 were about equally
`paired with 22:6(n-3). For diacyl species with 20:4 in
`the sn-2 position, sn-1 18:1 was more prevalent than
`16:0. The opposite was true in the alkenyl and alkyl
`subclasses. These relationships were not affected by
`diet.
`Because the HPLC detector response is propor-
`tional to the molar concentration of the glyceroben-
`zoate derivatives of the different molecular species, 21
`
`000005
`
`

`
`Diet and Retinal Phospholipid Molecular Species
`
`799
`
`TABLE 4. Diet-Induced Changes in the Major Molecular Species at Alkylacyl Ethanolamine
`Glycerophospholipid in Monkey Retina
`Deficient
`Molecular
`Control
`(Soy Oil)
`(Safflower Oil)
`Species
`33.4 ± 13.0b
`70.2 ± 3.la
`1.3 ± 0.4a
`0.2±0.2b
`1.7 ± 1.0a
`0.0 ± 0.0b
`28.3 ± 3.3a
`11.2±5.9b
`25.4 ± 3.5
`15.2 ±5.9
`13.5±0.7a
`6.9 ± 1.9b
`28.0 ± 2.4a
`58.6 ± 12.2b
`0.3 ±0.3
`1.0 ± 1.2
`0.6 ± 0.5a
`1.4 ± 1.0"
`1.1 ± 1.0
`1.4 ±0.6
`0.6 ±0.6
`1.5 ± 0.9
`8.1 ±2.1
`4.5 ± 2.1
`8.5 ± 1.8
`10.1 ± 1.2
`5.9 ± 3.0
`4.4 ± 2.7
`0.4 ±0.3
`3.5 ±2.1
`0.8 ±0.7
`5.4 ±3.4
`2.8 ± l.lb
`0.6 ± 0.3"
`1.0 ± 0.9a
`9.9 ± 5.2b
`0.0 ± 0.0a
`12.5 ± 5.8b
`2.0 ± 1.5
`5.9 ±5.3
`0.1 ±0.2
`1.4 ±2.8
`0.0 ±0.0
`0.1 ±0.3
`1.9 ± 1.6
`3.5 ±2.5
`0.0 ±0.0
`1.4 ± 2.8
`0.0 ± 0.0
`0.8 ± 1.7
`
`N-3
`16:0-22:5
`18:0-22:5
`16:0-22:6
`18:0-22:6
`18:1-22:6
`N-6
`16:0-18:2
`18:0-18:2
`16:0-20.3
`18:0-20:3
`16:0-20:4
`18:0-20:4
`18:1-20:4
`16:0-22:4
`18:0-22:4
`18:1-22:5
`16:0-22:5
`18:0-22:5
`N-9/Saturated
`16:0-16:0
`17:0-18:1
`16:0-18:1
`18:0-18:1
`18:1-18:1
`
`Repletion
`(DHA + Safflower Oil)
`48.6 ± 2.5b
`0.0 ± 0.0b
`0.0 ± 0.0b
`18.0 ± 4.9b
`17.3 ±0.0
`13.3±2.4;1
`46.9 ± 2.2b
`0.0 ±0.0
`6.7 ± 3.9b
`3.2 ±0.8
`4.7 ±0.4
`3.7 ± 1.6
`14.9 ± 5.4
`3.9 ± 4.2
`2.7 ±0.7
`6.3 ± 3.0
`1.0±0.4a
`0.0 ± 0.011
`0.0 ± 0.0a
`4.7 ± 0.4
`0.0 ± 0.0
`0.0 ± 0.0
`4.7 ± 0.4
`0.0 ± 0.0
`0.0 ± 0.0
`
`P Value
`
`0.049
`0.002
`0.031
`0.004
`
`0.008
`0.061
`
`0.024
`
`0.056
`0.041
`0.020
`
`Values are mean ± SD (mol %). Values with unlike superscripts within a given row are different at the indicated P value, which reflects a
`Bonferroni adjustment for all possible pairwise comparisons after ANOVA.
`
`we were able to calculate the relative concentrations of
`the three subclasses. These data are shown in Table 5.
`In the control (soybean oil) monkey retina, the diacyl,
`alkenylacyl, and alkylacyl subclasses of ethanolamine
`glycerophospholipids were present
`in
`ratios of
`63.9:30.6:5.5. This composition was not significantly
`changed by either the depletion or repletion diet.
`It is of interest that monkeys fed the control soy-
`bean oil diet had almost identical molecular species in
`the retina as those fed the commercial stock diet (Ta-
`ble 6). The stock diet, unlike the soybean oil diet, con-
`tained longer-chain polyunsaturated fatty acids of the
`
`TABLE 5. Distributon of the Subclasses of
`Retinal Ethanolamine Glycerophospholipid
`in Monkeys Fed Different Diets
`
`Phospholipid
`Subclass
`
`Control
`(Soy Oil)
`
`Deficient
`(Safflower Oil)
`
`Repletion
`(DHA +
`Safflower oil)
`
`Diacyl
`Alkenylacyl
`Alkylacyl
`
`63.9 ±9.7
`30.6 ± 10.7
`5.5 ±2.3
`
`63.3 ±5.2
`29.4 ± 7.2
`7.6 ± 1.9
`
`71.0 ± 2.0
`25.3 ±0.1
`3.7 ±2.1
`
`Values are mean ± SD (% of total).
`
`n-3 series, including 22:6(n-3), derived from fish meal.
`Although there was somewhat more of the molecular
`species 22:6(n-3)-22:6(n-3) in the chow group, this dif-
`ference did not attain statistical significance. The total
`dipolyenes were, however, identical at 15.6% and
`15.1% of total species.
`
`DISCUSSION
`
`The data in the present study characterize for the first
`time how the phospholipid molecular species of the
`primate retina may be modified by different diets. Be-
`cause the function of individual phospholipid molecu-
`lar species is unclear at this time, the precise implica-
`tions of our findings are not yet understood. However,
`phospholipids are the major component of the lipid
`bilayer of the cell membrane, and the distribution of
`molecular species has been found to affect cell mem-
`brane fluidity, function, and the activity of membrane-
`bound proteins. 12 Therefore, the large changes in mo-
`lecular species composition seen in the present study
`are likely to have a significant effect on cellular metab-
`olism and function. In this regard, n-3 fatty acid defi-
`cient monkeys were found to have impaired visual
`acuity and abnormal electroretinograms. 11"13 The defi-
`
`000006
`
`

`
`800
`
`Investigative Ophthalmology 8c Visual Science, March 1994, Vol. 35, No. 3
`
`Control
`Repletion
`Deficient
`
`15
`
`10
`
`60
`
`50
`
`40
`
`i> 30
`o
`
`20
`
`10
`
`18:0-22:6
`16:0-22:6 18:1-22:6 22:6-22:6
`FIGURE l. The proportion of some diacyl ethanolamine glycerophospholipids in the retinal
`molecular species containing 22:6(n-3). Data from three different diets are indicated: the
`control diet, the repletion diet and the n-3 deficient diet. *Different from control at P < 0.05.
`
`cient monkeys studied here showed abnormal electro-
`retinogram recovery functions by 3 to 4 months of
`age28 and altered background adaptation at 3 to 4
`years,29 within 2 months of these biochemical studies.
`Furthermore, one important behavior, fluid ingestion,
`was altered in these deficient monkeys.30
`After feeding weanling rats for 15 weeks with hy-
`drogenated coconut oil, safflower oil, or linseed oil
`diets, Wiegand et al31 found that there was replace-
`ment of 22:6(n-3) with 22:5(n-6) molecular species in
`the rod outer segment membrane phospholipids of
`the safflower oil fed animals. Although both the Wie-
`gand study and the current report show dietary effects
`on the molecular species of retina phospholipids,
`there are a few differences between the studies. First,
`they examined isolated rod outer segment membranes
`and found a much higher concentration of 22:6(n-3)-
`22:6(n-3) (13% versus 2% in the whole retinas of our
`control animals). Second, they found few ether-linked
`glycerophospholipids. Third, the effects of diet were
`far less pronounced in their study because the diets
`were fed for a shorter time and were initiated at wean-
`ing rather than at birth, as in our deficient group. For
`example, a safflower oil diet drastically decreased
`16:0-22:6(n-3) and 18:0-22:6(n-3) in monkey retina,
`whereas it had no effect on 16:0-22:6 and decreased
`18:0-22:6(n-3) and 18:l-22:6(n-3) only moderately in
`the rat retina. Fourth, we found that there was conser-
`vation of 18:l-22:6(n-3) in n-3 fatty acid deficient
`monkeys. This preferential sparing may indicate a spe-
`cial role for 18:l-22:6(n-3).
`To explore further the differences among the
`various 22:6(n-3)-containing and 22:5(n-6)-containing
`molecular species, we calculated the ratios of the spe-
`cies having different fatty acids in the sn-1 position.
`
`For example, in the control (soybean oil) group, the
`18:0-22:6(n-3)/16:0-22:6(n-3) ratios were 5.5, 2.9,
`and 0.9 in the diacyl, alkenylacyl, and alkylacyl sub-
`classes, respectively. These ratios were unchanged in
`the deficient (safflower oil) group. The analogous ra-
`tios for species containing 22:5(n-6), which rose in
`concentration in the deficient group, were similar at
`5.4, 2.5, and 1.7 for the deficient group. It is interest-
`ing that similar ratios were also obtained in the cere-
`bral cortex of monkeys fed these diets.16 On the other
`hand, the relative amounts of species containing
`22:6(n-3) and 22:5(n-6) paired with sn-1 18:1, as op-
`posed to sn-1 18:0 and 16:0, were different. As noted
`above, among all major species containing 22:6(n-3),
`we found that 18:l-22:6(n-3) showed the least change
`(indeed, no significant change) between the control
`and the deficient diets. In the diacyl subclass, there
`were 23%, 74%, and 78% decreases in the concentra-
`tions of 18:l-22:6(n-3), 16:0-22:6(n-3), and 18:0-
`22:6(n-3), respectively. Thus, deficiency did not affect
`these three 22:6(n-3)-containing species in an equal
`manner. Species with 18:1 in the sn-1 position were
`more conserved when 22:6(n-3) was in short supply. A
`similar but less striking differential change in 18:1-
`22:6(n-3) was observed in the cerebral cortex of mon-
`keys fed the safflower oil diet.14
`From an experiment of injecting rats intravitreally
`with 3H-glycerol, Stinson et al32 concluded that each
`glycerolipid molecular species has an unique rate of
`biosynthesis and turnover that determines the steady-
`state level of each species in rod outer segments. Simi-
`larly, Nakagawa and Horrocks33 reported that the in-
`corporation rates of intracerebrally injected 3H-ara-
`chidonic acid into the 18:0-20:4(n-6), 16:0-20:4(n-6),
`and 18:l-20:4(n-6) species of alkenylacyl, alkylacyl,
`
`000007
`
`

`
`Diet and Retinal Phospholipid Molecular Species
`
`801
`
`TABLE 6. Comparison of the Diacyl
`Ethanoamine Glycerophospholipid
`Molecular Species in the Retinas of Monkeys
`Fed a Soybean Oil-Based Control Diet or a
`Commercial Chow Diet
`
`Control Diets
`
`Soybean Oil*
`
`Commercial Diet
`
`Dipolyenes
`22:6-22:5(n-6)
`22:5(n-6)-22:5(n-6)
`20:3(n-6)-22:6
`20:4-22:6
`22:4(n-6)-22:6
`22:6-22:6
`Unknown
`N-3
`16:0-22:5
`18:0-22:5
`16:0-22:6
`18:0-22:6
`18:1-22:6
`N-6
`16:0-18:2
`18:0-18:2
`16:0-20:3
`18:0-20:3
`16:0-20:4
`18:0-20:4
`18:1-20:4
`16:0-22:4
`18:0-22:4
`18:1-22:5
`16:0-22:5
`18:0-22:5
`N-9 Saturated
`16:0-16:0
`17:0-18:1
`16:0-18:1
`18:0-18:1
`18:1-18:1
`
`15.6 ±3.6
`0.0 ±0.0
`0.0 ±0.0
`4.4 ± 1.7
`3.4 ±0.3
`3.9 ± 1.5
`2.4 ±0.7
`1.5 ± 1.3
`58.9 ± 8.7
`0.8 ±0.3
`0.9 ± 0.6
`7.8 ±0.8
`43.1 ± 8.8
`6.2 ±0.1
`20.7 ± 3.4
`0.2 ±0.2
`0.8 ±0.2
`0.8 ±0.8
`0.7 ±0.3
`1.9 ± 0.8
`8.3 ±0.3
`3.4 ±0.5
`0.2 ±0.3
`1.0 ± 0.5
`0.1 ±0.1
`0.6 ±0.6
`2.6 ± 1.8
`4.4 ± 2.6
`0.2 ±0.3
`0.0 ±0.0
`2.1 ±0.9
`1.2 ± 1.1
`0.9 ± 0.5
`
`* From Table 3, mean ± SD (mol %).
`f/5 < 0.01.
`
`15.1 ± 1.9
`0.0 ±0.0
`0.0 ±0.0
`3.4 ± 0.4
`2.8 ±0.6
`2.6 ±0.3
`5.1 ± 1.7
`1.2 ±0.7
`63.3 ± 2.1
`0.7 ±0.1
`1.2 ±0.1
`8.8 ±0.5
`47.4 ± 2.1
`5.3 ±0.7
`17.9 ± 1.3
`0.3 ±0.2
`0.2 ±0.If
`0.6 ±0.3
`0.7 ±0.2
`1.9 ±0.6
`7.1 ± 1.6
`4.0 ± 1.1
`0.2 ±0.2
`1.0 ± 0.7
`0.1 ±0.1
`0.5 ±0.5
`1.2 ±0.3
`3.9 ± 1.4
`0.6 ± 1.0
`0.0 ±0.0
`1.1 ±0.8
`1.5 ± 0.6
`0.7 ±0.4
`
`and diacyl ethanolamine glycerophospholipids of rat
`brain were different. They concluded that there must
`have been a different specificity toward the ethanol-
`amine glycerophospholipids with different fatty acid
`chains at the sn-1 position.
`Retinal membranes are dynamic structures whose
`components are constantly being renewed. Primate
`retinal outer segment membranes are completely re-
`placed every 10 to 14 days. Renewal of outer segment
`membrane lipids proceeds by two mechanisms: mem-
`brane replacement and molecular replacement.34 De-
`spite the rapid replacement of outer segment mem-
`branes, however, 22:6(n-3) is retained in the retinas of
`
`adult animals even when they are fed an n-3 fatty acid
`deficient diet for several months.31 This retention ap-
`parently results from mechanisms for the recycling of
`22:6(n-3) back to the photoreceptors from the retinal
`pigment epithelium